Potentiation of the respiratory burst of human neutrophils by cycloheximide: regulation of reactive oxidant production by a protein(s) with rapid turnover?

Incubation of human neutrophils with the protein biosynthesis inhibitor cycloheximide for > or = 90 min results in a decreased ability to generate reactive oxidants during the respiratory burst. This implies that active protein biosynthesis is required to sustain the ability of these cells to generate reactive oxidants. However, short term incubation of neutrophils (40-60 min) with either cycloheximide or puromycin results in a significant increase in oxidase activity stimulated by either fMet-Leu-Phe (> 60%) or by leukotriene B4 (> 30%). However, after incubation for 40-60 min with these inhibitors of protein biosynthesis, the respiratory burst stimulated by PMA was unaffected whilst that stimulated by the particulate stimuli opsonised zymosan or latex beads was significantly inhibited. The enhanced oxidase activity stimulated by the soluble agonists was not explained by changes in receptor expression, alterations in intracellular Ca2+ levels or by enhanced degranulation. These results suggest that oxidase activity stimulated by soluble agonists in neutrophils is normally regulated by a short-lived, actively-synthesised protein(s).

Sodium butyrate delays neutrophil apoptosis: role of protein biosynthesis in neutrophil survival.

Sodium butyrate, which directly affects chromatin structure and function in many cells, is as effective as granulocyte-macrophage colony-stimulating factor (GM-CSF) in delaying apoptosis in human neutrophils. Both butyrate and GM-CSF preserved the ability of neutrophils cultured for 22 h in vitro to generate reactive oxidants and express receptors such as CD16. They also delayed apoptotic morphology and DNA fragmentation, and stimulated de novo biosynthesis: newly-labelled polypeptides detected by 2D-polyacrylamide gel electrophoresis after treatment with GM-CSF and butyrate were very similar. Cycloheximide abrogated the effects of both GM-CSF and butyrate. Exposure to butyrate for 1 h did not prime oxidant production and did not up-regulate expression of CD11b. Hence, unlike GM-CSF, butyrate does not stimulate translocation of granules to the plasma membrane. These data suggest that active gene expression is involved in the regulation of neutrophil apoptosis and changes in chromatin structure and function may control apopotosis in these cells.

Regulation of neutrophil apoptosis by sodium butyrate.

Neutrophils have a very short half life because they constitutively undergo apoptosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay apoptosis, but this agent also primes functions such as the respiratory burst and receptor upregulation. Here, we show that sodium butyrate, which has been shown to increase gene expression and differentiation in a variety of cell types, is more effective than GM-CSF in delaying neutrophil apoptosis. Thus, sodium butyrate preserves cell morphology and function, and butyrate-treated cells express high levels of CD16 after overnight culture. However, neither GM-CSF nor sodium butyrate appear to affect mRNA levels for CD16.

Effect of cytotoxic drugs on mature neutrophil function in the presence and absence of granulocyte-macrophage colony-stimulating factor.

The effects of the cytotoxic drugs, adriamycin, cyclophosphamide, daunomycin (daunorubicin), prednisolone, actinomycin D, azacytidine and vincristine at concentrations of 1 microM on mature neutrophil function were examined. Up to 5 h incubation with adriamycin, azacytidine, cyclophosphamide, daunomycin and prednisolone had no effect on either luminol chemiluminescence or superoxide secretion. However, after 15 min or 1 h (but not 5 h) incubation vincristine enhanced fMet-Leu-Phe stimulated chemiluminescence, whilst after 5 h incubation with actinomycin D the ability of neutrophils to generate reactive oxidants in response to all stimuli tested was impaired: after 5 h incubation with adriamycin reactive oxidant production was also impaired, but only when fMet-Leu-Phe was used as stimulant. All of the drugs tested except azacytidine inhibited neutrophil oxidant production after 5 h incubation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Actinomycin D and cyclophosphamide also inhibited GM-CSF stimulated protein biosynthesis. These data indicate that cytotoxic drugs may compromise the potentially beneficial effects of CSFs on mature neutrophil function during therapy.

A phase I clinical evaluation of liposome-entrapped doxorubicin (Lip-Dox) in patients with primary and metastatic hepatic malignancy.

Liposome-entrapped doxorubicin (Lip-Dox) was evaluated in two phase I clinical trials in patients with hepatic malignancy. Patients with metastases from primary gastric or colonic tumours and patients with hepatoma were eligible. Lip-Dox was extremely well tolerated and acute toxicities such as nausea and vomiting were totally eliminated; no antiemetics were used even at doses of 80 mg/m2. Toxicities such as alopecia and myelosuppression were also ameliorated. There were tumor regressions and reductions in hepatomegaly in patients treated on both the weekly and 21-day studies. The maximum tolerated dose (MTD) in the weekly study was 22.5 mg/m2/week and in the 21-day trial the MTD was 70 mg/m2.