Abstract 1549 Neonatal Immunology & Hematology Poster Symposium, Monday, 5/3

Apoptosis of neutrophils (PMN) promotes their clearance by the reticuloendothelial system, a key process in the resolution of inflammation. In contrast, delayed apoptosis resulting in persistent tissue PMN may incite development of chronic inflammatory conditions. We previously observed a lower apoptosis rate in cord blood PMN than in those of adults (J.Leukoc. Biol., 1998). Since cord blood contains high numbers of immature marrow-derived PMN, we hypothesized that apoptotic capacity increasing during PMN maturation, and that therefore marrow PMN have lower rates of apoptosis than circulating PMN. To test this, paired samples of isolated peripheral blood (PB) and bone marrow (BM) PMN (n=4) were cultured in RPMI-1640/5% FCS for 0, 6 or 24 h. Apoptosis was induced by culturing PMN with the protein synthesis inhibitor, cycloheximide. The percentage of apoptotic PMN in cultures was determined by flow cytometric analysis (TUNEL assay). We observed a lower proportion of apoptotic PMN in 24 h BM cultures (X ± SD, 36 ± 17% vs. 57 ± 14% in PB cultures, P < 0.03). In addition, we observed that cycloheximide (6 h, 50 µg/mL) induced fewer numbers of apoptotic PMN in BM vs. PB cultures (50 ± 9% vs. 73% ± 4%, P < 0.02). These data, similar to those of previous cord blood studies, suggested to us that immature PMN might either lack or fail to synthesized pro-apoptotic protein(s). One major apoptotic pathway involves cysteine proteases belonging to the IL-1 converting enzyme (ICE) family, of which caspase-3 (CPP32) is a key member. We next investigated the expression of ICE-associated genes in PMN isolated from term cord blood or from healthy adult donors. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed on RNA extracted from freshly isolated cord blood or adult PMN to detect transcripts for the caspase-1, caspase-3 and ICE/REL2 genes. Amplification of GAPDH as an internal standard allowed relative comparison of PCR products. In other studies, a colorimetric assay was used to measure caspase-3 protease activity in timed samples of PMN cultured in media alone or in the presence of anti-Fas IgM (300 ng/mL). We detected transcripts of caspase-1, caspase-3 and ICE/REL2 in cord blood and adult PMN, although caspase-3 expression appeared reduced in some cord blood samples. Caspase-3 activity in adult PMN was increased 1.5-fold over baseline following a 6 h incubation with anti-Fas IgM or at 24 h in media alone (2.5-fold increase). In contrast, in cord blood PMN caspase-3 activity was not significantly increased over baseline values even at 24 h (P=0.09). We conclude that: 1) immature marrow PMN have a lower rate of apoptosis than their mature counterparts in the circulation and 2) cord blood PMN have diminished activity of the pro-apoptotic protein, caspase-3. We speculate that functionally immature PMN gain apoptotic capacity during maturation in association with an increasing activity of pro-apoptotic protein(s).