Modulation of neutrophil apoptosis by pharmacological agents.

Abstract

The human neutrophil is known to have a short half-life in circulation, estimated to be between 8 and 16 h. This lifespan is so short because circulating neutrophils constituitively undergo apoptosis. As neutrophils become apoptotic the chromatin becomes condensed and fragmented, expression of the low affinity receptor for IgG (CD16) on the cell surface falls and functional activities such as the ability to produce superoxide are diminished [ 11. Apoptotic circulating neutrophils are quickly cleared from the circulation by phagwytic cells, which recognise markers of apoptosis on the cell surface [2]. As the mature neutrophil has such a short lifespan, the production of neutrophils from the bone marrow must be maintained at -5 x every day to keep numbers of circulating neutrophils at levels that are able to protect against infections. If this production of neutrophils is disrupted, neutropaenia quickly develops, resulting in a decreased ability to fight off bacterial or fungal infections. Patients undergoing chemoor radio-therapy for the treatment of certain leukaemia's and solid tumours are known to suffer from neutropaenia during the periods of treatment due to damage to the bone marrow. The period and severity of the neutropeania vary greatly, but in all cases there is an increased risk of infection in these patients. This often results in a suspension of therapy and increased hospitalisation times. This therapyinduced neutropaenia may be managed by the use of colony stimulating factors such as Granulocyte-Colony Stimulating Factor (GM-CSF) to increase the production of neutrophils from the bone marrow. These treatments show a reduction in the period and severity of the neutropeania but side-effects such as increased adhesion of neutrophils to capillary walls and pulmonary accumulation, due to up-regulation of certain surface proteins, may be seen. These side-effects are a consequence of neutrophil priming. As an alternative to the use of colony stimulating factors to increase neutrophil production we have been studying the effects of three pharmacological agents (5azacytidine, hexarnethylene-bis-acetamide [HMBA] and sodium butyrate) on neutrophil apoptosis, with the aim of increasing the functional lifespan of the circulating neutrophil. Each of these drugs have previously been shown not to prime neutrophils [3] and so should not elicit the side effects that result from CSFinduced increases in adhesion. Neutrophils were isolated from venous blood of healthy volunteers to >98% purity, and suspended in RPMI 1640 medium supplemented with 0.25% (v/v) foetal calf serum and 2 mM L-glutamine as in [4]. Suspensions of 5 x lo6 celldml were incubated with no additions (controls), or were supplemented with 50 U/ml GM-CSF, 50 ph4 5-azacytidine, 1 mM hexamethylene-bis-acetamide, 0.4 mM sodium butyrate or a combination of GM-CSF with each of the other drugs. After incubation at 37 C for 22 h neutrophil aliquots were used for morphological assessment of apoptosis, ability to produce superoxide, and measurement of DNA fragmentation. Cytospins were prepared from 10' cells and stained with May-Griinwald solution; at least 500 cells were assessed per slide [4]. Superoxide production was measured from 5 x Id cells with 10 @I luminol using either 1 @I f-Met-Leu-Phe or 100 ng/d PMA [5]. DNA fragmentation was measured by separating fragmented and non-fragmented DNA by cenbifugation and determining DNA content colorimetrically using diphenylamine reagent [4]. Table 1. shows that sodium butyrate protects neutrophils from apoptosis more effectively than GM-CSF, confirming our previous results, and that this effect is enhanced when these two agents are used together. Sodium butyrate [6], 5-azacyt1dine [7] and HMBA [8] have all been shown to increase gene transcription in other cell types, and to stimulate protein biosynthesis in human neutrophils [3]. However neither HMBA or 5-azacyt1dine gave significant protection from apoptosis Table 1. &