Manabu Yanagita

Initiation of NALT organogenesis is independent of the IL-7R, LTβR, and NIK signaling pathways but requires the Id2 gene and CD3-CD4+CD45+ cells

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyers patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTalpha1beta2/LTbetaR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2(-/-) mice. NALT organogenesis is initiated following the adoptive transfer of CD3(-)CD4(+)CD45(+) cells into Id2(-/-) mice, constituting direct evidence that CD3(-)CD4(+)CD45(+) inducer cells can provide an IL-7R-, LTalpha1beta2/LTbetaR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.

Nasal immune system: distinctive Th0 and Th1/Th2 type environments in murine nasal-associated lymphoid tissues and nasal passage, respectively.

The nasal mucosa, an important arm of the mucosal immune system, is the first site of contact with inhaled antigens to induce an IgA response. A major aim of this study was to characterize the Th1 and Th2 cytokine expression of mucosal T cells residing in nasal‐associated lymphoid tissue (NALT) and nasal passages (NP) as IgA inductive and effector sites, respectively, at the transcription and cellular levels. An application of single‐cell reverse transcription‐PCR for analysis of Th1 (IFN‐γ) and Th2 (IL‐4 and IL‐6) cytokine‐specific mRNA revealed the presence of CD4+ T cells with a Th0 profile in NALT, while high numbers of Th2 cytokine‐specific mRNA expressed by CD4+ T cells were noted in NP followed by Th1‐type cells. NALT CD3+ CD4+ T cells of Th0 type have the capacity to become Th1‐ and/or Th2‐type cells since their activation via the TCR‐CD3 complex resulted in the expression of an array of Th1 and Th2 cytokines. CD3+ CD4+ T cells from NP, but not NALT, provide a helper function for the induction of antibody‐forming cells including IgA isotype in B cell cultures. These findings suggest that NALT is characterized by a Th0 environment which can gain a Th1 and/or Th2 phenotype. In contrast, NP is considered to be a Th2 dominant site with some Th1 cells that can support the induction of IgA‐producing cells.

IL-15 and IL-15 Receptor Selectively Regulate Differentiation of Common Mucosal Immune System-Independent B-1 Cells for IgA Responses

We show in this report a new regulatory role for IL-15 and IL-15R in the development of B-1 cells and their differentiation into IgA-producing cells. Mucosal IgA levels were found to be inhibited by anti-IL-15 mAb treatment in vivo, but enhanced by administration of rIL-15, while serum IgA levels remained unaffected. Mucosal B-1 cells preferentially proliferated in response to IL-15 in vitro. When mucosal B-1 and B-2 cells were separated into surface (s)IgM+sIgA− and sIgM−sIgA+ fractions, IL-15R-specific mRNA was found to be predominant in both sIgM+sIgA− and sIgM−sIgA+ B-1 cells at a much higher level than B-2 cells. Further, incubation of these different subsets of B-1 and B-2 cells with IL-15 resulted in greater enhancement of the corresponding receptor expression by B-1 subset when compared with B-2 fraction. Interestingly, de novo isolated sIgM+sIgA− B-1, but not sIgM+sIgA− B-2, cells were already class-switched cells because the germline Cα transcript was detected and was then further enhanced by IL-15. IL-15 also supported differentiation of both sIgM+sIgA− and sIgM−sIgA+ B-1 cells into IgA-producing cells. Taken together, these findings suggest that IL-15 is a critically important cytokine for the differentiation of both sIgM+,IgA− and sIgM−sIgA+ B-1 cells expressing IL-15R into IgA-producing cells in mucosal tissues.

Fibroblast growth factor-2 regulates the cell function of human dental pulp cells.

