INF-γ Enhances Nox2 Activity by Upregulating phox Proteins When Applied to Differentiating PLB-985 Cells but Does Not Induce Nox2 Activity by Itself

Abstract

Background: The cytokine and drug interferon-γ enhances superoxide anion production by the antimicrobicidal Nox2 enzyme of neutrophils. Because mature neutrophils have a short lifespan, we hypothesized that the effects of interferon-γ on these cells might be mediated by its prolonged exposure to differentiating neutrophil precursors in the bone marrow rather than its brief exposure to mature circulating neutrophils. Effects of INF-Γ on NOX2 activity: To address this possibility we exposed the myeloid PLB-985 cell line to interferon-γ for 3 days in the presence of dimethyl sulfoxide which induces terminal differentiation of these cells. Interferon-γ was found to enhance superoxide production by Nox2 in a concentration dependent manner. In contrast, application of interferon-γ alone for 3 days failed to induce detectible Nox2 activity. Additionally, application of interferon-γ for 3 hours to pre-differentiated PLB-985 cells, which models studies using isolated neutrophils, was much less effective at enhancing superoxide anion production. Effects of INF-Γ on phox protein levels: Addition of interferon-γ during differentiation was found to upregulate the Nox2 proteins gp91phox and p47phox in concert with elevated transcription of their genes. The p22phox protein was upregulated in the absence of increased transcription presumably reflecting stabilization resulting from binding to the elevated gp91phox. Thus, increased levels of gp91phox, p47phox and p22phox likely account for the interferon-γ mediated enhancement of dimethyl sulfoxide-induced Nox2 activity. In contrast, although interferon-γ alone also increased various phox proteins and their mRNAs, the pattern was very different to that seen with interferon-γ plus dimethyl sulfoxide. In particular, p47phox was not induced thus explaining the inability of interferon -γ alone to enhance Nox2 activity. Short application of interferon-γ to already differentiated cells failed to increase any phox proteins.

Conclusions: Our findings indicate that interferon-γ has complex effects on phox protein expression and that these are different in cells undergoing terminal differentiation. Understanding these changes may indicate additional therapeutic uses for this cytokine in human disorders.

Conflict of interest statement

Competing Interests: The authors have the following interests: This study was funded by Horizon Pharma Ireland Ltd. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Effect of IFN-γ on PLB-985 Cell Nox2 Activity in the Presence and Absence of Differentiation.

A; PLB-985 cells were pretreated for 72 hours with 1.3% DMSO, 30ng/ml IFN-γ or 1.3% DMSO plus 30ng/ml IFN-γ. The cells were stimulated with the Nox2-agonists PMA or fMLF, as indicated, and O2 - generation was followed over time (DMSO plus IFN-γ, black lines; DMSO, dots and dashes; IFN-γ, dashes). Representative traces are shown. When SOD was included no superoxide generation was detected for any combination of pretreatment or agonist (not shown). B; The amount of O2 - formed in 10 minutes after addition of PMA, or 5 minutes after addition of fMLF, was recorded during multiple experiments like those shown in A. The graph shows the average O2 - produced (error bars are +/- SEM) for at least 6 experiments carried out after at least 3 independent pretreatments with DMSO, IFN-γ or DMSO plus IFN-γ Statistical differences with in the three cell culture conditions subsequently treated with PMA or those treated with fMLF were confirmed by one way ANOVA and significance between each pair was then explored by post hoc-FDR analysis of paired t-tests. *, # and † indicate significant difference from IFN-γ, DMSO and DMSO+IFN-γ treatment respectively (p<0.05).

Fig 2. Stimulation of PLB-985 Nox2 Activity when IFN-γ was Applied After or During Cell Differentiation.

In the two graphs on the left, PLB-985 cells were treated for 72 hours with 1.3% DMSO (pre-differentiated) and were then treated for 3 hours with the indicated concentrations of IFN-γ. The cells were then stimulated with PMA (top graph) or fMLF (bottom graph) and the amount of O2 - generated in 10 minutes (for PMA) or 5 minutes (for fMLF) was measured. In the two graphs on the right, PLB-985 cells were cultured for 72 hours in the presence of 1.3% DMSO and simultaneously treated with the indicated concentrations of IFN-γ(co-application). The cells were then stimulated with the Nox2 agonists PMA (top graph) or fMLF (bottom graph) and the amount of O2 - generated after 10 minutes (for PMA) or 5 minutes (for fMLF) was measured. The error bars are +/- SEM and n = at least 12 measurements from at least 2 independently treated cultures of PLB-985 cells. For a given graph, * indicates a significant difference (p<0.05; two tailed t-test) and ** indicates a very significant difference (p<0.005; two tailed t-test) between O2 - produced at a given IFN-γ concentration and O2 - formed in the corresponding 0ng/ml-IFN-γ condition. For a given agonist (PMA or fMLF), ## indicates a very significant difference (p<0.005; two tailed t-test) between the increase in O2 - formed due to a given IFN-γ concentration and the equivalent increase in the alternative IFN-γ application protocol (co-application of IFN-γ and DMSO versus IFN-γ application to pre-differentiated cells).

Fig 3. PLB-985 phox Proteins in Cells Exposed to IFN-γ in the Presence or Absence of Differentiation.

A; The western blots are from representative experiments and show the indicated Nox2 proteins in PLB-985 cells that were cultured for 72 hours in the presence of media with, no additions (lane 1), 30ng/ml IFN-γ (lane 2), 1.3% DMSO (lane 3) or 1.3% DMSO plus 30ng/ml IFN-γ (lane 4). Blots were reprobed for GAPDH to establish controls for gel loading and transfer efficiency. For gp91phox the protein was detected over a broad range of molecular weights (indicated by the square bracket) due to glycosylation. B; The graphs show the average density of phox proteins measured in 4 experiments like the representative examples in A; the protein densities are expressed as a ratio to the corresponding GAPDH density. For densitometry of glycosylated and thus heterogeneously sized gp91phox, the lanes were scanned to the extent indicated by the square bracket in the representative gp91phox blot. The error bars are +/- SEM. For each protein, statistical differences with in the different cell culture conditions were confirmed by one way ANOVA and significance between each pair was then explored by post hoc-FDR analysis of paired t-tests. *, #, † and ‡ indicate significant difference from untreated cells (“no additions”), IFN-γ treatment, DMSO treatment and DMSO+IFN-γ treatment respectively (p<0.05). C; The western blots show the indicated Nox2 proteins in subcellular membrane preparations from PLB-985 cells that were cultured as described in panel A. The blots are representative of 2 independent experiments.

Fig 4. Changes in PLB-985 phox Proteins in Pre-Differentiated Cells Exposed to IFN-γ.

A; The western blots are from representative experiments and show the indicated Nox2 proteins in PLB-985 cells that were pre-differentiated for 72 hours in media containing 1.3% DMSO and were then exposed for 3 hours to nothing (lane 1) or 30ng/ml IFN-γ (lane2). Western blotting for GAPDH was done to establish controls for gel loading and transfer efficiency. For gp91phox the protein was detected over a broad range of molecular weights (indicated by the square bracket) due to glycosylation. B; The graphs show the average density of phox proteins measured in 4 experiments like the representative examples in A; the protein densities are expressed as a ratio to the corresponding GAPDH density. For densitometry of gp91phox the lanes were scanned to the extent indicated by the square bracket in the representative gp91phox blot. The error bars are +/- SEM. * indicates a significant difference (p