Yukie Murata

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.

Regulation of LPS induced IL-12 production by IFN-γ and IL-4 through intracellular glutathione status in human alveolar macrophages

Interleukin‐12 (IL‐12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon‐γ (IFN‐γ) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN‐γ and IL‐4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL‐12 production in human alveolar macrophages (AM). We used both AM from healthy non‐smokers obtained by bronchoalveolar lavage and the monocytic THP‐1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N‐acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)‐induced IL‐12 secretion by AM. In THP‐1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS‐induced IL‐12 mRNA, whereas l‐buthionine‐[S,R]‐sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS‐induced IL‐12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN‐γ or the helper T cell type 2 (Th2) cytokine IL‐4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)‐induced secretion of IL‐12 in AM was enhanced by IFN‐γ but inhibited by IL‐4. These results suggest that IFN‐γ and IL‐4 oppositely affect the GSH/GSSG balance, which may regulate IL‐12 secretion from AM in response to LPS.

IFN‐γ and pro‐inflammatory cytokine production by antigen‐presenting cells is dictated by intracellular thiol redox status regulated by oxygen tension

Murine mature splenic DC with elevated intracellular glutathione, pretreated with IL‐18, strikingly augmented the production of IFN‐γ in response to IL‐12, whereas intracellular glutathione deprivation ablated this effect of IL‐18. Likewise, macrophages with elevated intracellular glutathione augmented IFN‐γ production upon LPS or IL‐12+IL‐18 stimulation, whereas macrophages withreduced intracellular glutathione showed the reciprocal response. Under hypoxia, macrophages displayed a functional phenotype with decreased intracellular glutathione, i.e. decreased NO and IL‐12, and elevated IL‐10 production. However, mature DC and macrophages produced an elevated amount of IFN‐γ under hypoxia. Taken together, our results suggest that the intracellular redox status of DC and macrophages may play a pivotal role in local innate immunity, depending on local oxygen tension.

The skewing to Th1 induced by lentinan is directed through the distinctive cytokine production by macrophages with elevated intracellular glutathione content

In vivo lentinan (LNT)-elicited peritoneal macrophages (Mps) showed the reduced release of prostaglandins (PGs), IL-10 and IL-6, while it endowed Mps with the elevated capability to produce IL-12 and nitric oxide (NO) upon in vitro triggering, due to the elevated intracellular glutathione (GSH) content in Mps. Deprivation of intracellular GSH completely ablated the production of IL-12. Conversely, lipopolysaccharide (LPS) induced peritoneal Mps with the reduced intracellular GSH content and the reciprocal profile of mediator production. Mps with the elevated intracelluar GSH is arbitrarily termed as reductive Mp (RMp) and that with reduced amount as oxidative Mp (OMp). OMp was converted to RMp when GSH was replenished with glutathione monoethylester (GSH-OEt). The IL-2 administration in combination with LNT exerted the synergistic induction of RMp, resulting in synergistic augmentation of IL-12, NO and reduction of IL-6 production. It was also confirmed that CD4+T cells derived of LNT-administered mice showed augmented IFN-gamma and reduced IL-4 production upon in vitro anti-CD3 stimulation. Taken together it is concluded that skewing of Th1/Th2 balance to Th1 by a beta-(1-3)-glucan, LNT, is directed through the distinctive production of IL-12 versus IL-6, IL-10 and prostaglandin E2 (PGE2) by Mps, depending on intracellular GSH redox status. To the efficient tumor immunotherapy, it may be one of the critical elements to induce a reductive form of Mps in tumor stromal tissues to maintain Th1 response.

Intracellular thiol redox status of macrophages directs the Th1 skewing in thioredoxin transgenic mice during aging

We have been proposing the functional distinction of two classes of macrophages (Mp), namely the reductive macrophages (RMp) with high intracellular content of glutathione (GSH) and the oxidative macrophages (OMp) with reduced content. At the same time we have been investigating the variation of RMp/OMp balance during aging of mice, especially in relation to the age related onset of autoimmune diseases. In this paper we have investigated the Th1/Th2 balance of thioredoxin (TRX) transgenic (Tg) mice, with prolonged life longevity, during aging in the context of the intracellular redox status of Mp, which has been hypothesized to be crucial in regulating the Th1/Th2 balance. It was confirmed that peritoneal resident Mp of Tg mice showed the higher GSH/GSSG ratios compared with that of age matched wild type (WT) mice. The predominance of RMp was associated with the sustained maintenance of Th1 prevalence during aging until 2 years in Tg mice, whereas WT littermates showed rapid polarization to Th2 around the age of 8 months. The Tg mice showed elevation of IFN-gamma and reduction of IL-10 with moderate change of IL-4 produced by CD4+ T cells. The WT mice showed inverse changes of IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios during aging. In addition, IL-10 production by Mp was dramatically reduced in aged Tg mice. Thus, TRX Tg mice may be useful to investigate the contribution of the anti-oxidant defense mechanism during aging accompanied with increasing oxidative stress.

Sequential conversion of the redox status of macrophages dictates the pathological progression of autoimmune diabetes

The redox status of macrophages (Mϕ), indexed by intracellular content of glutathione (icGSH), varies sequentially along with disease progression in nonobese diabetic mice. At the stage ofearly insulitis, Mϕ skew to oxidative Mϕ (OMϕ) with decreased icGSH, then to reductive Mϕ (RMϕ) with elevated icGSH and to OMϕ after the occurrence of diabetes. RMϕ or OMϕ inducing agents either delayed or accelerated the onset of diabetes in a mutually inverse manner. RMϕ or OMϕ adoptively transferred exacerbated or ameliorated the disease progression depending on the redox status of Mϕ of recipient mice. The new paradigm that the sequential conversion of redox status of Mϕ dictates the pathological progression may provide a new insight on the mechanism underlying autoimmune diabetes.