Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappaB-dependent pathway
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Methotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it.
Caspase-3 activity, annexin-V binding/7-aminoactinomycin D (7-AAD) exclusion and cell-cycle analysis were used to measure steps in apoptosis of primary murine macrophages and cells of the RAW264.7 macrophage cell line that had been exposed to clinically-relevant concentrations of MTX and TNF-α.
MTX induces apoptosis in primary murine macrophages at concentrations as low as 100 nM in vitro. TNF-α, which has a context-dependent ability to increase or to suppress apoptosis, efficiently suppresses MTX-induced macrophage apoptosis. This depends on NF-κB signaling, initiated through TNF Receptor Type 1 ligation. Macrophage colony stimulating factor, the primary macrophage survival and differentiation factor, does not activate NF-κB or protect macrophages from MTX-induced apoptosis. A weak NF-κB activator, Receptor Activator of NF-κB Ligand (RANKL) is likewise ineffective. Blocking NF-κB in TNF-α-exposed macrophages allowed pro-apoptotic actions of TNF-α to dominate, even in the absence of MTX. MTX itself does not promote apoptosis through interference with NF-κB signaling.
These findings provide another mechanism by which TNF-α sustains macrophage numbers in inflamed tissue and identify a further point of clinical complementarity between MTX and anti-TNF-α treatments for rheumatoid arthritis.
KeywordsElectrophoretic Mobility Shift Assay AICAR NECA Parthenolide Primary Macrophage
bone marrow-derived macrophages
death-inducing signaling complex
Dulbecco's Modified Eagle Medium
extracellular signal-regulated kinase
foetal calf serum
- FLIP FLICE
growth arrest and DNA damage inducible protein
Granulocyte macrophage-colony stimulating factor
inhibitor of NF-κB
inhibitors of apoptosis
jun N-terminal kinase
mitogens-activated protein kinase
macrophage colony stimulating factor
manganese superoxide dismutase
nuclear factor-kappa-light-chain-enhancer of activated B cells
platelet-derived growth factor
phosphatidylinositol-3-kinase/protein kinase B
receptor activator of NF-κB
tumor necrosis factor-alpha
- TRAF TNF
receptor associated factor
vascular endothelial growth factor
X-linked inhibitor of apoptosis protein.
Synovial inflammatory macrophages have a central role in maintaining disease activity in rheumatoid arthritis (RA). Macrophage numbers in tissue are regulated by recruitment, local proliferation, local cell death and emigration to draining lymph nodes [1, 2]. Synovial macrophage apoptosis has also been observed in synovium in RA [3, 4]. A suppressed rate of apoptosis would contribute to maintaining inflammatory macrophage numbers, and thus clinical activity, in macrophage-dependent conditions. An enhanced rate of synovial macrophage apoptosis is reported in RA patients responding to anti-TNF-α treatments  as a delayed, rather than an early phenomenon . Macrophage apoptosis has also been reported in patients with Crohn's disease after anti-TNF-α treatment . These observations suggest that TNF-α directly, or indirectly, sustains macrophage survival in these conditions.
TNF-α activity supports recruitment of macrophages into RA synovium , but is not known to enhance the proliferation of macrophages or to prevent the emigration of macrophages through lymphatics . TNF-α, however, may deliver either apoptotic or survival signals, depending on the cell context. TNF-α is a ligand for two related receptors, TNF-R1 (p55) and TNF-R2 (p75). Macrophages and their bone marrow and blood-borne progenitors express both receptors . TNF-α ligation to TNF-R1 leads to assembly of a Death-Inducing Signaling Complex (DISC) and ultimately activation of downstream effector caspases-3/6/7. Where this is the dominant consequence of TNF-R1 ligation, apoptosis follows. In other circumstances, TNF-R1 initiates survival signaling  through activation of the NF-κB pathway. TNF-α ligation to TNF-R1 can thus bring apoptosis or survival, depending on the relative activities generated in the DISC-initiated and NF-κB pathways . Macrophage survival is notably dependent on NF-κB signaling. The transcriptionally active RelA/NF-κB1 (p65/p50) complex appears in macrophage nuclei constitutively during late differentiation and is important for continuing survival [10, 11]. In vivo, there is enhanced nuclear expression of RelA/NF-κB1 in synovial macrophages in RA , consistent with a role for NF-κB activity in maintaining macrophage survival.
