Abstract
Interleukin-12 (IL-12) is composed of two different subunits, p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the central nervous system of patients with multiple sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. However, the mode of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital role in the pathophysiology of MS, the present study was undertaken to explore the role of p40 in the induction of NO production and the expression of iNOS in microglia. Both IL-12 and p40(2), the p40 homodimer, dose-dependently induced the production of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of NO production by the expression of mouse p40 cDNA but not that of the mouse p35 cDNA suggests that the p40 but not the p35 subunit of IL-12 is involved in the expression of iNOS. In addition to BV-2 glial cells, p40(2) also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and p40(2) were unable to induce the production of NO in mouse primary astrocytes. Because activation of NF-kappaB is important for the expression of iNOS, we investigated the effect of p40(2) on the activation of NF-kappaB. Induction of the DNA binding as well as the transcriptional activity of NF-kappaB by p40(2) and inhibition of p40(2)-induced expression of iNOS by SN50, a cell-permeable peptide carrying the nuclear localization sequence of p50 NF-kappaB, but not by SN50M, a nonfunctional peptide mutant, suggests that p40(2) induces the expression of iNOS through the activation of NF-kappaB. This study delineates a novel role of IL-12 p40 in inducing the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.
Highlights
Nitric oxide (NO), derived in excessive amount from the activation of inducible nitric-oxide synthase1 in glial
To understand the role of IL-12 in the induction of induction of nitric-oxide synthase (iNOS), we examined the effect of IL-12 p70 on the production of NO in mouse BV-2 microglial cells
Consistent to the induction of iNOS by p402, expression of p40 cDNA but not that of p35 cDNA markedly induced the production of NO (Fig. 3A) and the expression of iNOS protein (Fig. 3B) suggesting that p40 but not the p35 subunit of IL-12 is involved in the induction of iNOS in microglial cells and that p40 induces iNOS independent of the so-called bioactive IL-12 p70
Summary
Nitric oxide (NO), derived in excessive amount from the activation of inducible nitric-oxide synthase (iNOS) in glial. Evidence from several laboratories emphasizes the involvement of NO in the pathophysiology of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), the animal model of MS (2, 6 –7). Increased levels of nitrotyrosine have been found in demyelinating lesions of MS brains as well as in spinal cords of mice with EAE [10, 11]. In mice with experimental allergic encephalomyelitis (EAE), an animal model of MS, the expression of p40 mRNA but not that of p35 mRNA increases in brain and spinal cord [22]. We report the first evidence that p402, the IL-12 p40 homodimer, markedly induces the production of NO and the expression of iNOS through the activation of NF-B in mouse microglia
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