IL-12 p40 homodimer, but not IL-12 p70, induces the expression of IL-16 in microglia and macrophages

Abstract

IL-16, a leukocyte chemoattractant factor (LCF), is involved in the disease process of multiple sclerosis and other autoimmune disorders. However, mechanisms by which this LCF is expressed are poorly understood. The present study underlines the importance of IL-12 p40 homodimer (p40(2)), the so-called biologically inactive molecule, in inducing the expression of IL-16 in primary mouse and human microglia, mouse BV-2 microglial cells, mouse peritoneal macrophages, and RAW264.7 cells. In contrast, IL-12 p70, the bioactive heterodimeric cytokine, was unable to induce the expression of IL-16 in any of these cell types. Similarly IL-12 p40(2) also induced the activation of IL-16 promoter in microglia. Among various stimuli tested, p40(2) was the most potent one followed by p40 monomer, IL-16 and IL-23 in inducing the activation of IL-16 promoter in microglial cells. Furthermore, induction of IL-16 mRNA expression by over-expression of p40, but not p35, cDNA and induction of IL-16 expression by p40(2) in microglia isolated from IL-12p35 (-/-) mice confirm that p40, but not p35, is responsible for the induction of IL-16. Finally, by using primary microglia isolated from IL-12Rbeta1 (-/-) and IL-12Rbeta2 (-/-) mice, we demonstrate that p40(2) induces the expression of this LCF via IL-12Rbeta1 but not IL-12Rbeta2. These results delineate a novel biological function of p40(2) and raise the possibility that biological function of IL-12 p40(2) may be different from IL-12 p70.

Fig. 1

Time- and dose-dependent induction of IL-16 expression by IL-12 p40 homodimer (p40₂) and p70 in mouse BV-2 microglial cells. Cells were stimulated with p40₂(10 ng/ml) and p70 (10 ng/ml) under serum-free conditions. At different time point of stimulation, total RNA was analyzed for the expression of IL-16 by semi-quantitative RT-PCR (A) and quantitative real time PCR (B). Cells were stimulated with different concentration of p40₂ and p70 under serum free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (C) and quantitative real time PCR (D). Results are means ± S.D. of three different experiments. ^ap < 0.001 vs control for p40₂; ^bp < 0.001 vs control for p70. (E) Cells (5 × 10⁵) were stimulated with p40₂ (10 ng/ml) and p70 (10 ng/ml) under serum-free conditions. After 24 h, supernatants were collected, concentrated, loaded onto SDS–PAGE, and immunoblotted with antibodies against IL-16. Two separate wells from each treatment group have been processed and shown. Results represent three independent experiments.

Fig. 2

Effect of p40₂ and p70 on the expression of IL-16 in primary mouse microglia. Microglia isolated from 7 to 9-day-old mouse pups were stimulated with different concentrations of p40₂ and p70 under serum-free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (A) and real-time PCR (B). ^ap < 0.001 vs control. After 18 h of stimulation, the expression of IL-16 protein was monitored by immunofluorescence (C). DAPI was used to visualize nucleus. Results represent three independent experiments.

Fig. 3

Effect of p40₂ and p70 on the expression of IL-16 in primary human microglia. Microglia isolated from 13 to 17-week-old human fetal brains were stimulated with 10 ng/ml p40₂ and p70 separately under the serum-free condition. (A) After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR. (B) After 18 h of stimulation, the expression of IL-16 protein was monitored by immunofluorescence using antibodies against mouse IL-16. Bottom panel represents labeling of p40₂-stimulated cells with isotype control antibodies. DAPI was used to visualize nucleus. Results represent three independent experiments.

