Interferon-gamma enhances phagocytosis , the production of reactive oxygen species and pro-inflammatory cytokines – implications for innate and acquired immunity

Neutrophils and interferon-γ (IFN-γ) are known critical components of innate and acquired immune responses. However, recent studies have demonstrated that these two immune functions are not entirely isolated. Treatment of neutrophils with IFN-γ elicits a variety of responses depending on the stimulus and on environmental conditions. In view of the physiological and pathophysiological importance of the regulatory activity of IFN-γ in neutrophil functions and the diverse, sometimes conflicting results reported, we decided to investigate the effects of this cytokine on phagocytosis, the production of reactive oxygen species (ROS) and the release of lysosome enzymes, as mediated by different types of immune receptors in mouse neutrophils. We found increased phagocytic capacity of mouse neutrophils, which was accompanied by the up-regulation of FcγR, CR, and the dectin-1 receptor. The increase in ROS production mediated by these receptors with the activation of NADPH oxidase is correlated with the increased expression of p47 and gp91 mRNA. In addition to enhanced phagocytosis, we also observed the release of some lysosomal enzymes. The increased production of reactive oxygen species may be important for the enhanced capacity for killing phagocytosed microorganisms but may also favor the induction of oxidative stress in adjacent cells. In agreement with this potential inflammatory role, we also demonstrated the increased release of the inflammatory cytokines TNF-α and IL-6 from neutrophils treated with recombinant IFN-γ. These results show that IFN-γ can act as a signaling molecule for neutrophils, modulating their functions in innate as well as acquired immunity. This Research Highlight discusses the findings of our recent study and active research endeavors.

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Neutrophils, which are a type of polymorphonuclear leukocyte, are well recognized as one of the major players during acute inflammation [1] .These cells are typically the first leukocytes to be recruited to an inflammatory site and

