c-Kit-stem cell factor signal-independent development of interstitial cells of Cajal in murine small intestine.

c-Kit receptor tyrosine kinase and its ligand stem cell factor (SCF) play critical roles in regulating the development and proliferation of various cells, including the interstitial cells of Cajal (ICC) in the gastrointestinal tract. Many subtypes of ICC are known to be lacking in c-Kit-SCF-insufficient mice, such as W/Wv and Sl/Sld, whereas ICC-deep muscular plexus (DMP) in small intestine are not lacking. In this study, we examine ICC-DMP development in normal and c-Kit-SCF signal-insufficient mice. In normal mice, numerous ICC-DMP labeled with c-Kit and neurokinin 1 receptor (NK1R) antibodies were observed only in the duodenum on the day of birth, in the duodenum and the jejunum on postnatal day 4 and throughout the small intestine after postnatal day 6. In W mutant mice (W/Wv, Wv/Wv, W/W), ICC-DMP investigated using c-Kit and NK1R immunoreactivities were similar to that in normal mice. c-Kit ligand SCF-deficient mice (Sl/Sl) also showed almost identical ICC-DMP development and proliferation as normal mice. These results show that the development and proliferation of ICC-DMP occur in the postnatal period independent of c-Kit-SCF signaling.

Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

The role of CCR7 in allergic airway inflammation induced by house dust mite exposure.

House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-ß, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.

Downregulation of msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2) in the developmental gut of KIT mutant mice.

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are the regulatory cells of gut movement. W/W mutant mice that have receptor tyrosine kinase KIT mutation lack ICC along the myenteric plexus layer of small intestine. The development and maintenance of the ICC phenotype have been related to KIT, but the other genes involved in ICC development during embryogenesis are not clear. Our aim was to identify ICC-specific genes in the embryonic stage. We examined genes that are expressed less in ICC-deficient W/W mice than in wild type (WT) at embryonic day 14 (E14) in order to clarify the genes associated with the ICC development using subtractive hybridization and microarray. Among them, we identified msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2). Using real-time PCR, msx2 and ntrk2 were found to be expressed at significantly lower levels in W/W than in WT during embryogenesis. Msx2 immunoreactivity was high in the WT small intestine. These data suggest that the gene expressions of ntrk2 and msx2 were significantly suppressed in KIT mutant mouse embryo and neonate and that these genes are likely to regulate ICC development.

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric alpha7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions.

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via alpha7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of alpha7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.

Interstitial cells of Cajal in Wsh/Wsh c-kit mutant mice.

The c-Kit receptor tyrosine kinase regulates the development and differentiation of several progenitor cells. In the gastrointestinal (GI) tract, the c-Kit regulates the development of the interstitial cells of Cajal (ICC) that are responsible for motility regulation of the GI musculature. W-sash (Wsh) is an inversion mutation upstream of the c-kit promoter region that affects a key regulatory element, resulting in cell-type-specific altered gene expression, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the GI tract of Wsh/Wsh mice using immunohistochemistry and electron microscopy. Although the musculature of the Wsh/Wsh mice did not show any c-Kit immunoreactivity, we detected intensive immunoreactivity for transmembrane member 16A (TMEM16A, anoctamin-1), another ICC marker. TMEM16A immunopositive cells were observed as ICC-MY in the gastric corpus-antrum and the large intestine, ICC-DMP in the small intestine, and ICC-SM in the colon. Electron microscopic analysis revealed these cells as ICC from their ultrastructural features, such as numerous mitochondria and caveolae, and their close contact with nerve terminals. In the developmental period, we examined 14.5 and 18.5 day embryos but did not observe c-Kit immunoreactivity in the Wsh/Wsh small intestine. From this study, ICC subtypes developed and maturated structurally without c-Kit expression. Wsh/Wsh mice are a new model to investigate the effects of c-Kit and unknown signaling on ICC development and function.

Investigation of Novel c-Kit-expressing Smooth Muscle Cells in Murine Cecum.

In the gastrointestinal tract musculatures, c-Kit receptor tyrosine kinase is specifically expressed in interstitial cells of Cajal (ICC). ICC are distributed among the smooth muscle cells and are either bipolar or multipolar in shape. Our previous and current study shows that c-Kit-immunopositive smooth muscle cells are present in the murine cecum. Here, we found that c-Kit-expressing smooth muscle cells (named Kit-SM cells) are situated at the submucosal surface of the circular muscle layer. These cells showed smooth muscle actin and myosin immunoreactivities and ultrastructural features such as thick and thin filaments and caveolae. Kit-SM cells also expressed TMEM16A and LRIG1, which are known to be expressed in ICC. Although the functional significance of Kit-SM cells has yet to be revealed, these cells can be considered to have proliferation or differentiation potential in the cecal musculature.

Expression of SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells.

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for alpha7 nicotinic acetylcholine (ACh) receptors (alpha7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-alpha (TNF-alpha) release from keratinocytes and macrophages via alpha7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-alpha release from macrophages in bronchial tissue.

c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor alpha in the murine gastrointestinal musculature.

