Sumiko Iho

Nicotine induces human neutrophils to produce IL‐8 through the generation of peroxynitrite and subsequent activation of NF‐κB

Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL‐8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil‐IL‐8 production in vitro; the causative role of NF‐κB in its production, in association with the possible production of reactive oxygen intermediates that activate NF‐κB; and the nicotinic acetylcholine receptors (nAChRs) involved in IL‐8 production. Nicotine stimulated neutrophils to produce IL‐8 in both time‐ and concentration‐dependent manners with a 50% effective concentration of 1.89 mM. A degradation of IκB‐α/β proteins and an activity of NF‐κB p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, nω‐Nitro‐l‐arginine methyl ester, prevented nicotine‐induced IL‐8 production, with an entire abrogation of DHR oxidation, IκB degradation, and NF‐κB activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF‐κB activity, thereby producing IL‐8. The nAChRs seemed to be involved in IL‐8 production. In smokers, blood IL‐8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil‐IL‐8 production via nAChR by generating peroxynitrite and subsequent NF‐κB activation, and the IL‐8 appears to contribute to leukocytosis in tobacco smokers.

CpG‐DNA‐induced IFN‐α production involves p38 MAPK‐dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors

Human plasmacytoid or CD4+CD11c− type 2 dendritic cell precursors (PDC) were identified as natural type I interferon (IFN)‐producing cells in response to viral and bacterial infection. They represent effector cells of innate immunity and link it to the distinct adaptive immunity by differentiating into mature DC. It has been reported that oligodeoxyribonucleotides containing unmethylated CpG motifs (CpG DNA) stimulate PDC to produce IFN‐α, but the molecular mechanisms involved remain unknown. We found that CpG‐DNA‐induced IFN‐α production in PDC was completely impaired by the inhibitor of the p38 mitogen‐activated protein kinase (MAPK) pathway. Expression of IFN regulatory factor (IRF)‐7 was enhanced by CpG‐DNA treatment, which was preceded by the phosphorylation of signal transducer and activator of transcription (STAT)1 on Tyr‐701, as well as its enhanced phosphorylation on Ser‐727. All of these events were also suppressed by the p38 MAPK inhibitor. STAT1, STAT2, and IRF‐9, components of IFN‐stimulated gene factor 3 (ISGF3), were recognized in the nuclear fraction of CpG‐DNA‐treated cells. Neither anti‐IFN‐α/β antibodies (Ab) nor anti‐IFNAR Ab suppressed STAT1 phosphorylation, enhancement of IRF‐7 expression, or IFN‐α production in the early phase of the culture. These results suggest that CpG DNA induces p38 MAPK‐dependent phosphorylation of STAT1 in a manner independent of IFN‐α/β, which may cause ISGF3 formation to increase the transcription of the IRF‐7 gene, thereby leading to IFN‐α production in human PDC.

Collaborative action of NF-kappaB and p38 MAPK is involved in CpG DNA-induced IFN-alpha and chemokine production in human plasmacytoid dendritic cells.

CpG DNA induces plasmacytoid dendritic cells (pDC) to produce type I IFN and chemokines. However, it has not been fully elucidated how the TLR9 signaling pathway is linked to these gene expressions. We examined the mechanisms involving the TLR9 and type I IFN signaling pathways, in relation to CpG DNA-induced IFN-α, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-κB subunits p65 and p50 were constitutively activated. pDC also constitutively expressed IRF-7 and CCL3, and the gene expressions seemed to be regulated by NF-κB. CpG DNA enhanced the NF-κB p65/p50 activity, which collaborated with p38 MAPK to up-regulate the expressions of IRF-7, CXCL10, and CCL3 in a manner independent of type I IFN signaling. We then examined the pathway through which IFN-α is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-α, in a NF-κB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-α in the presence of NF-κB/p38 MAPK inhibitor, and chloroquine abrogated this effect. With CpG DNA, IRF-7, both constitutively and newly expressed, moved to the nuclei independently of NF-κB/p38 MAPK. These findings suggest that, in CpG DNA-stimulated human pDC, the induction of IRF-7, CXCL10, and CCL3 is mediated by the NF-κB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-κB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-α.