RESEARCH HIGHLIGHT
are capable of eliminating pathogens through multiple mechanisms [2] .The antimicrobial function of phagocytes partially depends on the generation of superoxide anion (O2 -) and other highly reactive oxidants derived during the respiratory burst.The production of O2 -in response to a stimulant is potentiated by prior treatment of the phagocytes with activating or priming agents, such as interferon-gamma (IFN-γ).This cytokine has been used to treat several diseases and malignancies, such as chronic granulomatous disease (CGD), an inherited disorder of neutrophil function caused by defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme complex responsible for phagocyte superoxide generation [3] .IFN-γ has recently been produced by recombinant DNA techniques, providing a new opportunity to characterize the immunomodulatory properties this cytokine [4] .
In our recent study titled "In vitro activation of mouse neutrophils by recombinant human interferon-gamma: increased phagocytosis and release of reactive oxygen species and pro-inflammatory cytokines" [5] , we investigate the ability of IFN-γ to modulated the functions of mouse neutrophils in vitro.
Our results showed that phagocytosis by mouse neutrophils stimulated with immune complex (IC) or immune complex opsonized with complement (IC-C), as well as zymosan (Zy) or zymosan opsonized with complement (Opzy), was detectable after a 30-min incubation with IFN-γ, peaking at 2 h.Thus, IFN-γ enhances phagocytosis in neutrophils in response to these different stimuli.The activation of neutrophil functions also involves the modulation of cell surface molecules, a number of which have been well characterized.We assessed alterations in the surface expression of FcγR and CR on neutrophils in response to stimulation by IFN-γ.The treatment with IFN-γ had effect on the expression of FcγRII and FcγRIII.It is possible that the expression of FcγRI could explain the observed increased phagocytosis and ROS production with the different stimuli (immune complexes).Nevertheless, one cannot exclude the participation of the other two receptors in these effects.Our results shown that CD16, CD32, CR3, and dectin-1 receptors are present on the surface of mouse neutrophils and that, in agreement with other studies [6] , their expression can be enhanced by treatment with IFN-γ.
We showed that IFN-γ stimulates the release of O2 -, H2O2, and HOCl by mouse neutrophils.This effect was dose-dependent and peaked at 150 U/ml IFN-γ.Because the production of superoxide is a reflection of the activity of the NADPH oxidase complex, we investigated whether IFN-γ treatment could alter the gene expression of the components of this enzymatic complex in neutrophils.Our results showed that IFN-γ enhanced the mRNA expression of the genes that encode the p47 phox and gp91 phox subunits.We also examined the effect of IFN-γ on the release of the enzymatic content of granules (β-glucuronidase and alkaline phosphatase).Our results show that the release of both enzymes was significantly increased in the presence of IFN-γ.The effects on degranulation were not due to cell death because LDH release was the same in the presence or absence of IFN-γ.These results indicate that IFN-γ stimulates release of the contents of specific and azuriphilic granules.Our results also show that Zy, Opzy, and IC lead to the enhanced production of TNF-α and IL-6 by IFN-γtreated neutrophils.
Our research group has being greatly interested in understanding the role of neutrophils in the interplay between the innate and acquired immune responses [5,7,8,9,10,11] , addressing the functional similarities and differences with macrophages.It has been suggested that during inflammation or specific immune responses, mature myeloid cells may respond to humoral factors in a manner analogous to macrophages [12] .
In our experiments, we observed the effects of IFN-γ on mouse neutrophils using different agents, such as complement opsonized zymosan, which is a typical occurrence of the innate immune response, as well as IgG immune complexes, which in vivo would be largely expected in the secondary immune response.Thus, IFN-γ has been shown to be a pleiotropic cytokine, and its effects on neutrophils show the participation of these cells in the innate and acquired immune responses.
To date, neutrophils have emerged as an important component of effector and regulatory circuits in the innate and adaptive immune systems.In contrast to the traditional view of these cells as short-lived effectors, evidence now indicates that they have diverse functions.By responding to tissue-and immune cell-derived signals and undergoing polarization [13] , neutrophils are reminiscent of macrophages [14,15] .Neutrophils engage in bidirectional interactions with different components of both the innate and adaptive immune systems and can differentially influence the response depending on the context.Recent studies have also provided new insight into neutrophil effector functions [16, 17, 18]   .These gladiators of innate immunity can throw poisonous NETs (neutrophil extracellular traps) and produce components of the humoral arm of the innate immune response [19,20] .These new insights also raise new questions.Better tools (both genetic and antibody-based) are needed to dissect the function of neutrophils in vivo.The new perspectives opened by recent findings call for a reappraisal of the role of neutrophils in human pathology.Finally, it is now time to reconsider neutrophils as a valuable therapeutic target in inflammatory pathology.We look forward to reporting our outcomes in the near future.

Concluding remarks and future directions
Neutrophils are the most abundant circulating leukocytes, yet the full spectrum of their biological functions in innate and acquired immunity is only just beginning to emerge, likely because of the difficulties in manipulating this terminally differentiated, short-lived cell type [21] .Studies published in the last few years propose that neutrophils play a much more diverse role than previously appreciated.The novel roles described include the following: functions in viral infections and infections with intracellular pathogens; intricate interaction with various other components of the adaptive immune system, including marginal zone B cells, plasmacytoid dendritic cells, and T cell populations, and even controlling NK cell homeostasis; and roles in diseases, such as autoimmune diseases, anaphylaxis, diabetes, atherosclerosis, and thrombosis [21] .Although those studies have yet to be closely examined, they suggest exciting novel avenues for the understanding of how our immune system contributes to health and diseases.The function of neutrophils in immune defense and their role in various immune-mediated diseases [22] have resulted in significant interest in this cell type in clinical medicine.The emerging information on neutrophil functions and the mechanisms involved may hopefully be translated into important novel approaches for the diagnosis and treatment of diseases with a substantial neutrophil component.