Apoptosis: programmed cell death at a molecular level.

OBJECTIVES: To characterize cell surface receptors, their ligands, and their proteins in the 2 major pathways of apoptosis; the components that promote/suppress these interactions; the noninflammatory removal of apoptotic bodies by dendritic cells; and methods of assay in studies of cell death. To describe: how deregulation of apoptosis may contribute to autoimmunity, cancer, and neurodegenerative disorders and strategies some viruses have evolved that interfere with the host's apoptotic pathways. METHODS: The authors reviewed and compiled literature on the extrinsic (tumor necrosis factor [TNF] receptor superfamily and ligands) and intrinsic (mitochondria-associated) apoptotic pathways, the pro- and antiapoptotic proteins of the B-cell follicular lymphoma (Bcl)-2 family, the nuclear factor (NF)-kappaB family of proteins, commonly used laboratory methods to distinguish apoptosis from necrosis, the recognition and removal by phagocytosis of apoptotic cells by dendritic cells, and viral strategies to avoid a host's apoptotic response. RESULTS: The 2 major pathways of apoptosis are (1). FasL and other TNF superfamily ligands induce trimerization of cell-surface death receptors and (2). perturbated mitochondria release cytochrome c, the flavoprotein apoptosis-inducing factor, and second mitochondria-derived activator of caspases/DIABLO (a protein that directly neutralizes inhibitors of apoptotic proteins and activates proteases). Catalytically inactive cysteine proteases, called caspases, and other proteases are activated, ultimately leading to cell death with characteristic cellular chromatin condensation and DNA cleavage to fragments of approximately 180 bp. The inhibitory/promoting action of Bcl-2 family members is involved in the release of cytochrome c, an essential factor for the mitochondrial-associated pathway. A balance between inhibition/promotion determines a cell's fate. The NF-kappaB family in the cytoplasm of cells activates various genes carrying the NF-kappaB response element, such as members of the inhibitor of apoptotic proteins family. A few of the more common methods to detect apoptotic cell death are described, which use immunochemical, morphologic and flow cytometric methods, and genetic markers. Exposed phosphatidylserine at the outer leaflet of the plasma membrane of the apoptotic cell serves as a possible receptor for phagocytosis by immature dendritic cells. These cells phagocytize both apoptotic and necrotic cells, but only the latter induce maturation to become fully functional antigen-presenting cells. Viral inhibitors of apoptosis allow increased virus replication in cells, possibly resulting in their oncogenicity. CONCLUSIONS: Balanced apoptosis is crucial in development and homeostasis, and all multicellular organisms have a physiologically programmed continuum of pathways to apoptotic cell death. Further studies of the control at the molecular level of key components and promoters/suppressors of apoptosis may provide better approaches to treatment of autoimmune diseases, malignancies, and neurodegenerative disorders. Many important questions remain regarding the advantages of modifying apoptotic programs in clinical situations.

Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of human renal microvascular endothelial cells.

Capping and release of membranous, small (< 1.5 microm) endothelial microparticles were quantified by immunofluorescence microscopy and flow cytometry after treatment of cultures of human renal microvascular endothelial cells with agonists tumor necrosis factor-alpha (TNF-alpha) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-alpha and mitomycin C induced dissimilar appearing CD31-containing caps after 3 h, followed by endothelial microparticle release after 6 h. The degree of capping correlated with increasing counts of released microparticles. For lymphokine-inducible CD54, TNF-alpha also induced CD54-containing caps and microparticle release, but mitomycin C failed to induce the expression of either entity. Neither capping nor microparticle release caused by TNF-alpha was part of an apoptotic pathway that involved caspase 3. Mitomycin C treatment of endothelial cells caused capping and microparticle release with a time course similar to TNF-alpha induction for 15 to 24 h, but assays for caspase 3 were positive, confirming the apoptotic action of mitomycin C. Membrane capping and microparticle release from endothelial cells are a convenient experimental model for studying protein movement, release of microparticles, and their possible biological significance.

Inhibition of insulin-like growth factor binding protein 5 proteolysis in articular cartilage and joint fluid results in enhanced concentrations of insulin-like growth factor 1 and is associated with improved osteoarthritis.

OBJECTIVE: The complement component C1s is present in dog joint fluid in an activated state. Since C1s degrades insulin-like growth factor binding protein 5 (IGFBP-5), we undertook to determine whether inhibiting C1s in joint fluid would result in an increase in the amount of intact IGFBP-5 and IGF-1 in cartilage and joint fluid, and whether C1s inhibition would be associated with a reduction in cartilage destruction during the development of osteoarthritis (OA). METHODS: Twenty-two dogs were randomized to 3 treatment groups. All dogs underwent anterior cruciate ligament transection and were exercised. Dogs received 1 of 3 treatments: buffer alone (controls; n = 6); PB-145, a peptide derived from the sequence of antithrombin III (n = 9); and pentosan polysulfate (PPS; n = 7). PB-145 or saline was injected into the joint space 3 times per week for 3 weeks. PPS was injected intramuscularly weekly for 3 weeks. RESULTS: Joint histology showed preservation of chondrocytes and a smooth joint surface in the animals treated with PB-145 and PPS. Mankin scoring showed statistically significant reductions in joint destruction with PB-145 and PPS treatments (P < 0.01) compared with buffer control. Mean active collagenase concentrations were decreased by these two treatments. Immunoblotting of joint fluid showed that both treatments increased concentrations of intact IGFBP-5. Direct analysis of IGFBP-3 and IGFBP-5 protease activity showed that IGFBP-5 was degraded more rapidly and that PB-145 and PPS inhibited the degradation of both proteins. Total IGF-1 concentrations in joint fluid were increased 5.6-5.8-fold by these two treatments. Analysis showed that C1s was being activated in joint fluid and that its activation was inhibited by the addition of PB-145 or PPS. CONCLUSION: The findings suggest that direct inhibition of the serine protease C1s results in increased concentrations of intact IGFBP-5 and that proteolysis of IGFBP-3 is also inhibited, probably by the inhibition of some other protease. This increase in concentrations of intact IGFBP-3 and IGFBP-5 leads to an increase in IGF-1 which is associated with an improvement in joint architecture during the development of OA.