INTRODUCTION Homeostasis and tissue repair of dentin-pulp complex are attributed to dental pulp tissue and several growth factors. Dental pulp cells play a pivotal role in homeostasis of dentin-pulp complex and tissue responses after tooth injury. Among these cytokines, fibroblast growth factor (FGF)-2 has multifunctional biologic activity and is known as a signaling molecule that induces tissue regeneration. In this study, we examined the effects of FGF-2 on growth, migration, and differentiation of human dental pulp cells (HDPC). METHODS HDPC were isolated from healthy dental pulp. Cellular response was investigated by [(3)H]-thymidine incorporation into DNA. Cytodifferentiation was examined by alkaline phosphatase (ALPase) assay and cytochemical staining of calcium by using alizarin red. Migratory activity was determined by counting the cells migrating into cleared area that had introduced with silicon block. RESULTS FGF-2 activated HDPC growth and migration but suppressed ALPase activity and calcified nodule formation. Interestingly, HDPC, which had been pretreated with FGF-2, showed increased ALPase activity and calcified nodule formation when subsequently cultured without FGF-2. These results suggest that FGF-2 potentiates cell growth and accumulation of HDPC that notably did not disturb cytodifferentiation of the cells later. Thus, FGF-2 is a favorable candidate for pulp capping agent. CONCLUSIONS These results provide new evidence for the possible involvement of FGF-2 not only in homeostasis but also in regeneration of dentin-pulp complex.

HIV Mucosal Vaccine: Nasal Immunization with rBCG-V3J1 Induces a Long Term V3J1 Peptide-Specific Neutralizing Immunity in Th1- and Th2-Deficient Conditions

In the vaccine strategy against HIV, bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of potential vectors for mucosal delivery of vaccine Ag. We analyzed the induction of the Ag-specific Ab response by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that can secrete the V3 principal neutralizing epitope of HIV. Mice were nasally immunized with rBCG-V3J1 (10 μg) three times at weekly intervals. Four weeks after the initial immunization, high titers of V3J1-specific IgG Abs were seen in serum. These high levels of HIV-specific serum IgG responses were maintained for >12 mo following nasal immunization without any booster immunization. V3J1-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinated mice. Nasal rBCG-V3J1 also induced high levels of prolonged HIV-specific serum IgG responses in Th1 (IFN-γ−/−)- or Th2 (IL-4−/−)-immunodeficient mice. Further, IgG3 was highest among V3 peptide-specific IgG subclass Ab responses in these immunodeficient mice as well as in wild-type mice. In addition, this Ag-specific serum IgG Abs induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize clinical isolate of HIV in vitro. These results suggested that the nasal rBCG-V3J1 system might be used as a therapeutic vaccine in addition to a prophylaxis vaccine for the control of AIDS.

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011.

Nicotine modulates the immunological function of dendritic cells through peroxisome proliferator-activated receptor-γ upregulation.

We examined the effects of nicotine on differentiation and function of monocyte-derived human dendritic cells (DCs). NiDCs, which were the DCs differentiated in the presence of nicotine, showed lower levels of CD1a. Secretion of IL-12 and TNF-α by lipopolysaccharide (LPS)-stimulated NiDCs was significantly suppressed compared to monocyte-derived DCs grown without nicotine. NiDCs displayed a diminished capacity to induce allogeneic T cell proliferation with a reduced production of IFN-γ, and maintained/enhanced LPS-mediated expression of coinhibitory molecules. Interestingly, NiDCs enhanced the expression of nuclear receptor peroxisome proliferator-activated receptors γ (PPAR γ), which has immunomodulatory properties. Expression of PPAR γ and PPAR γ-target genes was significantly inhibited by pretreatment with d-tubocurarine, antagonist of non-selective nicotinic acetylcholine receptors (nAChR). In addition, reduction of Th1 responses was inhibited after blocking nAChR-mediated signal. These data suggest the effect of nicotine on altering DC immunogenicity by impeding Th1 immunity is partially mediated by upregulation of PPAR γ.

Nicotine Inhibits Mineralization of Human Dental Pulp Cells

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells ☆

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.

Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1β or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling.

OBJECTIVE Cigarette smoking is an important risk factor for periodontal disease. The aim of this study is to evaluate the effect of nicotine, a major component of cigarette smoke, on interleukin-8 (IL-8) production and cellular signalling via nicotinic acetylcholine receptors (nAChRs) in human gingival epithelial cells (HGECs). DESIGN Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1×10(-3)M nicotine in the presence or absence of IL-1β or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting. RESULTS HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1β or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator. CONCLUSION These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca(2+) signalling after nAChR activation.