Cellular Signalling 26 (2014) 683–690
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TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages☆ Gerhard Ruspi a, Emily M. Schmidt a, Fiona McCann a, Marc Feldmann a, Richard O. Williams a, A. Allart Stoop b, Jonathan L.E. Dean a,⁎ a Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road Campus, Roosevelt Drive, Headington, Oxford OX3 7FY, United Kingdom b Innovation Biopharm Discovery Unit, Biopharm R&D, GlaxoSmithKline, Cambridge CB4 0WG, United Kingdom
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Article history: Received 29 October 2013 Received in revised form 19 December 2013 Accepted 22 December 2013 Available online 27 December 2013 Keywords: TNF TNFR p55 p75 TRAF2 Macrophage
a b s t r a c t Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NFkappaB pathways was blocked in TNFR1−/− macrophages. In contrast, although TNFR2−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB proinflammatory signalling pathways. © 2013 Elsevier Inc. All rights reserved.
1. Introduction There are two separate TNF receptors: the type I receptor (TNFR1, p55, or p60) and the type II receptor (TNFR2, p75, or p80). Both receptors are widely expressed but TNFR1 is thought to be the major receptor required by a wide variety of cells for activation of the pro-inflammatory NF-κB and MAPK signalling pathways which in turn induce the expression of proteins of the inflammatory response (for reviews see [13,17,31]).
Abbreviations: TNF, tumour necrosis factor; TNFR, TNF receptor; TRAF2, TNF receptorassociated factor 2; MAPK, mitogen-activated protein kinase; P-p38, phospho-p38 MAPK; NF-κB, nuclear factor-κB; LPS, lipopolysaccharide; MCSF, macrophage colony stimulating factor; GM-CSF, granulocyte/macrophage colony stimulating factor; I-κB, inhibitor of κB; BMDM, bone marrow-derived macrophages; PBMC, peripheral blood mononuclear cells. ☆ The authors claim no conflict of interest. This study was funded by GlaxoSmithKline, Arthritis Research UK and the Kennedy Institute of Rheumatology Trust. MF was a consultant for GlaxoSmithKline. ⁎ Corresponding author. Tel.: +44 1865 612641. E-mail address: [email protected] (J.L.E. Dean). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.009
In contrast, TNFR2 displays cell-type specific expression and is the major TNFR expressed on activated T cells in which it signals the induction of apoptosis [9,35] in a process involving sensitisation to TNF [9]. TNFR2 is also constitutively expressed in regulatory T cells and plays a role in their activation, proliferative expansion, and survival [7]. Endothelial cells express more equal levels of both receptors [20], but in these cells, TNFR1 is thought to be the major receptor involved in the activation of the NF-κB and MAPK pathways and subsequent induction of inflammatory response proteins [4,21,24,36]. Immortalised macrophages are reported to express both receptors with TNFR1 being expressed weakly [23]. Both TNFR1- and TNFR2deficient macrophage cell lines display reduced activation of the NFκB and MAPK pathways in response to TNF compared to immortalised wild-type macrophages [23]. Experiments employing artificial ligands for the different receptors, also called muteins, have shown that TNFR2 cannot significantly activate pro-inflammatory signalling pathways independently of TNFR1 [6,8]. Physiological signalling functions of TNFR2 that are distinct from TNFR1 have proved elusive to identify. In cell lines, TNFR2 has been
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reported to induce TRAF2 [18,33] and ASK1 [34] protein degradation, and to regulate protein kinase B expression [16]. Recently, it has been shown in primary T cells and in tumour cell lines to activate the alternative NF-κB pathway [26]. Membrane-bound TNF has been shown to be a more potent activator of TNFR2 than the soluble form of the ligand [12,26]. In addition, TNFR2 shedding in response to TNF serves to neutralise TNF and inhibit signalling [25]. TNFR2 has been reported to regulate apoptosis via a mechanism of ‘ligand-passing’, shown to involve TNFR2-mediated facilitation of the activation of TNFR1 [29]. In this mechanism, TNF binds much more rapidly to TNFR2 than TNFR1. When both receptors are in close proximity, the presence of TNFR2 increases the association rate of TNF with TNFR1 thereby sensitising the cell to TNFR1-mediated cytotoxicity [29]. However, TNFR2 is able to drive apoptosis independently of TNFR1, and cooperation between TNFR1 and TNFR2 in activating the NF-κB pathway has also been found to be additive rather than synergistic [32]. Since the TNFR1 receptor has been more strongly implicated in the activation of pro-inflammatory signalling pathways than TNFR2, blockade of TNFR1 appears a logical choice for therapy of chronic inflammatory diseases. Indeed, TNFR1 expressed on mesenchymal cells has been shown to play an important role in arthritis in mice [1]. TNFR2 has also been suggested to be involved in diverse processes that may be of beneficial function and thus its blockade could have deleterious implications. These include diverse neuro-, cardio-, and osteo-protective effects of TNFR2 suggested from experiments in TNFR−/− mice. Activation of TNFR2 by TNF inhibits seizures [2] and attenuates cognitive dysfunction following brain injury [19]. TNFR2 appears not to affect myocardial infarct size, but does promote survival following myocardial infarction in mice [22]. Similarly, TNFR2 has been suggested to protect against myocardial ischaemia/reperfusion injury [10], and to reduce remodelling and hypertrophy following heart failure [14]. Furthermore, in experimental arthritis, TNFR2 has also been shown to protect against joint inflammation and erosive bone destruction through regulation of osteoclastogenesis [27]. Given the wide range of protective functions of TNFR2, and the lack of a clear consensus on the signalling function of this receptor, we sought to identify unique physiological TNFR2-dependent signalling processes that are distinct from TNFR1. We have focused on TRAF2 protein degradation, the TNFR2-dependent signalling event that is most strongly implicated from previous studies in cell lines. To see if TRAF2 degradation occurs in primary cells we have investigated the ability of TNFR2 to induce this process in primary wild-type and TNFR1- or TNFR2-deficient macrophages. LPS-treated macrophages strongly express TNF, and this allowed us to test the activation of the alternative NF-κB pathway by endogenous membrane-bound TNF. Since these cells express both TNF receptors we also investigated if, according to the ‘ligand-passing’ model, TNFR2 sensitises primary macrophages for activation of classical pro-inflammatory signalling pathways.
2. Materials and methods 2.1. Materials Lipopolysaccharide (LPS) (TLR-grade) was purchased from Alexis Biochemicals (Exeter, UK). Macrophage colony stimulating factor (MCSF) and TNF (human cells were treated with human TNF; murine cells were treated with murine TNF) and human granulocyte/ macrophage colony stimulating factor (GM-CSF) were from PeproTech (London, UK). Antagonistic anti-murine TNFR1 (55R-170) and antimurine TNFR2 (TR75-32.4) monoclonal antibodies were from BioLegend (Cambridge, UK). Antibodies for western blotting (phospho(Thr180/ Tyr182)-p38 MAPK, p38 MAPK, IκBα, TRAF2, p100/p52) were from Cell Signalling (Danvers, Massachusetts, USA) or (tubulin) Sigma-Aldrich (St. Louis, Missouri, USA). Anti-TNFR1 (FAB225P (Clone #16803)) and anti-TNFR2 (FAB226P (Clone #22235)) phycoerythrin-conjugated
antibodies for flow cytometry were from R&D Systems (Abingdon, UK). General lab reagents were from Sigma-Aldrich.
2.2. Mice Wild-type mice of C57BL/6 background were from Harlan Laboratories (Wyton, UK). TNFR1−/− and TNFR2−/− mice (C57BL/6 background) were maintained as heterozygotes and have been previously described [37]. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.