The observations of enhanced apoptosis during anti-TNF-α treatment imply that TNF-α predominantly antagonises apoptosis in RA and Crohn's disease [4, 6]. On the other hand, methotrexate (MTX), an anti-rheumatic drug that has demonstrable pro-apoptotic effects on monocyte/macrophage cells in vitro, does not appreciably alter the synovial membrane macrophage apoptosis rate in RA patients [3, 13]. This led us to question whether the presence of TNF-α in synovium was providing a survival signal to macrophages in MTX-treated patients, thus antagonizing one potential therapeutic function of MTX. That would provide additional explanation for the clinical complementarity of MTX and anti-TNF-α treatments in RA . In the study of Catrina et al., which demonstrated synovial macrophage apoptosis in response to anti-TNF-α therapy, the majority of patients were co-treated with MTX .
MTX is a folate analogue with numerous effects on inflammatory cell proliferation and survival, cytokine expression, angiogenesis, cell adhesion and reactive oxygen production . The known direct targets of MTX are dihydrofolate reductase, thymidylate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase . Consequences of inhibiting these enzymes include reduced availability of purines and pyrimidines for DNA and RNA synthesis and accumulation of AICAR. AICAR, by inhibiting enzymatic deamination of adenosine and adenosine monophosphate, is proposed to increase availability of adenosine extracellularly, for anti-inflammatory effect through adenosine cell surface receptors . These effects are generally believed to be mediated by long-lived polyglutamate forms of MTX, which are generated in many cell types, including normal myeloid precursor cells and myelocytic cancer cells .
Materials and methods
The Recombinant Macrophage-Colony Stimulating Factor (M-CSF) was obtained from R&D Systems (Sydney, NSW, Australia). Annexin V conjugated to phycoerythrin (Annexin V-PE) and 7-amino-actinomycin (7-AAD) were from BD Biosciences (Sydney, NSW, Australia). Parthenolide was purchased from Alexis Biochemicals (Lausen, Switzerland). Rabbit anti-IκBα (C-21) polyclonal antibody, anti-NF-κB p65, anti-NF-κB p50, anti-c-Rel and anti-RelB were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse anti-phospho-IκBα (Ser32/36) (5A5) and horseradish peroxidase-conjugated anti-rabbit were from Amersham Biosciences (Sydney, NSW, Australia). Methotrexate was provided by Mayne Pharma Pty Ltd. (Adelaide, South Australia, Australia). Etanercept came from Wyeth Australia Pty Ltd. (Sydney, NSW, Australia). Recombinant GST-rRANKL was kindly provided by Ming-Hao Zheng and Jiake Xu, Centre for Orthopaedic Research, University of Western Australia . All other reagents were sourced from Sigma-Aldrich (Sydney, NSW, Australia), unless otherwise indicated.
RAW264.7 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, California, USA) with 10% low endotoxin fetal calf serum (CSL, Melbourne, Australia), penicillin and gentamicin (DMEM-FCS). Bone marrow-derived monocytes/macrophages (BMDM) were collected from the femora and tibiae of eight-week old female C57BL or BALB/c mice, as previously described , with the approval of the Animal Ethics Committee of The University of Western Australia. Cells were expanded in M-CSF (30 ng/mL)-supplemented DMEM-FCS for seven days at 37°C/5% CO2/95% air. The experiments reported here were conducted with BMDM from C57BL mice, but results were comparable using BALB/c mice.
MTT cytotoxicity assay
Viability was estimated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay. BMDM (3 × 104 cells/well) were seeded in 24-well plates in M-CSF-supplemented DMEM-FCS. After 48 hr, cells were treated with TNF-α for 3 hr, followed by MTX exposure for 24 hr. At the end of treatments, 50 μL of MTT (5 mg/mL) was added to each well, and cells were incubated at 37°C for 2 hr. Formazan crystals were then solubilised with 150 μL of 44% dimethyl formamide/20% SDS at room temperature for at least 30 minutes on a rocking platform. A total of 100 μL aliquots were quantitated at 550 nm using a microplate reader (POLARstar OPTIMA, BMG, Germany). Triplicate assays were conducted for all conditions.
