Rumiko Takauji

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.

Evidence that Sema3A and Sema3F regulate the migration of GABAergic neurons in the developing neocortex

The ganglionic eminence (GE) supplies neurons containing γ‐aminobutyric acid (GABA) to the pallium of the telencephalon. We investigated the molecular guidance mechanisms of GE cell migration in the neocortex and found neuropilin‐1 (Npn‐1) or neuropilin‐2 (Npn‐2) on the GE cells. Ectopic Sema3A or ‐3F expression by COS1 cell clusters placed on embryo neocortical slices reduced the cell migration but did not block it completely. However, the cell migration was almost completely blocked by COS1 cell clusters expressing both Sema3A and ‐3F. The direction of cell migration could be reversed by placing Sema3A‐ and ‐3F‐coexpressing COS1 cell clusters at the distal cut end of the neocortical slices. Further slice experiments revealed that migration of half of the GE cells in the neocortex was regulated by Sema3A and that migration of the other half of the GE cells in the neocortex was regulated by Sema3F. When the cells responding to Sema3A were diverted by ectopic Sema3A expression in vivo, Dlx2‐positive cells were found predominantly in the lower intermediate zone (IZ). When the cells responding to Sema3F were diverted by ectopic Sema3F expression in vivo, Dlx2‐positive cells were found predominantly in the upper IZ. It was speculated that the semaphorin–neuropilin interactions distribute the GABAergic GE cells evenly in the neocortex as well as guide the GE cells from the GE to the neocortex. The Sema3A expression site under the subplate extended dorsally as the embryo developed. The Sema3A expression seemed to block the Npn‐1‐positive GE cells in the neocortex from entering the cortical plate (CP) and guide them to the dorsal cortex and the hippocampus. Sema3F expression in the CP continued through the embryonic stages. The expression seemed to block Npn‐2‐positive GE cells in the neocortex from entering the CP and make them migrate into the lower IZ. Finally, the semaphorin–neuropilin interactions sorted GABAergic inteneurons into the CP and white matter neurons into the IZ. J. Comp. Neurol. 455:238–248, 2003.

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.

Cell migration from the ganglionic eminence to the neocortex investigated by labeling nuclei with UV irradiation via a fiber-optic cable.

Recent studies have shown that the ganglionic eminence is one of the sources of tangentially migrating cells in the developing neocortex. Since the migration of the DiI-labeled cells from the ganglionic eminence to the neocortex was not monitored by videomicroscopy in these reports, we devised a novel method to study cell migration in vitro and in vivo. The new method involves ultraviolet (UV) irradiation of the cells through a fiber-optic cable and subsequent identification of the irradiated cells on the basis of the formation of thymine dimers in the nuclei. First, we tested the new method (UV-thymine dimer-labeling method) by applying it to monitor the cell migration of neuronal precursor cells in the rostral migratory stream in the neonatal rat telencephalon. In vitro, UV irradiation for 1 s through the fiber-optic cable resulted in the formation of sufficient thymine dimers as to allow immunohistochemical detection after 6 h of incubation; a significant proportion of the irradiated cells continued to migrate in the same direction and at the same speed as those before irradiation. There was no significant difference in the cell migration distance over 6 h between cells exposed and not exposed to the UV irradiation in vitro. In vivo, this method revealed that three times as many cells in the subventricular zone of the olfactory bulb migrated rostrally as caudally. The new method also allowed us to measure the speed of cell migration, which was estimated to be about 70 microm/h at the maximum in the rostral direction. After these examinations of reliability of the method, we applied it to the rat embryo brain. One day after UV irradiation of the ganglionic eminence, labeled migrating cells were found in the striatum, in the internal capsule, and in the intermediate zone of the neocortex. The observation period of cell migration to the neocortex was extended by the use of a xeroderma pigmentosum group A gene mutant mouse, which lacked an ability to remove thymine dimer from the UV-irradiated nuclei. Two days after the UV irradiation, labeled migrating cells from the ganglionic eminence of the mutant mouse embryos were found both in the cortical plate and in the intermediate zone of the neocortex.

Differential localization of high- and low-molecular-weight variants of microtubule-associated protein 2 in the developing rat telencephalon.

