Activated neutrophils induce prolonged DNA damage in neighboring cells

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Abstract
We have measured the capacity of highly-purified, paraffin oil-elicited neutrophils to induce DNA single-strand breaks in a newly established plasmacytoma cell line, RIMPC 2304, which was induced by a retrovirus containing the c- myc and V- Ha - ras oncogenes. This cell line effectively repairs DNA damage induced by γ-irradiation. DNA damage induced by neutrophils was correlated with the oxidative burst of the neutrophils. The levels of superoxide anion, H 2 O 2 and HOCl produced after stimulation of the neutrophils (6 × 10 5 /cm 3 ) with the tumor promoter phorbol myristate acetate (100 nM) were 33.8 μM, 12.8μM and 1.7 μM respectively in 15 mm, and 98 μM, 20 μM and 8.7 μM respectively in 90 mm.The results of alkaline elution experiments revealed that when the same concentration of neutrophils was co-incubated for15 min in serum-free medium with an equal number of radioactively labeled RIMPC 2304 cells, the latter incurred a level of damage that approximated that caused by 300 rad equivalents of γ-irradiation or by a 1-min treatment with 20 μM H 2 O 2 at 37δC. Damage from neutrophils was coincident with the oxidative burst; it was induced rapidly (within 5 min) but remained high for more than 90 min. The level of damage achieved was dependent upon the ratio of neutrophils: target cells and was clearly detectable at ratios as low as 0.25:1. Induction of single-strand breaks was completely inhibited by catalase and partially inhibited by superoxide dismutase, mannitol, and reduced glutathione but not by Na azide. Addition of the non-steroidal anti-inflammatory drug indomethacin either enhanced (at 50 μM) or had no effect (at 2 μM) on the damage detected. Finally, repair of strand breaks induced by neutrophils was significantly slower (half time ∼10 min) than that observed for repair of similar levels of damage induced by H 2 O 2 or γ-irradlatlon (half-times ∼3 min, each). The results indicate that neutrophils cause prolonged DNA damage in neighboring cells. Moreover, they indicate that although H 2 O 2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated. The data are discussed in relation to the possible role of neutrophils in chronic inflammation and in pristane-induced plasmacytoma formation in mice.