Caspase-3 protease activity
RAW264.7 cells and BMDM were cultured in six-well plates at a density of 0.3 × 106 cells/well for 48 hr. After treatments, trypsin-detached RAW264.7 cells and scraped BMDM were lysed and assayed for caspase-3 activity as described previously .
Flow cytometry analysis
Apoptotic cells were quantitated by staining with annexin V-PE and 7-AAD, as specified by the manufacturer (BD Biosciences). Flow cytometry was performed on populations of 5000-10,000 cells (Becton Dickinson FACSCalibur, Sydney, NSW, Australia), with fluorescence of annexin V-PE and 7-AAD measured with a 585/42 nm bandpass filter (FL2 channel) and a 670 nm longpass filter (FL3 channel), respectively. Data are expressed as percentages of apoptotic cells, as defined by annexin-V-PE positivity and 7-AAD negativity.
Cell cycle analysis
RAW264.7 cells and BMDM (0.25 × 106/well of a 12-well plate) were collected and washed in cold Dulbecco's Phosphate Buffered Saline (DPBS) before being fixed with 70% ethanol at -20°C. After 24 hr, cells were centrifuged at 500 × g for 10 minutes at 4°C and resuspended in 500 μL of staining buffer (50 μg/mL propidium iodide and 25 μg/mL RNase in 0.1% Triton X-100). After incubating at 37°C for 30 minutes, cells were analysed on a FACSCalibur flow cytometer (Becton Dickinson) using a doublet discrimination protocol.
Western blot analysis
RAW264.7 cells and BMDM (1 × 106 per treatment) were washed with ice-cold DPBS and lysed in RIPA buffer (50 mM Tris, pH 7.5, containing 150 mM NaCl, 1% IGEPAL, 1% sodium deoxycholate, 0.1% SDS and 10 mM EDTA) supplemented with 1X complete protease inhibitors (Roche Applied Science, Sydney, NSW, Australia). A total of 20 μg of total protein was separated by SDS-PAGE and transferred to Hybond-P PVDF membranes (Amersham Biosciences). Membranes were blocked in 1% BSA/5% non-fat milk in TTBS (10 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for one hour at room temperature, before being probed with specific antibodies to phospho-IκBα (1:1000 dilution), IκBα (1:2,000) or β-actin (1:8,000). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse were used at 1:8,000 and 1:20,000 dilutions, respectively. All antibodies were prepared in SignalBoost Immunoreaction Enhancer (Calbiochem, Darmstadt, Germany) and applied for one hour at room temperature. Blots were revealed with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences).
Electrophoretic Mobility Shift Assay (EMSA)
EMSA was performed on nuclear extracts from RAW264.7 cells (8 × 106 per treatment) that were treated with 10 ng/mL TNF-α for up to one hour, according to previously described protocols . A double-stranded NF-κB consensus oligonucleotide probe 5'-GGGCATGGGAATTTCCAACTC-3' (0.25 pmol) with 5'-G overhangs was filled in with labeled (α-32P)dCTP (Amersham Pharmacia Biotech, Buckinghamshire, England) using the Klenow fragment of E. coli DNA polymerase I (Promega, Madison, Wisconsin, USA). This DNA probe was incubated with 3 μg of nuclear proteins for 10 minutes at room temperature. Where indicated, antibodies (1 μg) to specific NF-κB factors or unlabelled oligonucleotide probes at 100-fold molar excess were also included for supershift EMSA and competition experiments, respectively. Samples were loaded onto a 4% polyacrylamide gel, containing 0.25X Tris-Borate-EDTA buffer, which had been pre-run for two hours in the same buffer. After separation, gels were exposed to Cronex X-ray film, using a single intensifying screen.
Transient transfection of RAW264.7cells and BMDM with an NF-κB reporter plasmid
RAW264.7 cells (1 × 107) were transiently transfected with 0.5 μg of pNFκB-TA-Luc (Mercury™ Pathway profiling system, BD Biosciences) by the DEAE-dextran procedure, as previously described . Transfected cells were resuspended in DMEM-FCS and distributed into a 24-well culture plate at a density of 3.3 × 105 cells/well. Cells were rested for 48 hr before experimentation. Firefly luciferase expression in transfected RAW264.7 cells was measured using the Promega Luciferase Assay System, according to the manufacturer's instructions. Promoter activity was quantified by measuring light production in a multifunctional microplate reader (POLARstar OPTIMA, BMG, Germany), and presented as relative light units (RLU).
