Takayuki Takahashi

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.

CLONING AND EXPRESSION OF A CDNA FOR RAT PROSTACYCLIN RECEPTOR

A cDNA clone for rat prostacyclin receptor was isolated. The cDNA encodes a protein of 416 amino acid residues (M(r) 44,662) with putative seven transmembrane domains, and belongs to the G protein-coupled receptor superfamily. Specific binding of [3H]iloprost was found in membrane of COS-7 cells transfected with the cDNA (Kd = 1.3 nM) and was displaced with unlabeled prostaglandins in the order of iloprost = cicaprost > PGE1 > STA2 = PGE2 = PGD2 > PGF2 alpha. Northern blot analysis demonstrated that rat prostacyclin receptor mRNA is expressed in the lung, spleen, heart, pancreas, thymus, stomach and aorta.

Erythropoietin receptor of a human leukemic cell line with erythroid characteristics.

A new cell line of human erythroleukemia cells differentiates spontaneously so that 30% of the cells are always hemoglobinized. Erythropoietin did not affect the percentage of such cells but stimulated the cell growth, indicating that the cells have functioning receptors. Binding of human recombinant radioiodinated erythropoietin to the receptors was specific. The bound ligand was internalized into cells at 37 degrees C but not at 15 degrees C. Scatchard analysis showed two classes of binding sites. Covalent binding of erythropoietin to its receptors yielded two products detected on sodium dodecyl sulfate-polyacrylamide gels electrophoresed under reducing conditions. Under non-reducing conditions, these species disappeared and larger products appeared.

Liver damage in patients with colony-stimulating factor-producing tumors

PURPOSE We have demonstrated that colony-stimulating factor (CSF)-producing tumor cell lines produce not only CSF but also interleukin-1 (IL-1) and interleukin-6 (IL-6). Clinically, we have observed that patients bearing such tumors present with liver dysfunction and fever in addition to marked leukocytosis. The purpose of this study was to determine whether or not the liver damage was specifically related to CSF-producing tumors. PATIENTS AND METHODS Clinicopathologic examinations were performed in six autopsied patients with CSF-producing tumors. We also transplanted two tumor cell lines (KHC287 and CHU-2), which produce granulocyte (G)-CSF, IL-1, and IL-6, to nude mice. RESULTS Of the six patients, five had G-CSF- and one had granulocyte/macrophage (GM)-CSF-producing tumors. IL-1 and IL-6 concentrations in plasma or culture supernatant were elevated in these patients. Biochemical examinations revealed high serum enzyme levels of the biliary system in contrast to normal or slight increases in transaminase levels in all patients studied. Serum direct bilirubin was elevated in five of the six patients. Three common pathologic changes of the liver were found: (1) focal necrosis associated with neutrophil infiltration in the centrilobular zones, (2) fibrous change and enlargement of the portal area associated with neutrophil infiltration, and (3) intrahepatic cholestasis. The same pathologic changes, except for cholestasis, were reproduced in the liver of mice transplanted with KHC287 or CHU-2. CONCLUSION These results indicate that patients with CSF-producing tumors have characteristic liver damage, and suggest a new paraneoplastic syndrome of leukocytosis and liver damage.

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.

The Serum Cytokine Profiles of Lymphoma-associated Hemophagocytic Syndrome: A Comparative Analysis of B-Cell and T-Cell/Natural Killer Cell Lymphomas

To elucidate the differences in pathogenesis between lymphoma-associated hemophagocytic syndromes (LAHS) of theT-cell/ natural killer cell (T/NK) and B-cell (B) types,we comparatively analyzed the clinical features and serum cytokine profiles of 33 patients with LAHS registered in the Kyoto University Hematology/Oncology Study Group.The serum cytokine levels of each patient group (B-LAHS versusT/NK-LAHS) were expressed as the ratio of the median to the upper normal values of the respective cytokines and were as follows: 19.05 versus 13.99 for soluble interleukin 2 (IL-2) receptor, 0.67 versus 0.67 for granulocytemacrophage colony-stimulating factor (GM-CSF), 0.64 versus 1.26 for G-CSF, 5.70 versus 3.61 for M-CSF, 1.54 versus 3.39 for interferon γ (IFN-γ), 13.17 versus 1.17 for IL-6, 6.88 versus 1.58 for tumor necrosis factor α (TNF-α), 0.71 versus 0.41 for IL-1, 1.99 versus 0.21 for IL-12, and 105.32 versus 29.65 for IL-10.The serum levels of IL-6,TNF-α, and IL-10 were significantly higher in the B-LAHS group,whereas those of IFN-γ were significantly lower. These differences between the 2 groups may reflect a difference in the pathogenesis. Higher serum levels of IL-6, TNF-α, and IL-10 may be derived at least partly from neoplastic B-cells themselves. In addition, the extremely high serum levels of IL-10 suggest that a compensatory anti-inflammatory process may operate in both groups and give rise to a profound immunosuppressive state and a poor outcome.

Expression of the erythropoietin receptor on a human myeloma cell line.

We demonstrated the expression of the erythropoietin (EPO) receptor by a human myeloma cell line (MM-S1) which was established in our laboratory. EPO dose-dependently stimulated the proliferation of MM-S1 cells. Binding of radioiodinated EPO (125I-Epo) to MM-S1 cells was competitively inhibited by unlabeled EPO, but not by other recombinant cytokines. Specific binding of 125I-Epo to MM-S1 cells was saturable, and the Scatchard analysis revealed 330 EPO binding sites per cell with a Kd of 0.56 nmol/L. Bound EPO was internalized by MM-S1 cells during incubation at 37 degrees C. This is the first report describing the expression of the EPO receptor by human cells other than those of the erythroid lineage.

Fulminant sepsis caused by Bacillus cereus in patients with hematologic malignancies: analysis of its prognosis and risk factors.

Bacillus cereus is a growing concern as a cause of life-threatening infections in patients with hematologic malignancies. However, the risk factors for patients with unfavorable outcomes have not been fully elucidated. At our institution, we observed the growth of B. cereus in blood culture in 68 patients with (23) or without (45) hematologic malignancies treated from September 2002 to November 2009. We defined a case as having sepsis when more than two blood culture sets were positive for B. cereus or only a single set was positive in the absence of other microorganisms in patients who had definite infectious lesions. We determined 12 of 23 patients with hematologic malignancies as having sepsis, as well as 10 of 45 patients without hematologic malignancies (p = 0.012). Of the 12 patients with hematologic malignancies, four patients with acute leukemia died of B. cereus sepsis within a few days. In our cohort, risk factor analysis demonstrated that a neutrophil count of 0/mm3, central venous (CV) catheter insertion, and the presence of central nervous system (CNS) symptoms were significantly associated with a fatal prognosis (p = 0.010, 0.010, and 0.010, respectively). Analysis of data from our cohort in conjunction with those from 46 previously reported patients with B. cereus sepsis identified similar risk factors, that is, acute leukemia, extremely low neutrophil count, and CNS symptoms (p = 0.044, 0.004, and 0.002, respectively). These results indicate that appropriate prophylaxis and early therapeutic intervention against possible B. cereus sepsis are crucially important in the treatment of hematologic malignancies.