Death of Monocytes through Oxidative Burst of Macrophages and Neutrophils: Killing in Trans

Abstract

Monocytes and their descendants, macrophages, play a key role in the defence against pathogens. They also contribute to the pathogenesis of inflammatory diseases. Therefore, a mechanism maintaining a balance in the monocyte/macrophage population must be postulated. Our previous studies have shown that monocytes are impaired in DNA repair, rendering them vulnerable to genotoxic stress while monocyte-derived macrophages are DNA repair competent and genotoxic stress-resistant. Based on these findings, we hypothesized that monocytes can be selectively killed by reactive oxygen species (ROS) produced by activated macrophages. We also wished to know whether monocytes and macrophages are protected against their own ROS produced following activation. To this end, we studied the effect of the ROS burst on DNA integrity, cell death and differentiation potential of monocytes. We show that monocytes, but not macrophages, stimulated for ROS production by phorbol-12-myristate-13-acetate (PMA) undergo apoptosis, despite similar levels of initial DNA damage. Following co-cultivation with ROS producing macrophages, monocytes displayed oxidative DNA damage, accumulating DNA single-strand breaks and a high incidence of apoptosis, reducing their ability to give rise to new macrophages. Killing of monocytes by activated macrophages, termed killing in trans, was abolished by ROS scavenging and was also observed in monocytes co-cultivated with ROS producing activated granulocytes. The data revealed that monocytes, which are impaired in the repair of oxidised DNA lesions, are vulnerable to their own ROS and ROS produced by macrophages and granulocytes and support the hypothesis that this is a mechanism regulating the amount of monocytes and macrophages in a ROS-enriched inflammatory environment.

Compromised DNA Repair and Signalling in Human Granulocytes

“…The purified granulocytes are biologically active as shown by their response to PMA triggering the ROS burst (Fig. 1b), which is a function of NADPH oxidase [9]. The expression of the BER proteins PARP-1, XRCC1 and ligase IIIα was analysed in Western blots.…”

“…Granulocytes were isolated by the Optiprep density method as described previously [9]. Briefly, two solutions of different densities (solution A 1.077 g/mL and solution B 1.095 g/mL) were prepared from Optiprep (density 1.32 g/mL, Progen Biotechnik GmbH, Heidelberg, Germany) diluted in 0.85% NaCl, 1 mM EDTA, 20 mM HEPES, pH 7.4.…”

“…The detection of extracellular ROS has been described elsewhere [9]. Briefly, 0.5 × 10 6 cells were resuspended in 500 µL of complete PBS (PBS with 0.5 mM MgCl 2 , 0.7 mM CaCl 2 and 0.1% glucose); 10 µg/mL horseradish peroxidase and 10 µM luminol were added to the cells before stimulation with 100 ng/mL PMA.…”

“…The phagocyte NADPH oxidase (NOX2) catalyses the production of superoxide radicals and its metabolite hydrogen peroxide can damage pathogens in the vicinity [6]. This so-called oxidative burst can also be observed in other phagocytes such as monocytes and macrophages [7–9]. The neutrophil extracellular traps (NET) represent another defensive mechanism [10–12].…”

“…This repair deficiency is abrogated if monocytes undergo cytokine-induced differentiation into macrophages or DC. Thus, monocyte-matured macrophages and DC re-express the relevant DNA repair proteins and therefore become resistant to genotoxins, including ionising radiation, chemical ROS and anticancer drugs [9, 13, 14]. Of note, T cells are repair competent similar to differentiated macrophages [16].…”