IFN-γ Alters the Expression of Diverse Immunity Related Genes in a Cell Culture Model of Maturing Neutrophils

The cytokine IFN-γ is used as a drug to treat chronic granulomatous disease (CGD), hyperimmunoglobulin E syndromes and osteopetrosis. The goal of this study was to better understand how IFN-γmight support PMN function in these diseases, and to obtain information that might expand potential uses for IFN-γ.

Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death; a range of human studies have estimated the neutrophil half-life at only 5-10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation toward a terminally differentiated state in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells which belong to a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we used microarrays and western blotting to compare transcription and protein expression in PLB-985 cells matured, using DMSO, in the presence or absence of IFN-γ. We observed changes in a significant number of mRNAs and proteins, that fall into the following categories, which could help explain the immune supportive effects of IFN-γ.

1 Genes known to be involved in classical aspects of neutrophil function, i.e. transmigration, chemotaxis, phagocytosis and pathogen killing

2 Genes involved in neutrophil clearance and homeostasis, including some involved in apoptosis.

3 Genes encoding innate immune receptors

4 Genes encoding guanylate binding proteins, a family of GTPases implicated in antimicrobial activity in different cell types but not, to our knowledge, neutrophils. These genes were among the most upregulated observed in this study, for example Gbp5 was increased 337 fold.

5 Genes involved in antigen presentation by MHCI and MHCII systems. Although neutrophils are generally considered to be involved in innate immunity a number of studies have suggested that they might act as antigen presenting cells and our finding of IFN-g induced upregulation of MHCII components is consistent with this. Some MHCI related genes were amongst the most upregulated genes in this study, for example HLA-DRA was increased 75 fold.

Next we explored if the effect of IFN-γ on expression of these genes depends on whether the cells are undergoing maturation. This was done by comparing the effects of IFN-γ on maturing cells (i.e. those cultured in the presence of DMSO) versus non-maturing cells. To do this, the fold changes in mRNAs for DMSO plus IFN-γ treated cells versus DMSO treated cells were compared to the fold changes for IFN-γ treated cells versus untreated cells. In general, for genes showing the largest changes in mRNA expression in response IFN-γ, it was found that even larger changes occurred in maturing cells. This suggests that the effects of IFN-γon maturing cells are due to more than just prolonged application of the chemokine and are enhanced by ongoing developmental differentiation of the cells.

Collectively the findings in this study enhance our understanding of the effects of IFN-γ on myeloid cells and indicate possible mechanisms by which this cytokine could support immune function.