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Published on: August 16, 2013
Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils
Lisa Schindler1, Leon C D Smyth2, Jürgen Bernhagen3
1Division of Vascular Biology, Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU), Munich, Germany; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
Background:
Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli.
Methods:
Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay.
Results:
Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA.
Conclusion:
Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.
Insights
Macrophage migration inhibitory factor (MIF) enhances neutrophil production of hypochlorous acid (HOCl) during infection. This finding suggests MIF plays a key role in inflammatory responses and host defense mechanisms.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophage migration inhibitory factor (MIF) is a key immuno-regulatory cytokine implicated in inflammatory conditions.
- Neutrophils are critical immune cells at infection sites, producing potent oxidants like hypochlorous acid (HOCl).
Purpose of the Study:
- To investigate the impact of MIF on HOCl production by neutrophils in response to phagocytic stimuli.
- To elucidate the role of MIF in modulating neutrophil-mediated oxidative responses.
Main Methods:
- Quantified HOCl production using fluorescent probes (R19-S) and flow cytometry during zymosan phagocytosis.
- Assessed HOCl production during Pseudomonas aeruginosa phagocytosis by measuring bacterial glutathione oxidation.
- Measured neutrophil extracellular trap (NET) formation using a SYTOX Green assay.
Main Results:
- MIF exposure doubled HOCl-producing neutrophils and increased HOCl concentration during zymosan phagocytosis.
- MIF treatment led to greater bacterial glutathione oxidation by neutrophils during P. aeruginosa phagocytosis.
- MIF enhanced NET formation and its effect on HOCl production was independent of phagocytosis rate and blocked by a MIF inhibitor.
Conclusions:
- MIF potentiates HOCl production in neutrophils upon phagocytic stimulation.
- This newly identified activity of MIF contributes to its role in inflammation and enhances host defense.
