Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells

Abstract

In this study we investigated the underlying mechanisms that confer resistance on mature macrophages with the use of macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDM). In the presence of M-CSF, immature precursor cells were induced to undergo proliferation and differentiation into mature macrophages in vitro with cell morphology similar to that of tissue macrophages by day 7-10. Immunoblot analyses showed that bone marrow precursors express appreciable levels of caspase-3 and caspase-9 but no or very low levels of c-fms (M-CSF receptor) and the apoptosis regulators X-linked inhibitor of apoptosis protein (XIAP), c-IAP-1, Bcl-2 and Bax. The differentiation of BMDM is associated with a steady and gradual increase in the levels of c-fms, XIAP, c-IAP-1, Bcl-2 and Bax, reaching maximal levels by day 7. However, the levels of caspase-3 and caspase-9 stayed essentially unchanged even after prolonged incubation (more than 10 days) with M-CSF. Unlike bone marrow precursor cells, mature BMDM (day 7-10) were resistant to apoptosis induced by M-CSF depletion, which includes the activation of caspase-3 and caspase-9 and the degradation of XIAP, Bcl-2 and Bax proteins in the process. Treatment of day 7 BMDM with XIAP anti-sense oligonucleotides (oligos), but not sense oligos, partly abolished their resistance to apoptosis. By using a gel-shift assay and a specific nuclear factor kappaB (NF-kappaB) inhibitor, we demonstrated that NF-kappaB activity is responsible for the up-regulation of XIAP in M-CSF-treated macrophages. In addition, treatment of starved macrophages with M-CSF induced a rapid phosphorylation of Akt kinase before the activation of NF-kappaB. Our results showed that XIAP is one of the anti-apoptotic regulators that confer resistance on mature macrophages by M-CSF.

A Requirement for the p85 PI3K Adapter Protein BCAP in the Protection of Macrophages from Apoptosis Induced by Endoplasmic Reticulum Stress

“…We attempted to induce apoptosis using various activators of the extrinsic pathway of apoptosis (e.g., TNF-a, FasL, TRAIL), but we found that these agents did not induce apoptosis in BMDMs, even when cultured in the absence or presence of serum and LCM (not shown). These results are consistent with earlier studies that report significant resistance to apoptosis in mature macrophages subjected to growth factor withdrawal or addition of activators of the extrinsic pathway of cell death (21)(22)(23). Because macrophages are known to be susceptible to apoptosis induced by ER stress (24-27), we determined whether expression of BCAP influenced survival after addition of thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor that activates ER stress and the intrinsic pathway of apoptosis (28).…”

Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

“…Ratio = (Nuclear intensity -background intensity) ÷ (Cytoplasmic intensity -background intensity) .7 seeded into 6 well plates were cultured in DMEM before being activated for 1 h, with or without isoflavone pretreatment, as described above. Nuclear extraction and the EMSA were performed as described by Lin et al (2001) using the NF-kB consensus oligonucleotide 5'-AGTTG AGGGG ACTTT CCCAG GC-3'. In preliminary experiments, supershift polyclonal antibodies for p65 and p50 were incubated with activated cell nuclear extracts.…”

The leaf extract ofSpondias mombinL. displays an anti-inflammatory effect and suppresses inducible formation of tumor necrosis factor-α and nitric oxide (NO)

“…Murine BM-MØ were generated from the BM cells of the tibia and femur of C57Bl/6 donor mice by a modification of previously described protocols (Lin et al, 2001;Weischenfeldt and Porse, 2008). BM cells were harvested and cultured in R-10 medium containing 10 ng/ml of recombinant murine colony-stimulating factor (rmCSF-1; Immunotools, Friesoythe, Germany) in T-75 cell culture flasks.…”