Cytosolic Phospholipase A2 Participates with TNF-α in the Induction of Apoptosis of Human Macrophages Infected withMycobacterium tuberculosisH37Ra

Abstract

Macrophage (MΦ) apoptosis, an important innate microbial defense mechanism induced by Mycobacterium tuberculosis (Mtb) H37Ra, depends on the induction of TNF-α synthesis. When protein synthesis is blocked, both infection with Mtb and addition of TNF-α are required to induce caspase 9 activation, caspase 3 activation and apoptosis. In this study, we show that the second protein synthesis-independent signal involves activation of group IV cytosolic phospholipase A2 (cPLA2). Apoptosis of Mtb-infected MΦ and concomitant arachidonic acid release are abrogated by group IV cPLA2 inhibitors (methyl arachidonyl fluorophosphate and methyl trifluoromethyl ketone), but not by inhibitors of group VI Ca2+-independent (iPLA2 ; bromoenol lactone) or of secretory low molecular mass PLA2. In MΦ homogenates, the predominant PLA2 activity showed the same inhibitor sensitivity pattern and preferred arachidonic acid over palmitic acid in substrates, also indicating the presence of one or more group IV cPLA2 enzymes. In concordance with these findings, MΦ lysates contained transcripts and protein for group IV cPLA2-α and cPLA2-γ. Importantly, group IV cPLA2 inhibitors significantly reduced MΦ antimycobacterial activity and addition of arachidonic acid, the major product of group IV cPLA2, to infected MΦ treated with cPLA2 inhibitors completely restored the antimycobacterial activity. Importantly, addition of arachidonic acid alone to infected MΦ significantly reduced the mycobacterial burden. These findings indicate that Mtb induces MΦ apoptosis by independent signaling through at least two pathways, TNF-α and cPLA2, which are both also critical for antimycobacterial defense of the MΦ .

Inhibition of Cytosolic Phospholipase A2 Suppresses Production of Cholesteryl Ester through the Reesterification of Free Cholesterol but not Formation of Foam Cells in Oxidized LDL-Stimulated Macrophages

“…While cPLA 2 a was expressed ubiquitously in the body, cPLA 2 g was expressed in heart, skeletal muscle, brain, 34) and monocytes from peripheral blood. 35) We also showed here that cPLA 2 g was expressed in MPM as well as THP-M (Figs. 1, 6).…”

Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis

“…Mononuclear cells from peripheral blood of healthy donors after obtained informed consent were isolated as previously described (7). M for the in situ TUNEL assay were cultured on plastic cover slips (Nunc, Rochester, NY) and were plated at 2.5 ϫ 10 6 mononuclear cells/ml/well in 24-well cluster plates (Costar, Cambridge, MA).…”

“…Counting of mycobacteria using the Bactec model 460TB system (BD Biosciences) was performed as described (7).…”

“…It is known that macrophages (M), the first line of defense against tuberculosis, undergo apoptosis when inoculated with Mtb and develop significant antimycobacterial activity (3)(4)(5). Mtb-induced M apoptosis requires a dual signal, the action of TNF-␣ (6), and the activation of cytosolic phospholipase A 2 (7), and is thought to result in activation of caspases, including caspase-9 and caspase-3. Caspase-9 is an initiator caspase of apoptosis and its activation, which leads to activation of the effector caspase caspase-3, requires formation of the "apoptosome", a complex formed by cytochrome c released from the mitochondria into the cytoplasm, the cytosolic factor Apaf-1, ATP, and procaspase-9 (8,9).…”

Induction of Nitric Oxide Synthase by Oldenlandia diffusa in Mouse Peritoneal Macrophages.

“…10) The proinflammatory cytokine tumor necrosis factor-a (TNF-a) regulates systemic responses to microbial infection or tissue injury. 11) These signals stimulate immune functions and induce expression of acute-phase reactants in the liver, among other effects. Production of TNF-a protein is enhanced by the presence of interferon-g (IFN-g).…”