Detection of antibodies to the Epstein-Barr virus nuclear antigens in the sera from patients with systemic lupus erythematosus

The sera from 65 patients with systemic lupus erythematosus (SLE) were examined by the immunoblotting method to detect antibodies to Epstein-Barr virus (EBV)-associated antigens, especially EBV nuclear antigens (EBNA), and compared with the sera from 66 healthy subjects roughly age- and sex-matched to the patients. Most sera from patients with SLE defined three major EBV-associated antigens with molecular weights (MW) of 70,000 (70K), 90K and 140K in Raji cells, which must correspond to the EBNA-1, 2, and 3, respectively. Approximately 70% of the sera from SLE patients demonstrated the antibodies to the 90K and 140K antigens, whereas the positive rates of these two antibodies were less than 10% in the sera from healthy subjects. The differences of these positive rates of the antibodies between SLE patients and healthy subjects were statistically highly significant. Antibody to EBNA-1 was conspicuously detected in the sera from both SLE patients and healthy subjects, although the difference between the two groups was still significant. The possible role of EBV infection was discussed on the basis of the pathogenesis of SLE.

The role of monocytes in the suppression of PHA-induced proliferation and IL 2 production of human mononuclear cells by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited phytohemagglutinin (PHA)-induced proliferation of human blood mononuclear cells (MNC) at concentrations of 10(-11) M or more. Interleukin 2 (IL 2) production of T cells activated with PHA was also inhibited by 1,25(OH)2D3. Furthermore, 1,25(OH)2D3 suppressed interleukin 1 (IL 1) production of monocytes (Mo), and the agent-treated Mo were unable to promote IL 2 production of non-adherent cells (NAC). Thus, the reduction of proliferative response of MNC to PHA by 1,25(OH)2D3 appeared to have resulted from the inhibitory effects of the agent on both IL 2 and IL 1 production. From these data, 1,25(OH)2D3 appears to play an important role in the immunoregulatory system.

Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

(Cancer Sci 2010; 101: 767–773)

Differentiation of Myeloid Cells and 1, 25-Dihydroxyvitamin D3

It is well established that 1,25(OH)2D3 induces monocyte/macrophage (Mo/Mphi) colonies when added to culture of granulocyte/macrophage progenitors. Recently, we demonstrated that one of the target cells of 1,25(OH)2D3 in Mo/Mphi differentiation is the neutrophilic promyelocyte that is believed to belong to the neutrophilic lineage. This fact overthrows the established theory that normal hematopoietic precursors are committed to respective cell lineages and do not deviate from their own lineage. The lineage switching from the promyelocyte to Mo/Mphi was suggested to be operating in vivo because 1,25(OH)2D3 is a physiological substance produced by Mphi. More recently, we have shown that transient exposure (24 h) of promyelocytes to 1,25(OH)2D3 causes Mo/Mphi differentiation. This strategy could be useful for examining the effects of 1,25(OH)2D3 on the growth and differentiation of normal myeloblasts and myeloid progenitor cells. Recent advances in molecular biology have enabled investigators to identify a number of genes involved in Mo/Mphi differentiation induced by 1,25(OH)2D3. Some of these may be the determinant genes for Mo/Mphi differentiation; however, further studies are required to determine the underlying mechanisms of Mo/Mphi differentiation.

Mechanism in 1,25(OH)2D3-induced suppression of helper/suppressor function of CD4/CD8 cells to immunoglobulin production in B cells

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.

CpG oligodeoxynucleotides as mucosal adjuvants

Bacterial DNA comprising palindromic sequences and containing unmethylated CpG is recognized by toll-like receptor 9 of plasmacytoid dendritic cells (pDCs) and induces the production of interferon-α and chemokines, leading to the activation of a Th1 immune response. Therefore, synthetic equivalents of bacterial DNA (CpG oligodeoxynucleotides) have been developed for clinical applications. They are usually phosphorothioated for in vivo use; this approach also leads to adverse effects as reported in mouse models.Mucosal vaccines that induce both mucosal and systemic immunity received substantial attention in recent years. For their development, phosphodiester-linked oligodeoxynucleotides, including the sequence of a palindromic CpG DNA may be advantageous as adjuvants because their target pDCs are present right there, in the mucosa of the vaccination site. In addition, the probability of adverse effects is believed to be low. Here, we review the discovery of such CpG oligodeoxynucleotides and their possible use as mucosal adjuvants.

Type I IFN Synthesis in Plasmacytoid Dendritic Cells

Kerkmann et al. ([1][1]) recently reported two distinct pathways for IFN-α induction in human plasmacytoid dendritic cells (PDC), one dependent on and one independent of the IFNR-mediated feedback loop. However, the mechanisms by which the IFNR-mediated pathway is initiated were not mentioned.