2.3. Cell culture Bone marrow-derived macrophages (BMDM) were isolated and differentiated as described previously by Hitti et al. [15]. Briefly, bone marrow cells were harvested from the femurs and tibiae of 10–12 week-old mice and were incubated for 7 days in the presence of MCSF (100 ng/ml). BMDM were cultured in DMEM supplemented with 10% (v/v) FCS, 2 mM L-glutamine (PAA, Yevil, UK), penicillin and streptomycin and 0.1% (v/v) 2-mercaptoethanol (Life Technologies, Paisley, UK). Centrifugal elutriation was used to isolate monocytes from human peripheral blood mononuclear cells (PBMC). Briefly, human PBMC were obtained by density centrifugation through ficoll/hypaque of a leukoreduction system chamber of apheresis instruments after routine platelet collection. The resulting PBMC were centrifugally elutriated in 1% (v/v) heat-inactivated FCS in RPMI in a Beckman JE6 elutriator (Beckman, High Wycombe, UK). The monocytes separated by this method were assessed for purity (≥75%) by flow cytometry (FACScan, BD, Oxford, UK) analysis of forward scatter and side scatter. Monocytes were differentiated into macrophages by treatment with human GM-CSF (50 ng/ml) for 6 days or human MCSF (100 ng/ml) for 5 days respectively. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.
2.4. Western blot In experiments analysing phospho-p38 MAPK, IκBα and p100/p52, cells were lysed for 10 min on ice with ≤200 μl of whole cell lysis buffer (50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% (v/v) Triton X-100, 0.5% (v/v) NP-40, 10% (v/v) glycerol) supplemented with protease inhibitor cocktail (1% v/v; Sigma-Aldrich), NaF (200 μM), NaVO3 (100 μM), microcystin (1 μM) and DTT (1 mM). Samples were clarified by centrifugation at 16,000 ×g, for 10 min at 4 °C. For TRAF2 degradation experiments cells were lysed on ice for 30 min with ≤ 100 μl of a mild lysis buffer (30 mM Tris–HCl, 120 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100) supplemented with protease inhibitors (1% v/v) and DTT (1 mM). Samples were clarified by centrifugation at 14,000 ×g for 30 min at 4 °C. Proteins in lysates (≥20 μg) were separated by electrophoresis through 10% (w/v) polyacrylamideSDS gels and transferred to a polyvinylidene fluoride membrane (DuPont, Stevenage, UK). Membranes were blocked in 5% (w/v) dried-fat milk powder (Premier International Foods, Spalding, UK) and incubated in primary antibody and secondary antibody coupled to horseradish peroxidase (Dako, Glostrup, Denmark). Protein detection was carried out by enhanced chemiluminescence (GE Healthcare, Amersham, UK). Films were scanned with ImageScanner III (GE Healthcare). Densitometry of digital images with background subtraction was performed using Quantity One software (BioRad) and data analysis was performed using Excel (Microsoft). Error bars are shown for all data except for normalised values but in some cases are too small to be visible.
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2.5. Assessment of TNF receptor expression by flow cytometry
detected in BMDM at 1 h and was sustained until 16 h. TRAF2 degradation was observed up to 4 h TNF treatment in two separate experiments (as shown graphically in Fig. 1A). Next, we examined TRAF2 degradation at 6 h over a range of TNF concentrations. The effect of TNF treatment was maximal at 30 and 100 ng/ml TNF (Fig. 1B).
Differentiated human macrophages were harvested by cell scraping. Cells were blocked with FcR block (Miltenyi Biotec, Bisley, UK), and treated isotype control IgGs (BD) or stained with fluorochromelabelled antibodies for 30 min at room temperature in PBS supplemented with 2% (v/v) FCS and 2 mM EDTA (final concentrations). Analysis was carried out on a FACS Canto II (BD) and using FlowJo™ software (Treestar, Ashland, USA).
3.2. TRAF2 protein degradation in BMDM is TNFR2-dependent To investigate which receptor is required for TRAF2 degradation induced by TNF, wild-type, TNFR1−/− and TNFR2−/−, BMDM were treated with 30 ng/ml TNF for 4 h and TRAF2 degradation was analysed by western blot, as before. TNF treatment resulted in TRAF2 degradation in wild-type and TNFR1−/− BMDM but not in TNFR2−/− cells (Fig. 1C). Thus in macrophages, TNFR2, but not TNFR1, is necessary for ligand-induced TRAF2 degradation, as reported previously in other cell types. In some blots, such as the ones shown in Fig. 1A and C, anti-TRAF2 antibody detected TRAF2 protein as a doublet. The precise reason for this is unclear but the data suggest that under certain gel running conditions two different forms of TRAF2 protein are resolved.
3. Results 3.1. Function of the TNFR2 in TNF-induced TRAF2 protein degradation: time and concentration dependence Signalling via TNFR2 induced by high concentrations of soluble TNF has previously been shown to cause TRAF2 protein degradation [18,33], but this has not been reported in macrophages. To determine if TRAF2 degradation occurs in murine macrophages, and to identify which receptor is responsible for this with the use of receptordeficient macrophages, it was firstly necessary to determine the time and concentration dependence of the process in BMDM. For this cells were treated with a high dose (100 ng/ml) of TNF and harvested at different times. Cells were then lysed using a mild lysis buffer containing a low concentration of sodium chloride and Triton X-100 to isolate the TNF-regulated pool of TRAF2. Cell lysates were analysed for TRAF2 protein by western blot. In the blot shown in Fig. 1A TRAF2 degradation was
3.3. TNF induces TRAF2 degradation in human macrophages but not monocytes To investigate whether TRAF2 degradation occurs in human macrophages in response to TNF, monocytes were isolated by elutriation and differentiated into M1- or M2-like macrophages using GM-CSF or
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Fig. 1. Timecourse and TNF concentration and TNFR dependence of TRAF2 protein degradation in BMDM. A, Wild-type (WT) BMDM were treated with TNF (100 ng/ml) for different times or B, at the concentrations indicated for 6 h. Cells were lysed with mild lysis buffer (See Materials and methods) and lysates analysed by western blot for TRAF2 (56 kDa) and tubulin (50 kDa). Dotted line indicates lanes removed from original blot for clarity of presentation. Relative molecular masses are shown in kilodaltons (kDa). Plots of data from densitometric analysis show mean TRAF2/tubulin protein ± SD for two independent experiments normalised for untreated cells. C, WT, TNFR1−/− (R1−/−) and TNFR2−/− (R2−/−) MCSFdifferentiated BMDM were treated with TNF (30 ng/ml) for 4 h or left untreated and western blots performed as above. Plots as for (A) but normalised for untreated WT, R1−/− and R2−/− cells. Data for R1−/− and R2−/− cells are indicated as hashed or black bars respectively.
