In Vivo Effects of Interferon-Gamma 1-b (IFN-γ) on Neutrophils

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 neutrophil function. Furthermore, the majority of studies with neutrophils evaluate the effects of INF-γ on differentiated cells. The clinical use of IFN-γ has been driven by these data and the clinical findings that administration of this cytokine to patients with Chronic Granulomatous Disease results in decreased incidence of severe infections although the defect in Nox2 activity is not altered. To determine the in vivoeffects on myeloid cells developed under the influence of this cytokine, we evaluated neutrophils from healthy human subjects receiving IFN-γ.

Methods: Healthy human volunteers were administered IFN-γ subcutaneously under an FDA approved IND and a protocol approved by the COMIRB at the University of Colorado. Healthy adults between 18 and 60 years of age with no history of recent infections were enrolled. IFN-γ, at single escalating doses of 10, 25, 50, and 100 mcg. /m², was given subcutaneously at weekly intervals. Blood samples were obtained before and 4, 8, 12, 24, 36, 48, 72, and 96 hrs. after the administration. Plasma was stored for IL-10 and Neopterin levels assayed by ELISA techniques. Neutrophils were isolated from heparinized whole blood by Dextran sedimentation, Ficoll Hypaque density gradient centrifugation, and hypotonic lysis of red blood cells. Superoxide anion generation after stimulation with PMA (200 ng/ml) and fMLF (1µM) was measured as SOD inhibitable cytochrome c reduction. RNA was isolated by standard techniques. Analysis of gene expression was completed after preparation of labelled cDNA and hybridization with microarrays (Affimetrix GeneChip), normalization, transformation and assignment of relative expression levels. Results for the first 5 subjects are summarized here.

Results: Superoxide anion production was enhanced in response to both PMA and fMLF. For PMA there was a peak response at 12-24 hours after IFN-γ then a reduction back to baseline at the lowest dose. For the other three doses there appeared a second increase, not as great as the first, which peaked at 36-48 hours and returned to baseline by 96 hrs. The fMLF response was similar to PMA, but the early peak occurred at 8 hours and returned to baseline at the lower two doses. At 50 and 100 µ/m2 doses a second peak was seen at 24-48 hours with return to baseline by 96 hours. Dose response increases in both peaks of activity were noted for the first two doses and then reached a plateau for the higher doses of both stimuli.

Plasma levels of IL-10 peaked at 4-8 hours returning to baseline by 12 hours for the first two doses of IFN-γ and 24 hours at the highest two doses. There was a clear cut dose response effect for peak levels achieved ranging from 60-80 pg./ml at the lowest dose of IFN-γ to 200-600 pg./ml at the highest. Neopterin levels peaked by 24 hours with all doses of IFN and continued to remain elevated to 72 hours moving back to baseline by 96 hours.

Evaluation of gene expression is ongoing with analysis by patient, time and dose of IFN-γ with the goal of summarizing the results for the group as a whole. We will evaluate genes which show a 2-fold or greater increase and focus on those genes showing significant expression in a previously analyzed in vitro system of IFN-γ effect during neutrophil maturation of a myeloid cell line with DMSO. These include genes known to be involved in classical aspects of neutrophil function, i.e. transmigration, chemotaxis, phagocytosis and pathogen killing; genes involved in neutrophil clearance and homeostasis, including apoptosis; genes encoding innate immune receptors; and genes encoding guanylate binding proteins, a family of GTPases implicated in antimicrobial activity in different cell types.

Conclusion: INF-γ has dramatic in-vivo effects on neutrophils. Dose and time related activities vary and results will help better define the optimal use of the drug. Understanding the nature of INF-γ related transcription activity will help define its clinical effects in CGD and extend the possible uses of this drug to other diseases.