Fig. 4

Effect of p40₂ and p70 on the expression of IL-16 in mouse primary macrophages and RAW cells. Mouse peritoneal macrophages were stimulated with 10 ng/ml p40₂ and p70 separately under the serum-free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (A) and quantitative real-time PCR (B). ^ap < 0.001 vs control. RAW cells were stimulated with different concentrations of p40₂ and p70 under the serum-free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (C) and quantitative real-time PCR (D). ^ap < 0.001 vs control. After 18 h of stimulation by 10 ng/ml p40₂ and p70, the expression of IL-16 protein was monitored by immunofluorescence in peritoneal macrophages (E). DAPI was used to visualize nucleus. Results represent three independent experiments.

Fig. 5

Effect of cycloheximide and actinomycin D on p40₂-induced expression of IL-16 in mouse BV-2 microglial cells. Cells preincubated with different concentrations of cycloheximide (CHX) and actinomycin D (ActD) for 30 min were stimulated with p40₂ (10 ng/ml) under serum-free conditions. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR. Results represent three independent experiments.

Fig. 6

Effect of different proinflammatory molecules on IL-16 promoter-driven luciferase activity in mouse BV-2 microglial cells. Cells plated at 50–60% confluence in twelve-well plates were transfected with 0.25 µg pIL-16-Luc and 25 ng pRL-TK (Renilla luciferase control) by Lipofectamine Plus (Invitrogen) as described Section 2. Twenty-four hours after transfection, cells were stimulated with different concentrations of p40₂ (A) or different stimuli (B) for 6 h under serum free condition. Firefly and Renilla luciferase activities were determined by Dual Luciferase Kit (Promega) following the manufacturer’s protocol. Data are mean ± S.D. of three separate experiments. ^ap < 0.001 vs control. Concentrations of different stimuli are as follows: p40₂, 10 ng/ml; LPS, 1 µg/ml; p70, 10 ng/ml; IL-23, 10 ng/ml; TNF-α, 20 ng/ml; IFN-γ, 12.5 mU/ml; gp120, 200 pg/ml; IL-16, 10 ng/ml; poly IC, 100 µg/ml; IL-1β, 10 ng/ml; p40, 10 ng/ml.

Fig. 7

Effect of functional blocking monoclonal antibodies (a3-1d) against p40₂ on p40₂-induced expression of IL-16 in mouse BV-2 microglial cells. Cells preincubated with different concentrations of either mAb a3-1d or normal hamster IgG for 30 min were stimulated with p40₂ (10 ng/ml)under serum-free conditions. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR. Results represent three independent experiments.

Fig. 8

Expression of p40, but not p35, cDNA induces the expression of IL-16 in BV2 microglial cells: Microglial cells plated in 12-well plates were transfected with different amounts of either p40 or p35 cDNA by LipofectAMINE Plus (Invitrogen). Empty vector (pCIneo mammalian expression vector from Promega) was used as control. After 24 h of transfection, cells were incubated in serum-free media. After 6 h, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (A) and quantitative real-time PCR (B). ^ap < 0.001 vs control. C) After 24 h of transfection, cells were incubated in serum-free media in the presence or absence of different concentrations of either p40₂ mAb a3-1d or normal hamster IgG. After 6 h, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR. Results represent three independent experiments.

Fig. 9

Effect of p40₂ and p70 on the expression of IL-16 in p35 (−/−) microglia. Microglia isolated from wild type and p35 (−/−) mice were stimulated with different concentrations of p40₂ under serum-free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by RT-PCR (A) and real time PCR (B). After 18 h of stimulation, cells were immunostained with antibodies against IL-16 (C). Results are means ± S.D. of three different experiments. ^ap < 0.001 vs control.

Fig. 10

Effect of p40₂ and p70 on the expression of IL-16 in primary microglia isolated from wild type, IL-12Rβ1 (−/−) and IL-12Rβ2 (−/−) mice: Microglia isolated from B6.129 wild-type, IL-12 Rβ1 (−/−) and IL-12Rβ2 (−/−) mice were stimulated with 10 ng/ml of p40₂ and p70 under serum free condition. After 6 h of stimulation, the mRNA expression of IL-16 was monitored by semi-quantitative RT-PCR (A) and quantitative real-time PCR (B). Results are means ± S.D. of three different experiments.