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MCSF, respectively. Cells were then treated with a high dose human TNF (30 ng/ml) for 4 h, or left untreated. Undifferentiated monocytes were treated in the same way. Both M1- and M2-like human macrophages displayed TRAF2 degradation in response to TNF, but human monocytes did not (Fig. 2A). Given that in murine macrophages TNFR2 is required for TRAF2 degradation (Fig. 1C), we decided to assess the expression of the two TNF receptors on human macrophages and monocytes. Cells were analysed by flow cytometry using antibodies to TNFR1 or TNFR2, and isotype control IgGs. This showed that monocytes differentiated into macrophages using either GM-CSF, or MCSF, express similar levels of both TNF receptors, but human monocytes display greater TNFR1 expression than macrophages (Fig. 2B). The predominance of TNFR1 as opposed to TNFR2 expression on human monocytes compared to macrophages (Fig. 2B) correlates with the apparent lack of TNF-induced TRAF2 degradation in these cells (Fig. 2A). 3.4. Role of TNFR2 in TNF-induced tolerance Since signalling by TNFR2 induces TRAF2 degradation, and because TRAF2 is required for the activation of MAPKs and the NF-κB pathway, we theorised that the phenomenon of tolerance of TNF signalling induced by TNF pre-treatment might involve TRAF2 protein degradation. This may identify TRAF2 degradation as a bonafide signalling event with
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functional consequences. To test this, wild-type and TNFR2−/− BMDM were treated with TNF (30 ng/ml) according to the regimens shown in Fig. 3A. Cells were left untreated at t = 0 and then treated with the same dose of TNF at 6 h to induce signalling, or cells were treated with TNF at t = 0 to induce tolerance and, either left untreated, or treated with TNF at 6 h. Some cells were left untreated and served as a control. All cells were harvested at 6 h 15 min. Treatment of wild-type or TNFR2−/− BMDM with a single high dose of TNF (30 ng/ml) resulted in strong p38 MAPK phosphorylation (Fig. 3B; lane 3), consistent with its activation, but this was inhibited by a prior 6 h TNF treatment (Fig. 3B; lane 4). The ability of the initial TNF treatment to block signalling was not impaired in TNFR2−/− BMDM (Fig. 3B; compare lanes 4 and 8). In both wild-type and TNFR2−/− BMDM treated with a single dose of TNF at t = 0, there was no detectable p38 MAPK activation following 6 h 15 min of treatment (Fig. 3B; lanes 2 and 6), consistent with resolution of signalling. TRAF2 degradation occurred in wild-type cells that were treated with TNF at t = 0 (Fig. 3B; lanes 2 and 4), but not in TNFR2−/− cells treated in the same way (Fig. 3B; lanes 6 and 8), as predicted. In order to rule out the involvement of TRAF2 degradation in TNFinduced tolerance, similar experiments were performed in wild-type cells using a low concentration of TNF which fails to induce TRAF2 degradation. TNF-induced tolerance of p38 MAPK activation was also
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Fig. 2. Induction of TRAF2 protein degradation by TNF in human monocytes differentiated into macrophages using GM-CSF or MCSF but not in undifferentiated human monocytes. A, Human monocytes were left untreated or treated with TNF (30 ng/ml) for 4 h. Monocytes were also differentiated into macrophages (MΦ) using GM-CSF or MCSF and treated as above. As described in Fig. 1, cells were lysed and lysates were analysed by western blot and data for normalised values for untreated cells for each cell type plotted. B, Monocytes were either left untreated, or differentiated into macrophages using GM-CSF or MCSF and TNFR1 and TNFR2 protein expressions assessed by FACS analysis with anti-TNFR1 or antiTNFR2 antibodies, or using isotype control IgGs. Similar results were obtained for (A) and (B) using cells from two separate donors.
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Fig. 3. TNFR2 is not required for tolerance of p38 MAPK activation induced by TNF in BMDM. A, Schematic of treatment protocol: Murine wild-type (WT) and TNFR2−/− (R2−/−) macrophages were left untreated (Lanes 1, 5), treated with TNF (30 ng/ml) at t = 0 (Lanes 2, 6), and then further treated with TNF at t = 6 h (lanes 3, 7) or, treated with TNF at both 0 and 6 h (Lanes 4, 8). Cells were not washed and culture medium was not changed in-between treatments. Cells were harvested and lysed at t = 6 h, 15 min. B, Wild-type and TNFR2−/− cells were treated with TNF according to lane numbers as indicated in (A). C, Wild-type BMDM cells were treated according to lane numbers with 1 ng/ml TNF as in (A) or left untreated. In (A) and (B) cells were lysed with whole cell lysis buffer (see Materials and methods) and lysates were analysed by western blot for phospho-p38 MAPK (P-p38); (41 kDa), TRAF2 and tubulin. Plots show mean phospho-p38/tubulin protein ± SD and mean TRAF2/tubulin protein ± SD normalised to cells treated with TNF for 6 h, or untreated cells, respectively from two independent experiments for (B) and two independent experiments, one of which was performed in duplicate for (C).
observed at a low concentration of TNF (1 ng/ml) (Fig. 3C; compare lanes 3 and 4) although in this case the inhibition of p38 MAPK activation induced by TNF pre-treatment was slightly less than that observed using 30 ng/ml TNF (Fig. 3B; lane 4). Western blot for TRAF2 protein confirmed that treatment of cells with 1 ng/ml TNF did not cause TRAF2 degradation (Fig. 3C; lanes 2 and 4). Overall the data suggest that TNF-induced tolerance of p38 MAPK activation occurs independently of TRAF2 degradation. 3.5. Neither TNF, nor LPS activate the alternative NF-κB pathway in BMDM We next sought to test the involvement of TNFR2 in another signalling event which has been previously reported to be specifically activated by this TNF receptor, namely the activation of the alternative NF-κB pathway. Ligand-induced activation of TNFR2 has previously been shown to activate the alternative NF-κB pathway by the processing of p100 to p52 [26]. To test whether TNF induces p100 processing in BMDM, cells were treated with a high concentration of TNF (20 ng/ml) for the times shown and lysates were analysed for p100 and p52 protein. No
reduction in p100 protein or accumulation of p52 protein could be detected for TNF treatment between 1 and 4 h (Fig. 4A), indicative of an inability of TNF to induce p100 processing in murine macrophages. Since membrane-bound TNF has been suggested to be a more potent stimulus for the TNFR2 receptor than soluble TNF [12,26], cells were also treated with LPS for 1–4 h to induce TNF expression and p100 processing was analysed as before. As for TNF, LPS treatment failed to induce activation of the alternative NF-κB pathway in BMDM (Fig. 4B). However, the antibody used to detect p100 and the cleaved product, p52, also detected a weakly stained band (indicated by *) representing a 40 kDa protein which was induced by both TNF and LPS (Fig. 4A and B). The identity of this protein is unclear but it may represent a cleaved species of p100, or p52, which is weakly induced following cell stimulation. 3.6. TNFR2 sensitises macrophages for ligand-induced activation of the p38 MAPK and NF-κB pathways We were also interested in examining the function of the TNFR2 receptor in activating pro-inflammatory signalling pathways at different
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Fig. 4. Neither TNF, nor LPS treatment, results in activation of the alternative NF-κB pathway in BMDM. A, MCSF-differentiated BMDM were treated with TNF (20 ng/ml) for the times shown, cells were lysed as in Fig. 1 and lysates analysed for p100 processing using an antibody that recognises both p100 (97 kDa) and the processed form p52 (50 kDa). An additional band representing a 40 kDa species (indicated by *) was also detected which was induced following cell stimulation. B, As for (A) but cells were treated with LPS (100 ng/ml).