Platelet-derived growth factor receptors (PDGFRs) belong to the same kinase group as c-Kit receptor tyrosine kinase that is specifically expressed in the interstitial cells of Cajal (ICC) in the gastrointestinal tract. In this study, we examined PDGFRalpha immunoreactivity in the murine gastrointestinal tract. PDGFRalpha-immunopositive (PDGFRalpha-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRalpha-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRalpha-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRalpha-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRalpha-ip cells in the ICC-deficient W(v)/W(v) mutant mice was the same as that in normal mice. PDGFRalpha-ip cells that showed intense immunoreactivity of SK3 potassium channel were considered to correspond to fibroblast-like cells or non-Cajal interstitial cells. Our observations suggest that PDGFRalpha-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits.

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BACKGROUND: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.

Interstitial cells of Cajal in the gastrointestinal musculature of W(jic) c-kit mutant mice.

Interstitial cells of Cajal (ICC) generate electrical rhythmicity and transduce neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and are identified morphologically by c-Kit immunoreactivity. The c-kit gene is allelic with the murine white-spotting locus W, and mutations of c-kit are known as W mutations. W mutations affect various developmental aspects of hematopoietic cells, germ cells, melanocytes, mast cells and ICC. We examined W(jic)/W(jic) mutant mice that have a mutation in the tyrosine kinase domain resulting in severe loss of protein function. W(jic)/W(jic) homozygotes exhibited white coats and black eyes. The gross morphology of the gastrointestinal tract showed no abnormality in mutant mice other than a forestomach papilloma. In the stomach, intramuscular ICC (ICC-IM) were missing, and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced; however there was a normal distribution of deep muscular plexus ICC (ICC-DMP). In the cecum, the numbers of ICC-IM and ICC-MY were severely depleted. ICC-IM were almost entirely absent in the colon, whereas ICC-MY loss was restricted to the distal colon. Patterns of ICC deficiency were generally similar between W(jic)/W(jic) mice and W/W(v) mutants, which lack a specific type of ICC. The enteric nervous system of the mutant mice appeared normal. From these findings, we conclude that W(jic)/W(jic) mice represent a distinct, novel genotype resulting in a lack of a specific type of ICC in the gastrointestinal musculature.

Interstitial cells of Cajal in the gastrointestinal musculature of W mutant mice.

Interstitial cells of Cajal (ICC) are important regulatory cells generating electrical rhythmicity and transducing neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and can be examined morphologically using the c-Kit antibody. The c-kit gene is allelic with the murine white-spotting locus W, and the c-kit mutation (W mutation) affects various aspects of hematopoietic cells, germ cells, melanocytes, mast cells, and ICC. Heterozygous W/W( v) mutant mice lack a specific type of ICC and have been used to reveal its function. To search for a new model that lacks a specific type of ICC, we examined homozygous W( v)/W( v) black-eyed-white mice that are viable with anemia. Results showed the principal patterns of ICC deficiency were the same between the W/W( v) and W( v)/W( v) mutants. In the stomach of both mice, intramuscular ICC (ICC-IM) were missing and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced in spite of a normal distribution of deep muscular plexus ICC (ICC-DMP). The cecum also exhibited fewer reduced. ICC-IM in the colon were almost entirely missing, whereas ICC-MY were reduced only in the distal colon. In the small intestine and colon, the number of remaining ICC-MY in W( v)/W( v) mice was greater than that in W/W( v) mice. The enteric nervous system of the two mutant mice showed normal characteristics. From these findings, we conclude that W( v)/W( v) mice represent a new genotype that lacks a part of the ICC in its gastrointestinal musculature.

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-alpha, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-kappaB 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-kappaB. CpG DNA enhanced the NF-kappaB 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-alpha is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-alpha, in a NF-kappaB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-alpha in the presence of NF-kappaB/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-kappaB/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-kappaB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-kappaB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-alpha.

Decreased enzyme activity of hepatic thioredoxin reductase and glutathione reductase in rabbits by prolonged exposure to inorganic arsenate.

Chronic exposure of humans to inorganic arsenic, mainly pentavalent arsenate (iAsV), results in drinking water-induced oxidative stress (Pi et al., 2002). Thioredoxin reductase (TR) and glutathione reductase (GR) are the two critical enzymes in the response to oxidative stress in vivo. In the present study we examined alterations in enzyme activities of hepatic TR and GR from prolonged exposure of male New Zealand white rabbits to iAsV. Exposure of rabbits to iAsV in drinking water (5 mg/L) for 18 weeks caused a significant suppression of hepatic TR and GR activities, of approximately 30% and 20%, respectively, below controls. In vitro experiments suggested that trivalent inorganic arsenic (iAsIII) but not pentavalent arsenicals including iAsV, monomethylarsonic acid (MMAsV), and dimethylarsinic acid (DMAsV) affected the hepatic TR activity of rabbit. So it was suggested that in the present study iAsV ingested via drinking water was metabolized to reactive trivalent arsenicals, such as iAsIII, which may play an important role in the decreased TR and GR activities from prolonged exposure to iAsV observed in vivo.