Macrophages Require Constitutive NF-κB Activation To Maintain A1 Expression and Mitochondrial Homeostasis

Abstract

NF-B is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophagesurvival has yet to be elucidated. Here, we demonstrate that constitutive NF-B activation is essential for macrophage survival. Blocking the constitutive activation of NF-B with pyrrolidine dithiocarbamate or expression of IB␣ induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-B activation induced a time-dependent loss of mitochondrial transmembrane potential (⌬⌿ m ) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD.fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on ⌬⌿ m collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to ⌬⌿ m loss and that ectopic expression of A1 protected against cell death following inactivation of NF-B. These data suggest that inhibition of NF-B in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-B activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.The mechanism(s) by which the pleiotropic transcription factor nuclear factor kappa B (NF-B) regulates cell survival remains unclear. Mice null homozygous for the p65 alleles or IB kinase ␤ are embryonic lethal due to extensive liver cell death (6, 40), demonstrating that NF-B p65 or its activating kinase is essential for development. Embryonic macrophages and fibroblasts from p65 null mice are susceptible to tumor necrosis factor alpha (TNF-␣)-induced apoptosis, which is rescued by overexpression of p65 but not p50 (5). Furthermore, inhibition of NF-B by IB␣ overexpression or by the chemical inhibitor pyrrolidine dithiocarbamate (PDTC) rendered many cell types normally resistant to the effects of TNF-␣ susceptible to TNF-␣-induced apoptosis (21,61,62). In addition, suppression of NF-B activation has been shown to enhance apoptosis following radiation or treatment with chemotherapeutic agents (59,64,66). Although many investigations have employed exogenous mediators to induce apoptosis following NF-B inactivation, few have reported the occurrence of apoptosis in response to NF-B inhibition in the absence of additional stimuli (16,36,38,67).Unlike monocytes, normal macrophages are long-lived cells resistant to many apoptotic stimuli, including Fas and TNF-␣ receptor ligation, ionizing radiation, and multiple antineoplastic or cytotoxic agents (32, 33, 49, 52). We recently demonstrated that expression of FLICE-inhibitory protein (Flip) protected differentiated macrophages from Fas-mediated apoptosis (52); however, the mechanisms responsible for macr...

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