concentrations of TNF as this would allow any sensitisation of signalling due to ligand-passing to TNFR1 to be detected. We focused on the NF-κB and p38 MAPK pathways since these are both strongly implicated in the expression of proteins of the inflammatory response. To investigate signalling in primary macrophages it was firstly necessary to check that the p38 MAPK and NF-κB pathways are activated at low concentrations as well as at high concentrations of TNF. To test this, wild-type BMDM were treated with different concentrations of TNF for 15 min and p38 MAPK activation was assessed by western blot as before. Treatment of wild-type BMDM with concentrations of TNF as low as 0.3 ng/ml resulted in activation of phosphorylation of p38 MAPK (Fig. 5). To investigate the involvement of either TNFR1 or TNFR2, BMDM from TNFR1- and TNFR2-deficient mice were used. TNF-induced activation of signalling at low doses of TNF (≤3 ng/ml) and at doses which are reported to activate TNFR2 most strongly (10 and 30 ng/ml) was examined. Little activation of p38 MAPK occurred in TNFR1−/− BMDM (Fig. 5). Surprisingly, at low doses, TNF-induced signalling was also impaired in TNFR2−/− cells in which the ligand concentration dependence of the activation of p38 MAPK was increased ~ 10-fold compared to wild-type cells (Fig. 5). The TNF concentration dependence of IκBα degradation in both wild-type and knockout cells was identical to that observed for p38 MAPK activation (Fig. 5). Thus at high doses of soluble TNF, TNFR1 is the main receptor required for activation of signalling and the presence of TNFR2 is not required for activation. However, at lower concentrations of TNF, TNFR2 is essential for full activation of signalling. A possible caveat in the above interpretation is that knockout cells may adapt in response to receptor deficiency and the results could be misleading. To address this we also investigated the signalling function of TNFR1 and TNFR2 using receptor-specific monoclonal antibodies. Wild-type BMDM were pre-treated with an IgG control, anti-TNFR1 mAb, anti-TNFR2 mAb and a murine form of soluble TNFR2 coupled to an Fc region (mTNFR2-Fc) as a control to block signalling by both
receptors. Cells were then stimulated with a low concentration of TNF (0.3 ng/ml) or left untreated. Anti-TNFR1 mAb strongly inhibited TNFinduced p38 MAPK signalling and IκBα degradation (Fig. 6A). In agreement with the result in TNFR2−/− cells, the anti-TNFR2 mAb also strongly inhibited signalling at a low concentration of TNF (Fig. 6A). Similar experiments were performed at a high concentration of TNF (10 ng/ml), and consistent with the results from receptor-deficient macrophages, the anti-TNFR1 mAb and mTNFR2-Fc blocked signalling, albeit rather weakly, but anti-TNFR2 mAb did not (Fig. 6B). Thus at high TNF concentrations, TNFR1 is the dominant receptor which activates signalling in the absence of TNFR2, whereas both receptors are essential for the activation of signalling at lower concentrations of TNF. 4. Discussion TNFR1 is known to activate the NF-κB and MAPK pathways in response to TNF in a wide range of cell types. In this study, induction by TNF of p38 MAPK phosphorylation and IκBα degradation was completely blocked in TNFR1−/− macrophages at all concentrations of TNF used, underscoring a dominant role for this receptor and showing that TNFR2 cannot significantly activate these pathways independently of TNFR1. The literature suggests that the role of TNFR2 in signalling is more complex. In macrophages, we find that at high concentrations of its ligand, TNFR2 is needed for TRAF2 protein degradation, whereas at low ligand concentrations, TNFR2 serves to sensitise the cells for activation of proinflammatory signalling. Our findings in primary cells differ from those in immortalised macrophage lines in which TNFR2 deficiency almost entirely blocked signalling induced by a wide range of TNF concentrations that encompassed the range used in this study [23]. A possible explanation is that, unlike in primary TNFR2-deficient macrophages, TNFR1 expression was reduced in the TNFR2-deficient line following extended passaging. Our observation that even sub-ng/ml concentrations of TNF are sufficient to strongly activate the p38 MAPK and NF-κB pathways in cells in culture suggests that our findings are very relevant to any situation in vivo where small amounts of TNF are present. The majority of previous studies reporting TNFR2-mediated TRAF2 degradation have utilised cell lines and receptor overexpression, although during the course of this work it has also been reported to occur in primary CD8 + T cells [30]. Treatment of both human and murine primary macrophages with high concentrations of TNF caused a reduction in TRAF2 protein. The loss of TRAF2 protein in murine macrophages was exclusively TNFR2-dependent as the TNF-induced reduction in TRAF2 protein was blocked by TNFR2, and not by TNFR1 deficiency. TRAF2 degradation was shown to occur in human macrophages, but the very high concentrations of soluble TNF needed for maximal TRAF2 degradation (30 ng/ml) in BMDM suggest that this process may be most relevant to physiological settings where localised TNF expression is extremely high or where presence of membrane TNF would favour TNFR2 signalling. TRAF2 plays an important role for the activation of the major signalling pathways downstream of TNF [3] including the p38 MAPK pathway [5]. The lack of a requirement for TNFR2-mediated TRAF2 degradation in TNF-induced tolerance of activation of p38 MAPK suggests that, in macrophages, TRAF2 degradation may serve some other purpose, such as the previously reported inhibition of the NF-κB pathway [11]. We failed to detect any evidence for activation of the alternative NF-κB pathway in response to soluble TNF or LPS in macrophages. The results suggest that under the conditions used either LPS fails to induce a significant amount of membrane-bound TNF in macrophages, or endogenous membranebound TNF does not activate the alternative NF-κB pathway in macrophages. To investigate TNF-induced gene expression, the cytokine is frequently reported to be used to treat cells at a concentration of ≥10 ng/ml which in BMDM lies well above the concentration that maximally activates signalling. Thus the important contribution of TNFR2 to signalling at sub-ng/ml concentrations of TNF suggests that many
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Fig. 5. TNFR1 is the major receptor needed for TNF-induced p38 MAPK and NF-κB pathway activation in BMDM but TNFR2 is also required at a low concentrations of TNF. Murine wildtype, TNFR1−/− and TNFR2−/−BMDM were treated with the concentrations of TNF indicated for 15 min. Similar results were obtained in two separate experiments. Western blot analysis of BMDM lysates (prepared as in Fig. 3) for phospho-p38 MAPK, IκBα (35 kDa) and tubulin is shown. Plots show mean phospho-p38/tubulin ± SD and mean IκBα/tubulin ± SD from duplicate blots normalised to values for 30 ng/ml TNF-treated wild-type cells, or untreated wild-type cells, respectively.
previous studies may have underestimated the importance of TNFR2 in the induction and regulation of gene expression in response to TNF. This may be particularly relevant for cell types such as endothelial cells which express both TNF receptors and in which TNF strongly induces the expression of a wide range of proteins, including inflammatory cytokines and adhesion molecules. It is noted that TNFR2 has been previously reported to play an important role in tissue factor expression in endothelial cells treated with low but not high concentrations of TNF [8]. Our data also suggest that under situations where the concentration of TNF is high, such as in inflammation, TNFR2 may have little role in the activation of pro-inflammatory signalling. However, where TNF levels are low, TNFR2 may be important for the initiation of inflammation and its subsequent autoregulated resolution. It is tempting to speculate that as opposed to chronic inflammation in which resolution may be dysregulated, TNFR2-dependent signalling induced by small amounts of TNF may activate signalling, its normal resolution, and the associated anti-inflammatory effects, thus providing a additional explanation for the previously reported anti-inflammatory functions of TNFR2 in mice. It has been reported recently that TNFR2-mediated TRAF2
degradation is needed for resolution of NF-κB pathway activation in 293 cells [28]. 5. Conclusions In macrophages high concentrations of soluble TNF induce TRAF2 protein degradation via TNFR2. TRAF2 degradation does not appear to be required for TNF-induced tolerance of p38 MAPK activation and TNF does not appear to induce TRAF2 degradation in human monocytes. However, in addition to its previously reported anti-inflammatory functions in mice, in macrophages TNFR2 may also act as an auxiliary receptor to TNFR1 to enable cells to activate pro-inflammatory signalling pathways in response to low levels of TNF which are likely to be within the range found in vivo. Acknowledgements We are grateful to GSK, Arthritis Research UK, and the Kennedy Institute of Rheumatology Trust for funding and members of the Joint
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Fig. 6. Effect of TNFR1 and TNFR2 blockade by receptor-specific monoclonal antibodies on p38 MAPK and NF-κB signalling at low and high concentrations of TNF. A, Wild-type macrophages were pre-treated for 1 h with isotype control IgG (10 μg/ml), anti-TNFR1 mAb (10 μg/ml), anti-TNFR2 mAb (10 μg/ml) or mTNFR2-Fc (1 nM) as a control to block signalling by both receptors, and then further treated with TNF (0.3 ng/ml) for 15 min. B, As for (A) but using 10 ng/ml TNF. Western blots are shown together with plots for mean phosphop38/tubulin ± SD and mean IκBα/tubulin ± SD from two independent experiments normalised to values for cells treated with TNF only, or untreated cells, respectively.