BMDM were transfected with Lipofectamine™ and Plus reagents (Invitrogen) according to manufacturer's instructions. Briefly, for each 24-well, 1 μg pNF-κB-TA-Luc was combined with 0.5 μL Plus reagent and 2 μL Lipofectamine™ in antibiotic-free medium. A total of 1.5 × 105 cells/well was added to the transfection mixture and plated in M-CSF-containing DMEM-FCS. After four to six hours of incubation at 37°C, wells were replaced with fresh growth medium. Luciferase expression was measured at least 48 hr after transfection.
Statistical significance (P < 0.05) between groups of experimental data was assessed using paired Student's t-tests.
TNF-α protects primary macrophages and a macrophage cell line from MTX-induced apoptosis
Cell cycle analysis of BMDM treated with TNF-α or its control, followed by MTX exposure for 24 hr or M-CSF withdrawal for 48 hr*.
Cell cycle distribution (% of cells)
3.5 ± 0.5
78.6 ± 2.9
6.0 ± 1.0
10.7 ± 2.7
MTX (10 μM)
7.5 ± 1.0a
62.7 ± 2.2a
16.0 ± 1.2a
12.0 ± 1.2
TNF-α (10 ng/mL)
3.6 ± 0.4
77.0 ± 2.2
7.5 ± 1.7
10.7 ± 2.0
TNF-α + MTX
5.8 ± 0.8b
65.7 ± 4.8
13.8 ± 3.2
13.1 ± 2.6
M-CSF (30 ng/mL)
1.5 ± 0.4
74.2 ± 2.6
8.4 ± 1.9
13.9 ± 2.2
15.4 ± 4.1a
74.7 ± 5.1
2.8 ± 0.7a
5.5 ± 0.9a
M-CSF + TNF-α
1.5 ± 0.2
71.8 ± 1.9
9.3 ± 1.3
15.3 ± 2.7
- M-CSF + TNF-α
4.4 ± 2.3b
82.3 ± 3.7b
1.9 ± 0.4
9.7 ± 2.7
Caspase-3 activation was evident at concentrations as low as 0.1 μM MTX in BMDM, with activity increasing dose-dependently up to 10 μM (Figure 1C). MTX, therefore, induces macrophage apoptosis at levels usually attained in treated human RA patients . This was replicated in the murine RAW264.7 macrophage cell line (Figure 1E). RAW264.7 macrophages do not require exogenous M-CSF to proliferate, having gained growth signaling through introduction of the Abelson leukaemia virus . They have been previously found to model primary macrophage apoptosis response . A concentration of 10 μM MTX was used in subsequent experiments.
TNF-α could also maintain the viability of macrophages exposed to 10 μM MTX. TNF-α, when introduced three hours before MTX, completely prevented the loss of cell numbers in primary BMDM cultures exposed to MTX (Figure 1A). TNF-α also almost completely inhibited MTX-induced caspase-3 activation (Figure 1B) and annexin-V binding (Figure 1B) and significantly suppressed the sub-G0 (apoptotic) fraction on flow cytometric cell cycle analysis of MTX-exposed BMDM (Table 1). TNF-α did not reverse the MTX-induced increase in cells in S-phase. TNF-α, therefore, protected from MTX-induced cell loss by countering apoptosis, not through any action on cell proliferation. TNF-α also reduced the sub-G0 population of M-CSF-deprived BMDM, without restoring S phase progression (Table 1). It is notable that TNF-α was protective even in the presence of M-CSF concentrations that are optimal for apoptosis prevention, suggesting distinct pathways for apoptosis protection. Further increases in M-CSF concentration in culture, up to 120 ng/mL, failed to substitute for the anti-apoptotic action of TNF-α (results not shown). This suggested an action that was independent of M-CSF intracellular signaling and removed the possibility that TNF-α was acting through autocrine induction of M-CSF secretion. At least three hours of pre-exposure to TNF-α was required for optimal protection from MTX-induced apoptosis (results not shown).
