The effect of reactive oxygen species on monocytes and macrophages
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Abstract
The myeloid immune system is the first line of defence against infections. The acute inflammatory response encompasses the recruitment of immune cells to the site of inflammation, the production of cytokines, phagocytosis of foreign material and the release of reactive oxygen species (ROS) to kill pathogens. The latter is accomplished by the NADPH oxidase which generates superoxide anions from molecular oxygen and releases them against pathogens.
Previously, it was shown that monocytes, but not macrophages and dendritic cells (DCs), lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 which are required for the efficient repair of damaged DNA bases and single-strand breaks (SSBs) during base excision repair (BER). In addition, the double-strand break repair protein DNA-PKcs is also not expressed (Bauer et al. 2011; Briegert and Kaina 2007). The attenuated BER and non-homologous end-joining pathways sensitise monocytes to DNA oxidising and alkylating agents while DNA repair competent macrophages and DCs are resistant.
As ROS are potent genotoxins, it was addressed whether the oxidative burst of myeloid cells led to DNA damage in the ROS-producing cells (auto-intoxication). It was shown that phorbol 12-myristate 13-acetate (PMA) can stimulate the NADPH oxidase via upstream activation of the protein kinase C. PMA-mediated stimulation of myeloid immune cells, i.e. granulocytes, macrophages and monocytes, led to an increase in intracellular ROS levels. The extracellular ROS burst was detected using a chemiluminescence-based assay. In contrast to T lymphocytes, myeloid immune cells produced massive ROS in a time-dependent manner. It was shown that monocytes, but not macrophages are killed by their own ROS; following PMA treatment and triggering the oxidative burst, cells exhibited oxidative DNA damage and SSB formation followed by cell death. Surviving monocytes were unable to differentiate into macrophages. Furthermore, it was shown that ROS from adjacent cells were also strong enough to damage monocytes (so called ‘killing in trans’). Co-culture experiments with stimulated macrophages or granulocytes and unstimulated monocytes led to DNA damage in monocytes, subsequent activation of the DNA damage response and ultimately monocytic cell death. DNA repair competent macrophages were resistant and did not die. These findings strengthen the hypothesis that the BER-defect of monocytes functions as a regulatory feedback loop in the acute immune response by removing monocytes from the site of inflammation and thus dampening the immune reaction.
The analysis of the regulation of the BER protein XRCC1 in monocytes and macrophages was another aspect of this work. It was shown that the demethylation inhibitor 2-hydroxy-glutarate led to an attenuated XRCC1 signal during cytokine-induced differentiation into macrophages. However, the XRCC1 promoter analysis showed no differential methylation pattern between the two cell types. Using a reverse chromatin immunoprecipitation assay coupled with mass spectrometry, potential transcription factors responsible for XRCC1 expression were screened for. Promising candidates were CTCF and AP-1 factors, which were also found by in silico analysis of the promoters of PARP-1, DNA-PK and ligase IIIα.
The findings of DNA repair defects in monocytes were extended to neutrophilic granulocytes. It was shown that neutrophils also display severe defects in DNA repair affecting BER and double-strand break repair. Similar to monocytes they do not express XRCC1, PARP-1 and ligase IIIα. Furthermore, DNA damage signalling factors like ATM, p53 and γH2AX were also not detectable. The data indicate a lineage-specific phenomenon.