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages☆ Gerhard Ruspi a, Emily M. Schmidt a, Fiona McCann a, Marc Feldmann a, Richard O. Williams a, A. Allart Stoop b, Jonathan L.E. Dean a,⁎ a Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road Campus, Roosevelt Drive, Headington, Oxford OX3 7FY, United Kingdom b Innovation Biopharm Discovery Unit, Biopharm R&D, GlaxoSmithKline, Cambridge CB4 0WG, United Kingdom
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 19 December 2013 Accepted 22 December 2013 Available online 27 December 2013 Keywords: TNF TNFR p55 p75 TRAF2 Macrophage
a b s t r a c t Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NFkappaB pathways was blocked in TNFR1−/− macrophages. In contrast, although TNFR2−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB proinflammatory signalling pathways. © 2013 Elsevier Inc. All rights reserved.
1. Introduction There are two separate TNF receptors: the type I receptor (TNFR1, p55, or p60) and the type II receptor (TNFR2, p75, or p80). Both receptors are widely expressed but TNFR1 is thought to be the major receptor required by a wide variety of cells for activation of the pro-inflammatory NF-κB and MAPK signalling pathways which in turn induce the expression of proteins of the inflammatory response (for reviews see [13,17,31]).
Abbreviations: TNF, tumour necrosis factor; TNFR, TNF receptor; TRAF2, TNF receptorassociated factor 2; MAPK, mitogen-activated protein kinase; P-p38, phospho-p38 MAPK; NF-κB, nuclear factor-κB; LPS, lipopolysaccharide; MCSF, macrophage colony stimulating factor; GM-CSF, granulocyte/macrophage colony stimulating factor; I-κB, inhibitor of κB; BMDM, bone marrow-derived macrophages; PBMC, peripheral blood mononuclear cells. ☆ The authors claim no conflict of interest. This study was funded by GlaxoSmithKline, Arthritis Research UK and the Kennedy Institute of Rheumatology Trust. MF was a consultant for GlaxoSmithKline. ⁎ Corresponding author. Tel.: +44 1865 612641. E-mail address: [email protected] (J.L.E. Dean). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.009
In contrast, TNFR2 displays cell-type specific expression and is the major TNFR expressed on activated T cells in which it signals the induction of apoptosis [9,35] in a process involving sensitisation to TNF [9]. TNFR2 is also constitutively expressed in regulatory T cells and plays a role in their activation, proliferative expansion, and survival [7]. Endothelial cells express more equal levels of both receptors [20], but in these cells, TNFR1 is thought to be the major receptor involved in the activation of the NF-κB and MAPK pathways and subsequent induction of inflammatory response proteins [4,21,24,36]. Immortalised macrophages are reported to express both receptors with TNFR1 being expressed weakly [23]. Both TNFR1- and TNFR2deficient macrophage cell lines display reduced activation of the NFκB and MAPK pathways in response to TNF compared to immortalised wild-type macrophages [23]. Experiments employing artificial ligands for the different receptors, also called muteins, have shown that TNFR2 cannot significantly activate pro-inflammatory signalling pathways independently of TNFR1 [6,8]. Physiological signalling functions of TNFR2 that are distinct from TNFR1 have proved elusive to identify. In cell lines, TNFR2 has been
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reported to induce TRAF2 [18,33] and ASK1 [34] protein degradation, and to regulate protein kinase B expression [16]. Recently, it has been shown in primary T cells and in tumour cell lines to activate the alternative NF-κB pathway [26]. Membrane-bound TNF has been shown to be a more potent activator of TNFR2 than the soluble form of the ligand [12,26]. In addition, TNFR2 shedding in response to TNF serves to neutralise TNF and inhibit signalling [25]. TNFR2 has been reported to regulate apoptosis via a mechanism of ‘ligand-passing’, shown to involve TNFR2-mediated facilitation of the activation of TNFR1 [29]. In this mechanism, TNF binds much more rapidly to TNFR2 than TNFR1. When both receptors are in close proximity, the presence of TNFR2 increases the association rate of TNF with TNFR1 thereby sensitising the cell to TNFR1-mediated cytotoxicity [29]. However, TNFR2 is able to drive apoptosis independently of TNFR1, and cooperation between TNFR1 and TNFR2 in activating the NF-κB pathway has also been found to be additive rather than synergistic [32]. Since the TNFR1 receptor has been more strongly implicated in the activation of pro-inflammatory signalling pathways than TNFR2, blockade of TNFR1 appears a logical choice for therapy of chronic inflammatory diseases. Indeed, TNFR1 expressed on mesenchymal cells has been shown to play an important role in arthritis in mice [1]. TNFR2 has also been suggested to be involved in diverse processes that may be of beneficial function and thus its blockade could have deleterious implications. These include diverse neuro-, cardio-, and osteo-protective effects of TNFR2 suggested from experiments in TNFR−/− mice. Activation of TNFR2 by TNF inhibits seizures [2] and attenuates cognitive dysfunction following brain injury [19]. TNFR2 appears not to affect myocardial infarct size, but does promote survival following myocardial infarction in mice [22]. Similarly, TNFR2 has been suggested to protect against myocardial ischaemia/reperfusion injury [10], and to reduce remodelling and hypertrophy following heart failure [14]. Furthermore, in experimental arthritis, TNFR2 has also been shown to protect against joint inflammation and erosive bone destruction through regulation of osteoclastogenesis [27]. Given the wide range of protective functions of TNFR2, and the lack of a clear consensus on the signalling function of this receptor, we sought to identify unique physiological TNFR2-dependent signalling processes that are distinct from TNFR1. We have focused on TRAF2 protein degradation, the TNFR2-dependent signalling event that is most strongly implicated from previous studies in cell lines. To see if TRAF2 degradation occurs in primary cells we have investigated the ability of TNFR2 to induce this process in primary wild-type and TNFR1- or TNFR2-deficient macrophages. LPS-treated macrophages strongly express TNF, and this allowed us to test the activation of the alternative NF-κB pathway by endogenous membrane-bound TNF. Since these cells express both TNF receptors we also investigated if, according to the ‘ligand-passing’ model, TNFR2 sensitises primary macrophages for activation of classical pro-inflammatory signalling pathways.
2. Materials and methods 2.1. Materials Lipopolysaccharide (LPS) (TLR-grade) was purchased from Alexis Biochemicals (Exeter, UK). Macrophage colony stimulating factor (MCSF) and TNF (human cells were treated with human TNF; murine cells were treated with murine TNF) and human granulocyte/ macrophage colony stimulating factor (GM-CSF) were from PeproTech (London, UK). Antagonistic anti-murine TNFR1 (55R-170) and antimurine TNFR2 (TR75-32.4) monoclonal antibodies were from BioLegend (Cambridge, UK). Antibodies for western blotting (phospho(Thr180/ Tyr182)-p38 MAPK, p38 MAPK, IκBα, TRAF2, p100/p52) were from Cell Signalling (Danvers, Massachusetts, USA) or (tubulin) Sigma-Aldrich (St. Louis, Missouri, USA). Anti-TNFR1 (FAB225P (Clone #16803)) and anti-TNFR2 (FAB226P (Clone #22235)) phycoerythrin-conjugated
antibodies for flow cytometry were from R&D Systems (Abingdon, UK). General lab reagents were from Sigma-Aldrich.
