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 18 . All other reagents were sourced from Sigma-Aldrich (Sydney, NSW, Australia), unless otherwise indicated.

RAW_264.7 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 19 , 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.

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.

RAW_264.7 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 RAW_264.7 cells and scraped BMDM were lysed and assayed for caspase-3 activity as described previously 20 .

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.

RAW_264.7 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.

RAW_264.7 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).

EMSA was performed on nuclear extracts from RAW_264.7 cells (8 × 10⁶ per treatment) that were treated with 10 ng/mL TNF-α for up to one hour, according to previously described protocols 21 . 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.

RAW_264.7 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 21 . 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 RAW_264.7 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.

M-CSF is the primary growth, differentiation and survival factor for macrophages under physiological conditions. Serum concentrations of M-CSF are elevated to approximately 0.6 ng/ml in patients with RA 22 , but concentrations in synovium are unknown. In vitro, a concentration of 30 ng/ml was optimal for sustaining the growth and survival of bone marrow-derived macrophages (BMDM) of C57BL/6 mice (results not shown). In the presence of 30 ng/mL M-CSF, exposure to 10 μM MTX for 24 hr caused an approximately 25% loss of cell numbers in BMDM cultures (Figure 1A). This was related to increased apoptosis, as estimated by caspase-3 activity (Figure 1B), annexin-V binding (early apoptosis; Figure 1B) and increased sub-G₀ (apoptotic) fraction on flow cytometric cell cycle analysis (Table 1). The proportion of cells in S-phase also increased with MTX, consistent with its known action on completing DNA synthesis 23 (Table 1).

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 24 . This was replicated in the murine RAW_264.7 macrophage cell line (Figure 1E). RAW_264.7 macrophages do not require exogenous M-CSF to proliferate, having gained growth signaling through introduction of the Abelson leukaemia virus 25 . They have been previously found to model primary macrophage apoptosis response 26 . 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 RAW_264.7 macrophage cell line. TNF-α exposure significantly (P < 0.05) suppressed spontaneous apoptosis of RAW_264.7 cells as measured by caspase-3 activity and annexin-V binding (Figure 1D). RAW_264.7 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 RAW_264.7 cells, allowing use of this cell line for studying the phenomenon.

Monocytes/macrophages express both TNF-R1 and TNF-R2 8 . To determine the receptor subtype(s) that mediated the survival effect of TNF-α, we exploited the species specificity of TNF-α action. Murine TNF-α (mTNF-α) activates both TNF-R1 and TNF-R2 on murine macrophages, while human TNF-α (hTNF-α) activates TNF-R1 only, thereby providing a means to distinguish effects that are mediated through different receptors 27 . mTNF-α and hTNF-α provided comparable protection from MTX-induced caspase-3 activation (Figure 2, P > 0.05), indicating that protection is mediated through TNF-R1, and does not require TNF-R2 signaling.

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 26 . 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.

Proteosomal degradation of the NF-κB inhibitory protein, IκBα, is an early indicator of activation in the canonical NF-κB pathway 30 . TNF-α (10 ng/mL) addition in RAW_264.7 cells induced the degradation of IκBα within 15 minutes (Figure 3A, P < 0.05). This corresponded to the appearance of DNA-binding NF-κB in the nucleus, with activity peaking at 30 minutes before returning to baseline levels by one hour (Figure 3B). Supershift analyses with specific antibodies to RelA, NF-κB₁, c-Rel and Rel-B identified RelA (p65) and NF-κB₁ (p50) as the main components of the NF-κB complex induced by TNF-α (Figure 3B). A light c-Rel supershifted band was also detected (Figure 3B). IκBα levels recovered by one hour (Figure 3A). The IKBA gene is also NF-κB-responsive, so early recovery of IκBα levels indicates functional NF-κB signaling 30 . Lastly, TNF-α stimulation of RAW_264.7 cells transiently expressing a NF-κB-responsive reporter construct showed that NF-κB transcriptional activity was enhanced in a time-dependent manner (Figure 3C). These results confirm that TNF-α is a rapid activator of NF-κB function in macrophages.

To investigate whether NF-κB activity is essential for TNF-α activity against MTX-induced apoptosis, the sesquiterpene lactone parthenolide (PAR) and BAY11-7085 (BAY) were used to specifically prevent NF-κB signaling 19 31 . PAR and BAY pre-treatment for 30 minutes abolished both constitutive and TNF-α-stimulated NF-κB transcriptional activity (Figure 4A). They also completely prevented TNF-α from rescuing RAW_264.7 cells (Figure 4B,C) and primary macrophages (Figure 4D-F) from MTX-induced apoptosis and caspase-3 activation. These findings indicate that NF-κB activation is required for TNF-α protection against apoptosis induced by MTX. Notably, TNF-α became pro-apoptotic, rather than anti-apoptotic when NF-κB signaling was blocked, when estimated by either annexin-V binding (P < 0.05) or caspase-3 activity (P < 0.05).

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).

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 32 . 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).

The observation that TNF-α protected macrophages from MTX-induced apoptosis led us to question whether other cytokines present in rheumatoid synovium or erosions may also protect through NF-κB induction. Receptor Activator of NF-κB Ligand (RANKL) circulates at elevated concentration in RA and is demonstrable in rheumatoid synovium and erosions 33 . It is required for osteoclast formation from monocyte/macrophage precursors. Unlike TNF-α, however, RANKL did not suppress caspase-3 activation in MTX-exposed primary macrophage cultures at concentrations up to 400 ng/mL (Figure 5A). A dose of 200 ng/mL of RANKL is sufficient to elicit classical osteoclastogenesis in our hands 34 , so was used for other experiments. Comparing effects on NF-κB activation, we found that RANKL (six hours) stimulated an approximately three-fold increase in NF-κB-luciferase reporter expression in transfected BMDM, well short of the approximately 22-fold increase with TNF-α (Figure 5B). Western blot analyses also indicated weaker phosphorylation and degradation of IκBα after RANKL treatment, compared to TNF-α (Figure 5C). Therefore, RANKL does not protect differentiated primary macrophages from MTX, possibly due to insufficient activation of NF-κB.

The observation that NF-κB induction countered the apoptotic action of MTX led us to test whether MTX itself acted through inhibiting NF-κB. This has been previously observed in Jurkat T-cells and in the poorly differentiated myelo-monocytic U937 cell line 35 . However, MTX, at concentrations sufficient to induce apoptosis, failed to suppress either basal or TNF-α-induced NF-κB activity in RAW_264.7 cells transiently transfected with a specific NF-κB reporter construct (pNF-κB-TA-Luc) (Figure 6A). In BMDM, it also failed to prevent the TNF-α-stimulated phosphorylation of IκBα, an essential step in IκBα degradation and RelA/NF-κB₁ complex release (Figure 6B). MTX, therefore, does not exert its apoptotic actions 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.