TNF-α provided comparable protection from MTX-induced apoptosis in the RAW264.7 macrophage cell line. TNF-α exposure significantly (P < 0.05) suppressed spontaneous apoptosis of RAW264.7 cells as measured by caspase-3 activity and annexin-V binding (Figure 1D). RAW264.7 cells shared the susceptibility of primary macrophages to MTX, demonstrating enhanced caspase-3 activity and annexin-V binding after six hours of exposure (Figure 1D). This increase was markedly suppressed when TNF-α was added three hours before MTX (Figure 1D). Thus, the anti-apoptotic effect of TNF-α is replicated in RAW264.7 cells, allowing use of this cell line for studying the phenomenon.
TNF-α protection from apoptosis is mediated through TNF-R1 (p55), not TNF-R2 (p75) receptor activation
NF-κB is required for the survival effect of TNF-α in macrophages
TNF-R1 ligation activates several pro-survival pathways via its association with TRAF2 (TNFR-Associated Factor 2), RIP (Receptor-Interacting Protein) c-Src and Jak2. These include the NF-κB pathway, Phosphatidylinositol-3-Kinase/Protein Kinase B (PI3K/AKT) and Mitogen-Activated Protein Kinase pathways [28, 29]. Constitutive NF-κB activity is critical to macrophage survival . Therefore, we next examined the kinetics of NF-κB activation in TNF-α-stimulated macrophages and investigated whether NF-κB activity was required for the anti-apoptotic effect of TNF-α.
The involvement of PI3K/AKT, ERK, JNK and p38 MAP kinases was also explored using their specific inhibitors, but they were found to be dispensable for TNF-α-induced survival of macrophages, as assessed by annexin-V staining and caspase-3 activity (results not shown).
Autocrine TNF-α signaling is not required for basal survival of M-CSF-maintained macrophages
Macrophages are also sources of TNF-α, raising the possibility that constitutive NF-κB activity (and thus survival) depended on autocrine TNF-α stimulation. We, therefore, treated BMDM cultures with etanercept, a chimeric protein comprising Fc domains of human IgG1 and TNF-α-binding domains of the Type 2 TNF receptor, which neutralises both murine and human TNF-α . An irrelevant human myeloma-derived IgG1 served as a control. Etanercept, however, did not increase caspase-3 activation, indicating that autocrine TNF-α stimulation was not important for survival of macrophages cultured with optimal concentrations of M-CSF (results not shown).
RANKL, a weak activator of NF-κB in macrophages, does not protect from MTX-induced apoptosis
MTX itself does not induce apoptosis through NF-κB suppression
Anti-inflammatory actions of MTX on macrophages have been attributed to enhanced extracellular adenosine generation. However, neither adenosine itself (Figure 6C), or the pan-adenosine receptor agonist NECA (Figure 6D), affected caspase-3 activity. Thus, adenosine accumulation alone is not a sufficient explanation for MTX-induced macrophage apoptosis.
TNF-α protects macrophages from apoptosis induced by MTX. This offers an explanation for the clinical observation that anti-TNF-α treatments cause apoptosis of monocyte/macrophage lineage cells in peripheral blood and synovium of patients with RA , in a study where most participants were also taking MTX. The survival signal from TNF-α is transduced through TNF-R1 and the canonical NF-κB pathway. M-CSF, which directs macrophage differentiation, growth and survival, but does not activate NF-κB signaling, cannot substitute for TNF-α. This is notable, because M-CSF is present in peripheral blood and synovial fluid in RA , and might have been expected to render anti-apoptotic effects of TNF-α redundant. M-CSF signals for growth and survival primarily through the PI3K/AKT pathway.
TNF-α can activate both pro-apoptotic and anti-apoptotic pathways, with outcomes that differ between cell types and conditions. The importance of NF-κB signaling in survival is highlighted by the outcomes of PAR and BAY exposure, which converted TNF-α from a dominantly pro-survival signal to an apoptotic signal. NF-κB-regulated proteins that have been linked to inhibition of apoptotic signaling include TRAF-1, TRAF-2, cIAP-1, cIAP-2, XIAP, FLIP, A20, GADD45β, the antioxidant Mn-SOD, and anti-apoptotic members of the Bcl-2 family, such as A1 and Bcl-xL .