2.2. Mice Wild-type mice of C57BL/6 background were from Harlan Laboratories (Wyton, UK). TNFR1−/− and TNFR2−/− mice (C57BL/6 background) were maintained as heterozygotes and have been previously described [37]. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.
2.3. Cell culture Bone marrow-derived macrophages (BMDM) were isolated and differentiated as described previously by Hitti et al. [15]. Briefly, bone marrow cells were harvested from the femurs and tibiae of 10–12 week-old mice and were incubated for 7 days in the presence of MCSF (100 ng/ml). BMDM were cultured in DMEM supplemented with 10% (v/v) FCS, 2 mM L-glutamine (PAA, Yevil, UK), penicillin and streptomycin and 0.1% (v/v) 2-mercaptoethanol (Life Technologies, Paisley, UK). Centrifugal elutriation was used to isolate monocytes from human peripheral blood mononuclear cells (PBMC). Briefly, human PBMC were obtained by density centrifugation through ficoll/hypaque of a leukoreduction system chamber of apheresis instruments after routine platelet collection. The resulting PBMC were centrifugally elutriated in 1% (v/v) heat-inactivated FCS in RPMI in a Beckman JE6 elutriator (Beckman, High Wycombe, UK). The monocytes separated by this method were assessed for purity (≥75%) by flow cytometry (FACScan, BD, Oxford, UK) analysis of forward scatter and side scatter. Monocytes were differentiated into macrophages by treatment with human GM-CSF (50 ng/ml) for 6 days or human MCSF (100 ng/ml) for 5 days respectively. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.
2.4. Western blot In experiments analysing phospho-p38 MAPK, IκBα and p100/p52, cells were lysed for 10 min on ice with ≤200 μl of whole cell lysis buffer (50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% (v/v) Triton X-100, 0.5% (v/v) NP-40, 10% (v/v) glycerol) supplemented with protease inhibitor cocktail (1% v/v; Sigma-Aldrich), NaF (200 μM), NaVO3 (100 μM), microcystin (1 μM) and DTT (1 mM). Samples were clarified by centrifugation at 16,000 ×g, for 10 min at 4 °C. For TRAF2 degradation experiments cells were lysed on ice for 30 min with ≤ 100 μl of a mild lysis buffer (30 mM Tris–HCl, 120 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100) supplemented with protease inhibitors (1% v/v) and DTT (1 mM). Samples were clarified by centrifugation at 14,000 ×g for 30 min at 4 °C. Proteins in lysates (≥20 μg) were separated by electrophoresis through 10% (w/v) polyacrylamideSDS gels and transferred to a polyvinylidene fluoride membrane (DuPont, Stevenage, UK). Membranes were blocked in 5% (w/v) dried-fat milk powder (Premier International Foods, Spalding, UK) and incubated in primary antibody and secondary antibody coupled to horseradish peroxidase (Dako, Glostrup, Denmark). Protein detection was carried out by enhanced chemiluminescence (GE Healthcare, Amersham, UK). Films were scanned with ImageScanner III (GE Healthcare). Densitometry of digital images with background subtraction was performed using Quantity One software (BioRad) and data analysis was performed using Excel (Microsoft). Error bars are shown for all data except for normalised values but in some cases are too small to be visible.
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2.5. Assessment of TNF receptor expression by flow cytometry
detected in BMDM at 1 h and was sustained until 16 h. TRAF2 degradation was observed up to 4 h TNF treatment in two separate experiments (as shown graphically in Fig. 1A). Next, we examined TRAF2 degradation at 6 h over a range of TNF concentrations. The effect of TNF treatment was maximal at 30 and 100 ng/ml TNF (Fig. 1B).
Differentiated human macrophages were harvested by cell scraping. Cells were blocked with FcR block (Miltenyi Biotec, Bisley, UK), and treated isotype control IgGs (BD) or stained with fluorochromelabelled antibodies for 30 min at room temperature in PBS supplemented with 2% (v/v) FCS and 2 mM EDTA (final concentrations). Analysis was carried out on a FACS Canto II (BD) and using FlowJo™ software (Treestar, Ashland, USA).
3.2. TRAF2 protein degradation in BMDM is TNFR2-dependent To investigate which receptor is required for TRAF2 degradation induced by TNF, wild-type, TNFR1−/− and TNFR2−/−, BMDM were treated with 30 ng/ml TNF for 4 h and TRAF2 degradation was analysed by western blot, as before. TNF treatment resulted in TRAF2 degradation in wild-type and TNFR1−/− BMDM but not in TNFR2−/− cells (Fig. 1C). Thus in macrophages, TNFR2, but not TNFR1, is necessary for ligand-induced TRAF2 degradation, as reported previously in other cell types. In some blots, such as the ones shown in Fig. 1A and C, anti-TRAF2 antibody detected TRAF2 protein as a doublet. The precise reason for this is unclear but the data suggest that under certain gel running conditions two different forms of TRAF2 protein are resolved.
