IL-12 p40 homodimer, the so-called biologically inactive molecule, induces nitric oxide synthase in microglia via IL-12R beta 1

Abstract

Earlier we have demonstrated that IL-12 p40 homodimer (p40(2)) induces the expression of inducible nitric oxide synthase (iNOS) in microglia. This study was undertaken to investigate underlying mechanisms required for IL-12 p40(2)- and IL-12 p70-induced expression of iNOS in microglia. IL-12 p40(2) alone induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, the ERK pathway coupled p40(2) to iNOS expression via C/EBP beta, but not NF-kappaB, whereas the p38 pathway relayed the signal from p40(2) to iNOS expression via both NF-kappaB and C/EBP beta. Furthermore, by using microglia from IL-12R beta 1 (-/-) and IL-12R beta 2 (-/-) mice or siRNA against IL-12R beta 1 and IL-12R beta 2, we demonstrate that p40(2) induced the expression of iNOS in microglia via IL-12R beta 1-(ERK+p38)-(NF-kappaB +C/EBP beta) pathway. In contrast, both IL-12R beta 1 and IL-12R beta 2 were involved for IL-12 p70-induced microglial expression of iNOS. Although IL-12R beta 1 coupled p70 to NF-kappaB and C/EBP beta, IL-12R beta 2 was responsible for p70-mediated activation of GAS. This study delineates a new role of IL-12R beta 1 and IL-12R beta 2 for the expression of iNOS and production of NO in microglia that may participate in the pathogenesis of neuroinflammatory diseases.

Fig. 1

IL-12 p40 homodimer (p40₂) induces the expression of iNOS in primary microglia isolated from p35 (−/−) mice. Microglia isolated from wild type and p35 (−/−) mice were stimulated with different concentrations of p40₂ under serum-free condition. A: After 6 h of stimulation, the mRNA expression of iNOS was monitored by RT-PCR. B: Microglia isolated from p35 (−/−) mice were stimulated with 10 ng/mL p40₂ for 24 h followed by immunostaining with antibodies against iNOS. Results represent three independent experiments.

Fig. 2

Effect of serum albumin on p40₂-induced expression of iNOS in primary mouse microglia. Microglia were stimulated with different concentrations of BSA, carrier (BSA)-free (CF) p40₂ and carrier (BSA)-containing p40₂ (p40₂). After 6 h of stimulation, the mRNA expression of iNOS was monitored by RT-PCR. Results represent three independent experiments.

Fig. 3

Effect of siRNA knockdown of IL-12Rβ1 and IL-12Rβ2 on the expression of iNOS in BV-2 microglial cells. A: Cells were transfected with different amounts of the empty vector (pSilencer) and siRNA against IL-12Rβ1 (si-β1). Forty-eight hours after transfection, cells were analyzed for the expression of IL-12Rβ1 and IL-12Rβ2 by RT-PCR. B: Cells were transfected with the empty vector and siRNA against IL-12Rβ2 (si-β2). Forty-eight hours after transfection, cells were analyzed for the expression of IL-12Rβ1 and IL-12Rβ2. Twenty-four hours after transfection, cells were stimulated with 10 ng/mL p40₂ and p70 under serum-free condition. After 6 h of stimulation, the expression of iNOS mRNA was examined by RT-PCR (C) and quantitative real-time PCR (D). After 24 h of stimulation, concentration of nitrite (E) was measured in supernatants. Data are mean + SD of three separate experiments.

Fig. 4

Effect of p40₂ and p70 on the production of nitrite and the expression of iNOS mRNA in primary microglia isolated from wild type, IL-12Rβ1 (−/−) and IL-12Rβ2 (−/−) mice. Microglia were stimulated with 10 ng/mL p40₂ and p70. A: After 24 h of stimulation, the production of nitrite was monitored by ‘Griess’ reagent. Data are mean + SD of three separate experiments. *P < 0.001 vs. control. After 6 h of stimulation, the expression of iNOS mRNA was monitored by RT-PCR (B) and quantitative real-time PCR (C). *P < 0.001 vs. control.

Fig. 5

Effect of p40₂ and p70 on the level of iNOS protein in primary microglia isolated from wild type, IL-12Rβ1 (−/−) and IL-12Rβ2 (−/−) mice. Microglia were stimulated with 10 ng/mL p40₂ and p70. After 24 h of stimulation, iNOS protein was monitored by immunofluorescence. DAPI was used to visualize nucleus. Results represent three independent experiments.

Fig. 6

Role of Erk and p38 MAP kinases in p40₂-induced expression of iNOS in mouse BV-2 microglial cells. A: Cells were stimulated with 10 ng/mL p40₂ for different minute intervals. Activities of ERK (upper panel) and p38 (lower panel) MAP kinases were assayed by immuno-complex kinase assay. Cells preincubated with different concentrations of SB203580 and PD98059 for 30 min were stimulated with 10 ng/mL p40₂. B: After 24 h, supernatants were used for nitrite assay. **P < 0.05 vs. p40₂; *P < 0.001 vs. p40₂. C: After 6 h of incubation, Northern blot analysis for iNOS mRNA was carried out. D: After 24 h, viability was monitored by MTT assay. Data are mean + SD of three different experiments.