The canonical NF-κB pathway is activated in macrophages of rheumatoid synovium , consistent with a role in maintaining cell survival in untreated patients [10, 11]. Other products of the inflamed synovium, including IL-1, RANKL, GM-CSF, VEGF, PDGF and leukotrienes  can promote NF-κB signaling in responsive cells and may contribute, with TNF-α, to NF-κB activation in macrophages of rheumatoid synovium. However, the experiments with RANKL indicated that not all NF-κB activators protect from apoptosis. RANKL failed to protect primary murine macrophages from MTX-induced apoptosis, even at concentrations that promote osteoclastogenesis in our hands  and which exceed the concentrations reported in rheumatoid synovial fluid . The difference may arise because RANKL activates NF-κB less intensely, or because of differences in other pathways recruited by TNF-R1 and RANK. Finding that RANKL is not a survival factor for macrophages mirrors findings with osteoclast precursors, where RANKL promotes activation and differentiation, but not survival . The mature osteoclast, however, is protected from apoptosis by RANKL with mixed reports of NF-κB involvement . The finding that RANKL-induced NF-κB activation is insufficient to protect primary macrophages from apoptosis is also in accord with the conclusion from clinical studies, that RANKL is not an important driver of synovial inflammation in RA, even though it is critical for erosion formation .
MTX has been reported to suppress NF-κB activity in a different cell context . However, NF-κB suppression could not be demonstrated in MTX-exposed macrophages, so cannot be the mechanism of MTX-induced macrophage apoptosis. MTX anti-inflammatory response has also been linked with the extracellular accumulation of adenosine. Adenosine is reported to promote apoptosis of colon cancer cells in vitro , but we could find no evidence that adenosine alone causes apoptosis in macrophages. The experiments could not disprove a permissive role for adenosine. The experiments do, however, prove that MTX and TNF-α do not just act conversely on NF-κB signaling. MTX has many potentially apoptotic actions [3, 13, 15] and NF-κB has many anti-apoptotic actions . It, therefore, seems more likely that the fate of TNF-α and MTX-exposed macrophages is decided by the late effectors of apoptosis signaling, particularly at mitochondrial steps, that integrate and balance multiple converging inputs . This requires better definition.
Macrophage NF-κB signaling is an attractive therapeutic target in RA because of roles in inflammation, differentiation and survival. However, NF-κB pathways are employed in diverse tissues, with broad biological consequences. Anti-TNF-α treatments suppress NF-κB activity in circumstances where it is promoted by TNF-α, such as in the synovial macrophage. Currently available NF-κB-suppressing small molecules, however, lack such cellular specificity. Glucocorticoids suppress macrophage NF-κB activity, but with poor specificity . The proteosomal inhibitor, bortezomib, suppresses NF-κB signaling by preventing IκBα degradation and is reported to reduce synovial macrophage inflammatory activity in experimental murine collagen arthritis  but, again, is not specifically targeted to the macrophage. Thalidomide likewise suppresses clinical activity of systemic onset juvenile RA , and leads to monocyte apoptosis in vitro through actions on AKT-1  and NF-κB signaling , but with the attendant threat of teratogenesis and other serious off-target effects. Developing macrophage-specific NF-κB suppressants may also have to contend with internal redundancy, with RelA and cRel able to serve similar functions for apoptosis suppression in haematopoietic cells .
We were able to confirm that the potent NF-κB activator, TNF-α, counters MTX-induced apoptosis in primary murine macrophages. A less potent activator, RANKL, could not. Neither could the primary macrophage differentiation and survival factor, M-CSF, which we found did not activate NF-κB signaling. However, MTX did not suppress either constitutive or induced NF-κB signaling, ruling out a direct apoptotic action through NF-κB inhibition. These findings provide an additional explanation for the clinical complementarity between MTX and anti-TNF-α treatments for rheumatoid arthritis.
The authors acknowledge the facilities, scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, a facility funded by The University, State and Commonwealth Governments. We thank Dr. Kathy Hill-Miller for technical support with flow cytometry.
Supported by National Health and Medical Research Council (Australia) Project Grant No. 572638 and an Australian Commonwealth Postgraduate Scholarship awarded to Susan ZY Lo.
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