Microtubule‐associated protein 2 (MAP2) occurs in developing mammalian neuronal tissue as both high‐ and low‐molecular‐weight forms with temporally regulated expression. We studied the MAP2 expression in the developing rat telencephalon with monoclonal antibodies that recognized both the high‐ and low‐molecular‐weight forms of MAP2 variants or that specifically recognized high‐molecular‐weight forms of MAP2 variants. Differences in the staining patterns of these antibodies reflected differences in the distribution of the high‐ and low‐molecular‐weight MAP2s. The immunoreactive sites of high‐ and low‐molecular‐weight MAP2 had a more widespread distribution in the embryonic telencephalon than those of high‐molecular‐weight MAP2. Many bipolar cells in the ganglionic eminence (GE) and in the intermediate zone (IZ) of the neocortex showed low‐molecular‐weight MAP2 immunoreactivity, but they showed weak or no high‐molecular‐weight MAP2 immunoreactivity. Expression of mRNA containing exons common to high‐ and low‐molecular‐weight MAP2 was detected in the tangentially ellipsoidal cells in the IZ, but expression of mRNA containing an exon specific to high‐molecular‐weight MAP2 was not detected in these cells by in situ hybridization. We interpreted these observations as indicating that the bipolar cells contained MAP2c preferentially, but contained MAP2a and MAP2b (MAP2a/b) at a very low or negligible level. The cells that expressed MAP2c preferentially among the MAP2 splicing variants composed 50% of the preplate cells, most of the MAP2‐positive cells in the hippocampus and the corpus callosum. Double labeling by DiI staining and Dlx2 immunohistochemistry, or by Dlx2 and MAP2 immunohistochemistry, revealed that most of the Dlx2‐positive cells in the IZ expressed MAP2c preferentially at embryonic day 16. Another double‐labeling study revealed that most GAD‐positive cells in the preplate were MAP2a/b positive, whereas most GAD‐positive cells in the IZ expressed MAP2c preferentially, with only a negligible level of MAP2a/b immunoreactivity. We conclude that MAP2 immunoreactivity in the IZ was localized in the tangentially migrating neurons. The tangentially migrating neurons seemed to acquire MAP2a/b immunoreactivity as they entered the preplate or cortical plate and developed into mature neurons. Radially migrating neurons in the IZ were MAP2 negative. After entering to the preplate or the cortical plate, they became MAP2a/b positive as they developed into mature neurons. J. Comp. Neurol. 449:330–342, 2002.

A Procedure for In Situ Hybridization Combined with Retrograde Labeling of Neurons: Application to the Study of Cell Adhesion Molecule Expression in DiI-labeled Rat Pyramidal Neurons

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer DiI. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry. (J Histochem Cytochem 45:455–459, 1997)

Enhancement of Ca2+-dependent endonuclease activity in L1210 cells during apoptosis induced by 1-β-D-arabinofuranosylcytosine : possible involvement of activating factor(s)

Internucleosomal DNA fragmentation and morphological changes in nuclei typical of apoptosis were observed in L1210 cells incubated with 1.0 μg/ml of 1‐β‐D‐arabinofuranosylcytosine (ara‐C). To investigate the mechanisms involved, we examined the activities of endogenous endonucleases in nuclei and cytoplasm. Both fractions of control cells contained Ca2+ ‐dependent endonuclease which was capable of mediating internucleosomal DNA fragmentation. The assay system using two kinds of target substrates, i.e., nuclear chromatin of CCRF‐CEM cells and naked DNA purified from the same cells, revealed that the activity of Ca2+‐dependent endonuclease was enhanced in the crude nuclear extracts of cells treated with 1.0 μg/ml of ara‐C for 24 h or 48 h. The activity was extracted more easily from ara‐C‐treated cells than control cells without sonication of the nuclear fraction. On the other hand, in the cytoplasmic fraction of the cells, the activity towards naked DNA was unchanged, whereas that towards nuclear chromatin was clearly enhanced. These results suggest that internucleosomal DNA fragmentation induced by ara‐C treatment is associated with enhancement and activation of constitutively expressed Ca2+ ‐dependent endonuclease in L1210 cells.

Intermediate Zone and Intermediate Zone Neurons

It is assumed that pioneer neurons will predominantly show path finding ability, and that other neurons only follow the pioneer fibers. Here, tracers were injected into the newborn rat spinal cord fol

1001 Migration of IZ neurons originating in the lateral ganlionic eminence to the neocortex

Nobuaki Tamamaki’, Kiyoji Tanaka”, Kazuhiro E. Fujimori’, Rumiko Takauji’ Neuroblasts produced in the ventricular zone of the neocortex migrate radially and form neocortical plate, but the tangentially migrating cells in the intermediate zone (IZ) were not negligible in the embryonic neocortex. Recently we proposed that most of the tangentially migrating cells in the IZ, the IZ neurons are not incorporated into the cortical plate and originate in the lateral ganglionic eminence (LGE). Here we used a new method to investigate the cell migration in vivo and in vitro. The migrating cells from the LGE were marked by UV radiation through optic fiber. Thymine dimer was formed in the DNA molecule by the radiation and detected by immunohistochemistry with anti-thymine-dimer antibody. Labeled migrating cells in the subventricular zone of the LGE crossed the boundary between the LGE and the neocortex. To investigate the final destination of the cell migration, a trial to use this method in the XPA mutant mouse was started.