Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain–like interleukin 1β–converting enzyme)–inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense–treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain–containing 5′ inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-xL. Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-κB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-κB, or Bad.

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...