Fig. 7

Role of IL-12Rβ1 and IL-12Rβ2 on the activation of Erk and p38 MAP kinases in p40₂-stimulated mouse microglia. Mouse BV-2 microglial cells were transfected with the empty vector (pSilencer) and siRNA against IL-12Rβ1 (si-β1) and IL-12Rβ2 (si-β2). Twenty-four hours after transfection, cells were stimulated with 10 ng/mL p40₂ under serum-free condition. At different minute intervals, activities of Erk (A) and p38 (B) MAP kinases were assayed by immuno-complex kinase assay. Primary microglia isolated from wild type, IL-12Rβ1 (−/−) and IL-12Rβ2 (−/−) mice were stimulated with 10 ng/mL p40₂. At different minute intervals, activation status of ERK (C) and p38 (D) was monitored by western blot using antibodies against either phospho-MAP kinases or total MAP kinases. Results represent three independent experiments.

Fig. 8

Role of NF-κB and C/EBPβ in p40₂- and p70-induced expression of iNOS in mouse BV-2 microglial cells. A: Cells were cotransfected with 0.2 μg of either pNF-κB-Luc or pC/EBPβ-Luc and 10 ng of pRL-TK. After 24 h of transfection, cells were stimulated with p40₂ and p70 for 6 h. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract. *P < 0.001 vs. control. B: Cells were transfected with an empty vector, Δp65 or ΔC/EBPβ. Twenty-four hours after transfection, cells were stimulated with 10 ng/mL p40₂ and p70. Nitrite was assayed after 24 h. Data are mean + SD of three different experiments. *P < 0.001 vs. p40₂; **P < 0.001 vs. p70.

Fig. 9

Effect of PD98059 and SB203580 on p40₂- and p70-induced activation of NF-κB and C/EBPβ in mouse BV-2 microglial cells. Cells were cotransfected with 0.2 μg of either pNF-κB-Luc (A) or pC/EBPβ-Luc (B) and 10 ng of pRL-TK. After 24 h of transfection, cells were treated with different concentrations of PD, SB for 30 min. Then cells were stimulated with 10 ng/mL p40₂ and p70 for 6 h followed by analysis of firefly and Renilla luciferase activities. Data are mean + SD of three different experiments. *P < 0.01 vs. p40₂; **P < 0.01 vs. p70.

Fig. 10

Effect of siRNA knockdown of IL-12Rβ1 and IL-12Rβ2 on the activation of NF-κB and C/EBPβ in p40₂- and p70-stimulated mouse BV-2 microglial cells. Cells were co-transfected with siRNA constructs against IL-12Rβ1 or IL-12Rβ2 and reporter constructs for NF-κB (A) or C/EBPβ (B). Twenty-four hours of transfection, cells were stimulated with 10 ng/mL p40₂ and p70 for 6 h followed by analysis of luciferase activities. Data are mean + SD of three different experiments. *P < 0.001 vs. pSilencer-p40₂; **P < 0.001 vs. pSilencer-p70.

Fig. 11

Role of GAS activation in p70-induced expression of iNOS in mouse BV-2 microglial cells. Cells were cotransfected with 0.2 μg of either pGAS-Luc (an STAT-dependent reporter construct) (A) or pISRE-Luc (an IRF-dependent reporter construct) (B) and 10 ng of pRL-TK. After 24 h of transfection, cells were stimulated with different concentrations of p40₂ and p70 for 6 h followed by analysis of firefly and Renilla luciferase activities. C: Cells were cotransfected with pGAS-Luc and pRL-TK. After 24 h of transfection, cells were treated with different concentrations of AG490 for 30 min followed by stimulation with 10 ng/mL p70. After 6 h of stimulation, luciferase activities were analyzed. D: Cells preincubated with different concentrations of AG490 for 30 min were stimulated with 10 ng/mL p70. After 6 h of stimulation, the expression of iNOS was monitored by RT-PCR. E: After 24 h of stimulation, concentration of nitrite was measured in supernatants. *P < 0.001 and **P < 0.05 vs. p70. F: Cells were co-transfected with siRNA constructs against IL-12Rβ1 or IL-12Rβ2 and pGAS-Luc. The pRL-TK was also used as transfection efficiency control. Twenty-four hours after transfection, cells were stimulated with 10 ng/mL p70 for 6 h followed by analysis of luciferase activities. Data are mean ± SD of three different experiments. *P < 0.001 vs. pSilencer-p70.

Fig. 12

Schematic presentation exhibiting the involvement of IL-12Rβ1 and IL-12Rβ2 in microglial expression of iNOS.