Nucleotidase and alkaline phosphodiesterase We activities were considered as markers for the plasma membrane portion and showed an approximately 45-fold enrichment compared to the whole homogenate

Nucleotidase and alkaline phosphodiesterase We activities were considered as markers for the plasma membrane portion and showed an approximately 45-fold enrichment compared to the whole homogenate. membrane is an early event of the apoptotic cellular program that may represent an trigger for the generation of aCL. Keywords: cardiolipin, apoptosis, anticardiolipin antibodies, COH000 antiphospholipid antibody syndrome Introduction Programmed cell death (PCD) or apoptosis, a common phenomenon that issues any nucleated cell type, is usually a cardinal element of immune system homeostasis [1,2]. Apoptosis may be defined morphologically and biochemically [3]. In most cases, PCD is accompanied by characteristic ultrastructural alterations (cell shrinkage, cytoplasmic compaction, membrane blebbing, nuclear chromatin condensation) [4] and internucleosomal ladder-type DNA fragmentation [5,6]. Most cells undergoing apoptosis are removed by phagocytosis before the death process culminates in low-molecular-weight DNA fragmentation and apoptotic modification of nucleus morphology [7]. It has recently been suggested that phosphatidylserine (PS), which is normally almost totally confined to the inner leaflet of the plasma membrane, represents a membrane flag on apoptotic cells [8,9] SPN and thereby functions as acknowledgement transmission for phagocytosis [10]. Changes in the asymmetric distribution of PS precedes the loss of membrane integrity by several hours. The investigation of lipid topology in eukaryotic cells demonstrates a physiological asymmetrical distribution of phospholipids in cell membranes [11,12]. Phosphatidylcholine (PC) and sphingomyelin (SM) are almost exclusively located in the outer leaflet of the lipid bilayer, while phosphatidylserine (PS) and 70% of phosphatidylethanolamine (PE) are located in the inner leaflet of the plasma membrane. Cardiolipin (CL) is mostly confined to the mitochondrial membrane. This asymmetrical distribution, which results from transbilayer movements, can be perturbed, transiently or permanently, by a variety of events [13C15], including apoptosis [8,9]. Increased levels of apoptosis of MRL-lpr/lpr T lymphocytes [16], as well as of peripheral blood lymphocytes (PBL) from patients with systemic lupus erythematosus (SLE), have recently been reported [17]. Because of accelerated apoptosis, increased amounts of nucleosomes are released into the extracellular space. However, the role of apoptosis in SLE pathogenesis is still controversial. In apparent contrast with the observation of an increased rate of apoptosis in PBL, overexpression of bcl-2 has been explained in T lymphocytes and related to disease activity [18]. In HIV contamination, a correlation between serum antiphospholipid antibodies and the level of apoptosis has been suggested [19]. We recently developed a highly selective method for detection of antiphospholipid reactivity [20]. This method allows the selection of highly specific anticardiolipin antibodies (aCL) from sera of patients with antiphospholipid antibody syndrome (APS) [20]. Using this method, we show that affinity-purified highly specific aCL, isolated from patients with APS, target CL molecules uncovered around the cell surface of apoptotic cells. Exposure of CL on plasma membrane of apoptotic cells may be a relevant contributing factor to the generation of aCL in the course of APS or HIV contamination. Materials and methods Cells and reagents Human promonocytic U937 cells were cultured in RPMI COH000 1640 (Gibco-BRL, Life technologies Italia srl, Milano, Italy) made up of foetal calf serum (FCS) 10% at 37C in a humified 5% CO2 athmosphere. Apoptosis was induced by incubating the cells at a concentration of 5 105 cells/ml in serum-free medium supplemented with insulin (5 mg/l) and transferrin (5 mg/l), and by adding either Tumor Necrosis Factor (TNF, Genzyme Diagnostics, Cambridge MA, USA), 1000 IU/ml for 0 min, 30 min, 1 h, 2 h and 3 h), or anti-Fas (CD95) IgM mAb (Immunotech, Marseille, France), 100 ng/ml for 0 min, 30 min, 1 h, 2 h and COH000 4 h. Phospholipid analysis of isolated plasma membrane fractions Plasma membrane fractions from U937 cells were isolated as previously reported [21]. Briefly, after homogenization, untreated and anti-FAS treated cells (100 ng/ml for 4 h) were sedimented at 200 000 for 20 min and the pellet was resuspended in 65% sucrose. This resuspended pellet was throughly homogenized. It was then brought to a refractive index of 1430 (68% w/v) with chilly saturated sucrose. The sample (15 ml) was placed COH000 on the bottom of 15 90 mm cellulose nitrate tubes. Continuous 25C65% (w/v) density gradients of sucrose were created above the particulate homogenate. The gradients were then centrifuged at 90 000 for 16 h in a SW41 rotor. The membranes and organelles in the homogenate were separated into bands of differing density. The bands corresponding to the plasma membranes were removed and the material from these layers was diluted with 016 m NaCl and sedimented at 200 000 for 40 min. The purity of plasma membrane preparations COH000 was evaluated by testing specific enzymatic activities [21]. Nucleotidase and alkaline phosphodiesterase I activities.

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