Myeloid Cells Differentiated in the Presence of Interferon-γ (INF-γ) Compared to Mature Cells Exposed to This Cytokine Exhibit Different Phox Protein Expression and Nox2 Activity

Introduction: Most information about the immunoregulatory functions of INF-γ has focused on the interaction with the adaptive immune system and less is reported about neutrophils. The majority of studies with neutrophils evaluate the effects of INF-γ on differentiated cells. Since an important issue INF-γ treatment is the function of neutrophils developed under the influence of this cytokine, we investigated the effects of INF-γ on a myeloid cell line that can be differentiated into neutrophil-like cells.

Methods: PLB-985 cells were grown under standard culture conditions (5% CO₂, 37^oC) with RPMI and 10% bovine serum and were matured with the addition of 1.3% DMSO in the presence and absence of various concentrations of INF-γ-1b (Vidara Therapeutics Ltd.). After 72 and 96 hours, the cells exhibited neutrophil-like phenotype. As controls, cells were incubated with media or media plus INF-γ alone.

Superoxide anion (O₂^-) production was measured as S00 inhibitable cytochrome c reduction after exposure to PMA (200 ng/mL) or FMLP (1 µM). Cell lysates were prepared in the presence of 0.1% SDS, 1% Triton X 100 and a protease inhibitor mix. Proteins from the lysate were separated on 10-15% SDS PAGE, blotted onto PVDF or nitrocellulose, exposed to primary antibodies for phox proteins overnight at 4^oC and then appropriate peroxidase labeled secondary antibodies. Protein/antibody complexes were detected with an ECL detection system and were quantitated by densitometry with Image J software.

Results: PLB-985 cells cultured for 4 days with 30ng/ml ( 600 IU/ml) INF-γ, DMSO or DMSO+ INF-γ were stimulated with PMA or fMLF and O₂^- production was measured over 5 minutes (for fMLF) or 10 minutes (for PMA). INF-γ alone resulted in minimal O₂^- generation. For fMLF a significant (p<0.05) increase in O₂^- was seen when DMSO was included in the culture (2.4+/- 0.3 nmol O₂^-, mean+/-SEM) and for PMA a significant (p<0.005) increase (17.9+/-1.3) was seen. For both agonists an additional increase (p<0.005) was documented when INF-γ was included with DMSO during the culture, fMLF, 8.5+/-0.53 and PMA 45.8+/-2.9.

When a range of INF-γ concentrations (5-60 ng/ml) were co-applied during DMSO mediated PLB-985 differentiation over 3 days, the increase in O₂^-production was concentration dependent and reached a plateau at 30ng/ml for both stimuli.

Culture of cells with INF-γ alone resulted in dramatic increases in p22phox and gp91phox but failed to cause more than minimal increases in the initially low levels of p40phox, p47phox and p67phox accounting for the inability of INF-γ alone to induce oxidase activity. Differentiation of the cells with DMSO caused a small decrease in p22phox and a small increase in gp91phox but also resulted in large increases in p40phox, p47phox and p67phox which would explain the ability of DMSO to induce Nox2 activity. Inclusion of INF-γ with DMSO further increased p47phox, and increased p22phox and gp91phox relative to DMSO alone, potentially explaining the enhanced Nox2 activity noted above.

In striking contrast, when INF-γ was applied to cells first differentiated with DMSO, a minimal increase in fMLF induced O₂^- production was seen and only a small increase in PMA induced O₂^-generation was determined. Consistent with this, INF-γ applied to pre-differentiated PLB-985 cells did not enhance p40phox, and gp91phox, minimally increased p22phox, and decreased p47phox and p67phox slightly.

Conclusions: These studies demonstrate that INF-γ induces very different phenotypes in myeloid cells depending on the differentiation level of the cell. Dramatic differences were noted whether the INF-γ was present during maturation of these myeloid cells as opposed to after differentiation and include major differences in expression of phox proteins as well as activity of the Nox2 enzyme system. These data not only support the administration of INF-γ for specific neutrophil dysfunction disorders, but also suggest, in theory, expanded uses of this cytokine.