3. Results 3.1. Function of the TNFR2 in TNF-induced TRAF2 protein degradation: time and concentration dependence Signalling via TNFR2 induced by high concentrations of soluble TNF has previously been shown to cause TRAF2 protein degradation [18,33], but this has not been reported in macrophages. To determine if TRAF2 degradation occurs in murine macrophages, and to identify which receptor is responsible for this with the use of receptordeficient macrophages, it was firstly necessary to determine the time and concentration dependence of the process in BMDM. For this cells were treated with a high dose (100 ng/ml) of TNF and harvested at different times. Cells were then lysed using a mild lysis buffer containing a low concentration of sodium chloride and Triton X-100 to isolate the TNF-regulated pool of TRAF2. Cell lysates were analysed for TRAF2 protein by western blot. In the blot shown in Fig. 1A TRAF2 degradation was
3.3. TNF induces TRAF2 degradation in human macrophages but not monocytes To investigate whether TRAF2 degradation occurs in human macrophages in response to TNF, monocytes were isolated by elutriation and differentiated into M1- or M2-like macrophages using GM-CSF or
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MCSF, respectively. Cells were then treated with a high dose human TNF (30 ng/ml) for 4 h, or left untreated. Undifferentiated monocytes were treated in the same way. Both M1- and M2-like human macrophages displayed TRAF2 degradation in response to TNF, but human monocytes did not (Fig. 2A). Given that in murine macrophages TNFR2 is required for TRAF2 degradation (Fig. 1C), we decided to assess the expression of the two TNF receptors on human macrophages and monocytes. Cells were analysed by flow cytometry using antibodies to TNFR1 or TNFR2, and isotype control IgGs. This showed that monocytes differentiated into macrophages using either GM-CSF, or MCSF, express similar levels of both TNF receptors, but human monocytes display greater TNFR1 expression than macrophages (Fig. 2B). The predominance of TNFR1 as opposed to TNFR2 expression on human monocytes compared to macrophages (Fig. 2B) correlates with the apparent lack of TNF-induced TRAF2 degradation in these cells (Fig. 2A). 3.4. Role of TNFR2 in TNF-induced tolerance Since signalling by TNFR2 induces TRAF2 degradation, and because TRAF2 is required for the activation of MAPKs and the NF-κB pathway, we theorised that the phenomenon of tolerance of TNF signalling induced by TNF pre-treatment might involve TRAF2 protein degradation. This may identify TRAF2 degradation as a bonafide signalling event with
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functional consequences. To test this, wild-type and TNFR2−/− BMDM were treated with TNF (30 ng/ml) according to the regimens shown in Fig. 3A. Cells were left untreated at t = 0 and then treated with the same dose of TNF at 6 h to induce signalling, or cells were treated with TNF at t = 0 to induce tolerance and, either left untreated, or treated with TNF at 6 h. Some cells were left untreated and served as a control. All cells were harvested at 6 h 15 min. Treatment of wild-type or TNFR2−/− BMDM with a single high dose of TNF (30 ng/ml) resulted in strong p38 MAPK phosphorylation (Fig. 3B; lane 3), consistent with its activation, but this was inhibited by a prior 6 h TNF treatment (Fig. 3B; lane 4). The ability of the initial TNF treatment to block signalling was not impaired in TNFR2−/− BMDM (Fig. 3B; compare lanes 4 and 8). In both wild-type and TNFR2−/− BMDM treated with a single dose of TNF at t = 0, there was no detectable p38 MAPK activation following 6 h 15 min of treatment (Fig. 3B; lanes 2 and 6), consistent with resolution of signalling. TRAF2 degradation occurred in wild-type cells that were treated with TNF at t = 0 (Fig. 3B; lanes 2 and 4), but not in TNFR2−/− cells treated in the same way (Fig. 3B; lanes 6 and 8), as predicted. In order to rule out the involvement of TRAF2 degradation in TNFinduced tolerance, similar experiments were performed in wild-type cells using a low concentration of TNF which fails to induce TRAF2 degradation. TNF-induced tolerance of p38 MAPK activation was also
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Fig. 2. Induction of TRAF2 protein degradation by TNF in human monocytes differentiated into macrophages using GM-CSF or MCSF but not in undifferentiated human monocytes. A, Human monocytes were left untreated or treated with TNF (30 ng/ml) for 4 h. Monocytes were also differentiated into macrophages (MΦ) using GM-CSF or MCSF and treated as above. As described in Fig. 1, cells were lysed and lysates were analysed by western blot and data for normalised values for untreated cells for each cell type plotted. B, Monocytes were either left untreated, or differentiated into macrophages using GM-CSF or MCSF and TNFR1 and TNFR2 protein expressions assessed by FACS analysis with anti-TNFR1 or antiTNFR2 antibodies, or using isotype control IgGs. Similar results were obtained for (A) and (B) using cells from two separate donors.
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Fig. 3. TNFR2 is not required for tolerance of p38 MAPK activation induced by TNF in BMDM. A, Schematic of treatment protocol: Murine wild-type (WT) and TNFR2−/− (R2−/−) macrophages were left untreated (Lanes 1, 5), treated with TNF (30 ng/ml) at t = 0 (Lanes 2, 6), and then further treated with TNF at t = 6 h (lanes 3, 7) or, treated with TNF at both 0 and 6 h (Lanes 4, 8). Cells were not washed and culture medium was not changed in-between treatments. Cells were harvested and lysed at t = 6 h, 15 min. B, Wild-type and TNFR2−/− cells were treated with TNF according to lane numbers as indicated in (A). C, Wild-type BMDM cells were treated according to lane numbers with 1 ng/ml TNF as in (A) or left untreated. In (A) and (B) cells were lysed with whole cell lysis buffer (see Materials and methods) and lysates were analysed by western blot for phospho-p38 MAPK (P-p38); (41 kDa), TRAF2 and tubulin. Plots show mean phospho-p38/tubulin protein ± SD and mean TRAF2/tubulin protein ± SD normalised to cells treated with TNF for 6 h, or untreated cells, respectively from two independent experiments for (B) and two independent experiments, one of which was performed in duplicate for (C).
observed at a low concentration of TNF (1 ng/ml) (Fig. 3C; compare lanes 3 and 4) although in this case the inhibition of p38 MAPK activation induced by TNF pre-treatment was slightly less than that observed using 30 ng/ml TNF (Fig. 3B; lane 4). Western blot for TRAF2 protein confirmed that treatment of cells with 1 ng/ml TNF did not cause TRAF2 degradation (Fig. 3C; lanes 2 and 4). Overall the data suggest that TNF-induced tolerance of p38 MAPK activation occurs independently of TRAF2 degradation. 3.5. Neither TNF, nor LPS activate the alternative NF-κB pathway in BMDM We next sought to test the involvement of TNFR2 in another signalling event which has been previously reported to be specifically activated by this TNF receptor, namely the activation of the alternative NF-κB pathway. Ligand-induced activation of TNFR2 has previously been shown to activate the alternative NF-κB pathway by the processing of p100 to p52 [26]. To test whether TNF induces p100 processing in BMDM, cells were treated with a high concentration of TNF (20 ng/ml) for the times shown and lysates were analysed for p100 and p52 protein. No
reduction in p100 protein or accumulation of p52 protein could be detected for TNF treatment between 1 and 4 h (Fig. 4A), indicative of an inability of TNF to induce p100 processing in murine macrophages. Since membrane-bound TNF has been suggested to be a more potent stimulus for the TNFR2 receptor than soluble TNF [12,26], cells were also treated with LPS for 1–4 h to induce TNF expression and p100 processing was analysed as before. As for TNF, LPS treatment failed to induce activation of the alternative NF-κB pathway in BMDM (Fig. 4B). However, the antibody used to detect p100 and the cleaved product, p52, also detected a weakly stained band (indicated by *) representing a 40 kDa protein which was induced by both TNF and LPS (Fig. 4A and B). The identity of this protein is unclear but it may represent a cleaved species of p100, or p52, which is weakly induced following cell stimulation. 3.6. TNFR2 sensitises macrophages for ligand-induced activation of the p38 MAPK and NF-κB pathways We were also interested in examining the function of the TNFR2 receptor in activating pro-inflammatory signalling pathways at different
