Figure 1.

IFNγ Induces Ca²⁺ Influx and Shapes M1 Functional Phenotype Development in Macrophage In Vitro

(A) BM macrophages were generated in vitro (20 ng/mL GMCSF) and cultured in the presence or absence of 20 ng/mL IFNγ (-phenotype inducer). Whole-cell patch-clamp and imaging analysis on these cells were performed to measure IFNγ-induced effects on Ca²⁺ release and influx. M1-associated mediators were measured in cells cultured in the presence or absence of 50 μM 2APB (Ca²⁺ entry inhibitor) by western blot, RT-PCR, and colorimetric assay.

(B and C) BM macrophages were pulsed with medium alone (M0) or IFNγ (M1) for 2 and 24 hr and loaded with Fura-2AM. 1 μM Tg was added (first arrow) to the Fura-2AM-loaded cells bathed in Ca²⁺-free medium to measure the internal Ca²⁺ release (first peak); thereafter 2 mM external Ca²⁺ was added (second arrow) to measure Ca²⁺ entry/influx through PM (second peak). Average analog plots of the fluorescence ratio (340/380 nm) from an average of 40–50 cells are shown. (B′ and C′) The corresponding bar graphs represent the mean ± SEM of Ca²⁺ release (first peak) and store-operated Ca²⁺ entry (SOCE) (second peak) under these conditions.

(D) Representative time course of Ca²⁺ current at −80 mV with 0 mV holding potential from BM macrophages pulsed with medium alone (M0) or IFNγ. Whole-cell patch-clamp was performed with Tg in the pipette solution.

(E) Comparison of NOS2 mRNA expression by qPCR analysis of BM macrophages cultured in medium alone (M0) or with IFNγ (M1) in the presence or absence of 2APB. The bars are representative of three independent experiments.

(F) Comparison of NO levels in culture supernatant of BM macrophages cultured with medium alone (M0) or IFNγ (M1) in the presence or absence of 2APB. Data shown are Mean ± SEM.

(G) The level of pNF-κB p65 (pp65) (Cell Signaling, 3033S), pSTAT1 (Cell Signaling, 9167S), GAPDH, p65, or STAT1 in BM macrophages cultured with medium alone (M0) or IFNγ (M1) in the presence or absence of 2APB by immunoblot. Data shown are representative of three independent experiments with similar results. The average pixel intensity of pSTAT1 or pp65 bands was measured and expressed in bar graphs as mean ± SEM (G′).

*p ≤ 0.05, ***p ≤ 0.001 (Student's t test).

See also Figure S1.