Figure 3.

Effect of IFN-γ on SLPI levels. A: PBMCs from HDs and TB patients were cultured for 48 hours in the presence or absence of 10 μg/mL Mtb-Ag, ±7.5 ng/mL rIFN-γ, and ±15 μg/mL blocking monoclonal antibody against IFN-γ (30 minutes) or 15 μg/mL isotype control. B: PBMCs from IFNGR1^∗ patients and age-matched HDs were cultured for 5 days in the presence or absence of Mtb-Ag. A and B: After culture, medium was removed and cell-free supernatants were collected and assayed for SLPI. Data are expressed as the means ± SEM in HDs (n = 6) and TB patients (n = 6) (A) and in HDs (n = 4) and IFNGR1^∗ patients (n = 2) (B). Analysis of variance post hoc Dunnett’s multiple comparisons test. C and D: Real-time PCR for IFN-γR expression on PBMCs. C: PBMCs were obtained from patients with TB classified as mild (n = 6), moderate (n = 6), and severe (n = 6)²¹ and HD subjects (n = 6). IFN-γR expression was determined by quantitative real-time PCR. Values are represented as fold of increase using the comparative method for relative quantification. Expression of IFN-γR was calculated as follows (a comparative method for relative quantification after normalization to GAPDH expression): $\text{Fold}\text{increase}=2^{-\Delta \Delta \text{Ct}},$ where $\Delta \text{Ct}=[\text{Ct}(\text{IFN}-\text{γ}\text{R})-\text{Ct}\left(\text{GAPDH}\right)]$ and $\Delta \Delta \text{Ct}=[\text{ΔCtTB}-\text{ΔCtHD}].$ Mann-Whitney test. C: Significant differences between mild and severe TB patients. D: PBMCs from patients with TB (n = 9) and HDs (n = 11) were cultured by 16, 24, or 48 hours in the presence or absence of Mtb-Ag. Cells were harvested, and IFN-γR expression was determined as in C. Expression of IFN-γR was calculated as follows (a comparative method for relative quantification after normalization to GAPDH expression): $\text{Fold}\text{increase}=2^{-\Delta \Delta \text{Ct}},$ where $\text{ΔCt}=[\text{Ct}(\text{IFN}-\text{γR})-\text{Ct}\left(\text{GAPDH}\right)]$ and ΔΔCt = [ΔCtstimulated−ΔCtunstimulated]. Unpaired t-test, for significant differences between HDs and TB patients at 16 hours and for significant differences between 16 and 48 hours in TB patients. ^∗P < 0.05 (A–D); ^††P < 0.01, for significant differences between 16 and 48 hours in HDs (D).