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concentrations of TNF as this would allow any sensitisation of signalling due to ligand-passing to TNFR1 to be detected. We focused on the NF-κB and p38 MAPK pathways since these are both strongly implicated in the expression of proteins of the inflammatory response. To investigate signalling in primary macrophages it was firstly necessary to check that the p38 MAPK and NF-κB pathways are activated at low concentrations as well as at high concentrations of TNF. To test this, wild-type BMDM were treated with different concentrations of TNF for 15 min and p38 MAPK activation was assessed by western blot as before. Treatment of wild-type BMDM with concentrations of TNF as low as 0.3 ng/ml resulted in activation of phosphorylation of p38 MAPK (Fig. 5). To investigate the involvement of either TNFR1 or TNFR2, BMDM from TNFR1- and TNFR2-deficient mice were used. TNF-induced activation of signalling at low doses of TNF (≤3 ng/ml) and at doses which are reported to activate TNFR2 most strongly (10 and 30 ng/ml) was examined. Little activation of p38 MAPK occurred in TNFR1−/− BMDM (Fig. 5). Surprisingly, at low doses, TNF-induced signalling was also impaired in TNFR2−/− cells in which the ligand concentration dependence of the activation of p38 MAPK was increased ~ 10-fold compared to wild-type cells (Fig. 5). The TNF concentration dependence of IκBα degradation in both wild-type and knockout cells was identical to that observed for p38 MAPK activation (Fig. 5). Thus at high doses of soluble TNF, TNFR1 is the main receptor required for activation of signalling and the presence of TNFR2 is not required for activation. However, at lower concentrations of TNF, TNFR2 is essential for full activation of signalling. A possible caveat in the above interpretation is that knockout cells may adapt in response to receptor deficiency and the results could be misleading. To address this we also investigated the signalling function of TNFR1 and TNFR2 using receptor-specific monoclonal antibodies. Wild-type BMDM were pre-treated with an IgG control, anti-TNFR1 mAb, anti-TNFR2 mAb and a murine form of soluble TNFR2 coupled to an Fc region (mTNFR2-Fc) as a control to block signalling by both
receptors. Cells were then stimulated with a low concentration of TNF (0.3 ng/ml) or left untreated. Anti-TNFR1 mAb strongly inhibited TNFinduced p38 MAPK signalling and IκBα degradation (Fig. 6A). In agreement with the result in TNFR2−/− cells, the anti-TNFR2 mAb also strongly inhibited signalling at a low concentration of TNF (Fig. 6A). Similar experiments were performed at a high concentration of TNF (10 ng/ml), and consistent with the results from receptor-deficient macrophages, the anti-TNFR1 mAb and mTNFR2-Fc blocked signalling, albeit rather weakly, but anti-TNFR2 mAb did not (Fig. 6B). Thus at high TNF concentrations, TNFR1 is the dominant receptor which activates signalling in the absence of TNFR2, whereas both receptors are essential for the activation of signalling at lower concentrations of TNF. 4. Discussion TNFR1 is known to activate the NF-κB and MAPK pathways in response to TNF in a wide range of cell types. In this study, induction by TNF of p38 MAPK phosphorylation and IκBα degradation was completely blocked in TNFR1−/− macrophages at all concentrations of TNF used, underscoring a dominant role for this receptor and showing that TNFR2 cannot significantly activate these pathways independently of TNFR1. The literature suggests that the role of TNFR2 in signalling is more complex. In macrophages, we find that at high concentrations of its ligand, TNFR2 is needed for TRAF2 protein degradation, whereas at low ligand concentrations, TNFR2 serves to sensitise the cells for activation of proinflammatory signalling. Our findings in primary cells differ from those in immortalised macrophage lines in which TNFR2 deficiency almost entirely blocked signalling induced by a wide range of TNF concentrations that encompassed the range used in this study [23]. A possible explanation is that, unlike in primary TNFR2-deficient macrophages, TNFR1 expression was reduced in the TNFR2-deficient line following extended passaging. Our observation that even sub-ng/ml concentrations of TNF are sufficient to strongly activate the p38 MAPK and NF-κB pathways in cells in culture suggests that our findings are very relevant to any situation in vivo where small amounts of TNF are present. The majority of previous studies reporting TNFR2-mediated TRAF2 degradation have utilised cell lines and receptor overexpression, although during the course of this work it has also been reported to occur in primary CD8 + T cells [30]. Treatment of both human and murine primary macrophages with high concentrations of TNF caused a reduction in TRAF2 protein. The loss of TRAF2 protein in murine macrophages was exclusively TNFR2-dependent as the TNF-induced reduction in TRAF2 protein was blocked by TNFR2, and not by TNFR1 deficiency. TRAF2 degradation was shown to occur in human macrophages, but the very high concentrations of soluble TNF needed for maximal TRAF2 degradation (30 ng/ml) in BMDM suggest that this process may be most relevant to physiological settings where localised TNF expression is extremely high or where presence of membrane TNF would favour TNFR2 signalling. TRAF2 plays an important role for the activation of the major signalling pathways downstream of TNF [3] including the p38 MAPK pathway [5]. The lack of a requirement for TNFR2-mediated TRAF2 degradation in TNF-induced tolerance of activation of p38 MAPK suggests that, in macrophages, TRAF2 degradation may serve some other purpose, such as the previously reported inhibition of the NF-κB pathway [11]. We failed to detect any evidence for activation of the alternative NF-κB pathway in response to soluble TNF or LPS in macrophages. The results suggest that under the conditions used either LPS fails to induce a significant amount of membrane-bound TNF in macrophages, or endogenous membranebound TNF does not activate the alternative NF-κB pathway in macrophages. To investigate TNF-induced gene expression, the cytokine is frequently reported to be used to treat cells at a concentration of ≥10 ng/ml which in BMDM lies well above the concentration that maximally activates signalling. Thus the important contribution of TNFR2 to signalling at sub-ng/ml concentrations of TNF suggests that many
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Fig. 5. TNFR1 is the major receptor needed for TNF-induced p38 MAPK and NF-κB pathway activation in BMDM but TNFR2 is also required at a low concentrations of TNF. Murine wildtype, TNFR1−/− and TNFR2−/−BMDM were treated with the concentrations of TNF indicated for 15 min. Similar results were obtained in two separate experiments. Western blot analysis of BMDM lysates (prepared as in Fig. 3) for phospho-p38 MAPK, IκBα (35 kDa) and tubulin is shown. Plots show mean phospho-p38/tubulin ± SD and mean IκBα/tubulin ± SD from duplicate blots normalised to values for 30 ng/ml TNF-treated wild-type cells, or untreated wild-type cells, respectively.
previous studies may have underestimated the importance of TNFR2 in the induction and regulation of gene expression in response to TNF. This may be particularly relevant for cell types such as endothelial cells which express both TNF receptors and in which TNF strongly induces the expression of a wide range of proteins, including inflammatory cytokines and adhesion molecules. It is noted that TNFR2 has been previously reported to play an important role in tissue factor expression in endothelial cells treated with low but not high concentrations of TNF [8]. Our data also suggest that under situations where the concentration of TNF is high, such as in inflammation, TNFR2 may have little role in the activation of pro-inflammatory signalling. However, where TNF levels are low, TNFR2 may be important for the initiation of inflammation and its subsequent autoregulated resolution. It is tempting to speculate that as opposed to chronic inflammation in which resolution may be dysregulated, TNFR2-dependent signalling induced by small amounts of TNF may activate signalling, its normal resolution, and the associated anti-inflammatory effects, thus providing a additional explanation for the previously reported anti-inflammatory functions of TNFR2 in mice. It has been reported recently that TNFR2-mediated TRAF2
degradation is needed for resolution of NF-κB pathway activation in 293 cells [28]. 5. Conclusions In macrophages high concentrations of soluble TNF induce TRAF2 protein degradation via TNFR2. TRAF2 degradation does not appear to be required for TNF-induced tolerance of p38 MAPK activation and TNF does not appear to induce TRAF2 degradation in human monocytes. However, in addition to its previously reported anti-inflammatory functions in mice, in macrophages TNFR2 may also act as an auxiliary receptor to TNFR1 to enable cells to activate pro-inflammatory signalling pathways in response to low levels of TNF which are likely to be within the range found in vivo. Acknowledgements We are grateful to GSK, Arthritis Research UK, and the Kennedy Institute of Rheumatology Trust for funding and members of the Joint
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Fig. 6. Effect of TNFR1 and TNFR2 blockade by receptor-specific monoclonal antibodies on p38 MAPK and NF-κB signalling at low and high concentrations of TNF. A, Wild-type macrophages were pre-treated for 1 h with isotype control IgG (10 μg/ml), anti-TNFR1 mAb (10 μg/ml), anti-TNFR2 mAb (10 μg/ml) or mTNFR2-Fc (1 nM) as a control to block signalling by both receptors, and then further treated with TNF (0.3 ng/ml) for 15 min. B, As for (A) but using 10 ng/ml TNF. Western blots are shown together with plots for mean phosphop38/tubulin ± SD and mean IκBα/tubulin ± SD from two independent experiments normalised to values for cells treated with TNF only, or untreated cells, respectively.
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