A new synthetic compound, 2-OH, enhances interleukin-2 and interferon-gamma gene expression in human peripheral blood mononuclear cells

Abstract

A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H)-one (2-OH), was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC) proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 microg/mL) stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC(50)) for 2-OH was 4.4+/-0.1 microM. In addition, effects of 2-OH on interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-gamma production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR indicated that IL-2 and IFN-gamma mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-gamma production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.

Figure 1

The structure of 2-OH (C₁₆H₁₃NO₄S; M.W. 315).

Figure 2

Effects of 2-OH on PBMC proliferation. PBMC (2×10⁵/well) were treated with 0.1% DMSO (control), PHA (5 µg/mL) or indicated concentrations of 2-OH (1.5, 3.13, 6.25, 12.5, and 25 μM) for three days. The proliferation of cells were detected by tritiated thymidine uptake. After a 16 hr incubation, the cells were harvested by an automatic harvester then radioactivity was measured by a scintillation counting. Each bar represents the mean±S.D. of three independent experiments with PBMC from different individuals. *P < 0.05, **P< 0.01, as compared with control group.

Figure 3

The IL-2 and IFN-γ production in PBMC cultures treated with 2-OH. PBMC (2×10⁵/well) were treated by 0.1% DMSO (control), PHA (5 µg/mL) or indicated concentrations of 2-OH (1.5, 3.13, 6.25, 12.5, and 25 μM) for three days. Then the cell supernatants were collected and (A) IL-2 and (B) IFN-γ concentrations were determined by EIA. Each bar is the mean±S.D. of three independent experiments with PBMC from different individuals. *P < 0.05, **P< 0.01, ***P< 0.001, as compared with control group.

Figure 4

Effects of 2-OH on: (A) IL-2 and (B) IFN-γ mRNA expression in PBMC, as detected with RT-PCR. PBMC (5 x 10⁶) were cultured with PHA (5 µg/mL) or 6.25 and 25 μM 2-OH for 18 hr. The total cellular RNA was isolated from PBMC and aliquots of RNA (1 μg) were reverse-transcribed for synthesis of cDNA. PCR was done as described in Materials and Methods. Following the reaction, the amplified product was taken out of the tubes and run on 2% agarose gel. Lane 1: Control (0.1% DMSO), Lane 2: PHA, Lane 3: 6.25 μM 2-OH, Lane 4: 12.5 μM 2-OH. Each band was quantitated using laser scanning densitometer SLR-2D/1D (Biomed Instruments Inc., Fullerton, CA, USA). The ratio of IL-2 or IFN-γ mRNA to GAPDH mRNA was calculated. Each bar is the mean±S.D. of three independent experiments with PBMC from different individuals. **P < 0.01, ***P < 0.001, as compared with control group.