Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages.

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This "engulfment synapse" can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.

Bcl-2 antisense oligonucleotide inhibits the proliferation of childhood leukemia/lymphoma cells of the B-cell lineage.

An 18-mer phosphorothioate bcl-2 antisense oligonucleotide (ASO) inhibited colony formation of three B-cell leukemia/lymphoma cell lines in a dose dependent manner in the range of 0.125-0.5 micromol/l. The scrambled cogener had no detectable effect. A decrease in BCL-2 protein and apoptotic DNA fragmentation was detected in the studied cell lines and primary blast cells of two children with acute lymphoblastic leukemia. Neither BCL-2 protein level, nor DNA integrity was affected by the scrambled control indicating the specific effect ASO. As far as we know, this is the first report on the effects of bcl-2 ASO on childhood leukemia/lymphoma cell samples.

Deoxy-adenosine-monophosphate (dAMP) di-n-butylester induces apoptosis by increasing the dATP level in HL-60 cells.

Deoxy-ATP is a potent inducer of apoptosis. We intended to synthesize a lipophilic dAMP derivative which, according to our working hypothesis penetrates into the cell, is converted to dAMP by intracellular esterases and to dATP by nucleotide kinases. We synthesized dAMP-di-n-butylester (DAB) and tested it. We found that it fulfills the above-described expectations. DAB treatment decreases the viability of HL-60 cells, increases the dATP concentration and induces apoptogenic cytochrome c release from mitochondria with concomitant elevation of caspase-9 activity. Our results indicate that use of dAMP derivatives with masked phosphate may be a feasible approach for pharmacological elevation of intracellular dATP and induction of apoptosis.