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.

Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice.

Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease.

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.

Activation through toll-like receptor 2 on group 2 innate lymphoid cells can induce asthmatic characteristics.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. OBJECTIVES: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. METHODS: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed. RESULTS: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. CONCLUSIONS & CLINICAL RELEVANCE: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease.

DNA methylation is a key epigenetic modification that can regulate gene expression. Genomic DNA hypomethylation is commonly found in many gastrointestinal (GI) diseases. Dysregulated gene expression in GI smooth muscle cells (GI-SMCs) can lead to motility disorders. However, the consequences of genomic DNA hypomethylation within GI-SMCs are still elusive. Utilizing a Cre-lox murine model, we have generated SMC-restricted DNA methyltransferase 1 (Dnmt1) knockout (KO) mice and analyzed the effects of Dnmt1 deficiency. Dnmt1-KO pups are born smaller than their wild-type littermates, have shortened GI tracts, and lose peristaltic movement due to loss of the tunica muscularis in their intestine, causing massive intestinal dilation, and death around postnatal day 21. Within smooth muscle tissue, significant CpG hypomethylation occurs across the genome at promoters, introns, and exons. Additionally, there is a marked loss of differentiated SMC markers (Srf, Myh11, miR-133, miR-143/145), an increase in pro-apoptotic markers (Nr4a1, Gadd45g), loss of cellular connectivity, and an accumulation of coated vesicles within SMC. Interestingly, we observed consistent abnormal expression patterns of enzymes involved in DNA methylation between both Dnmt1-KO mice and diseased human GI tissue. These data demonstrate that DNA hypomethylation in embryonic SMC, via congenital Dnmt1 deficiency, contributes to massive dysregulation of gene expression and is lethal to GI-SMC. These results suggest that Dnmt1 has a necessary role in the embryonic, primary development process of SMC with consistent patterns being found in human GI diseased tissue.

The anti-inflammatory pathway regulated via nicotinic acetylcholine receptors in rat intestinal mesothelial cells.

Regulation of inflammation in intestinal mesothelial cells in the abdominal cavity is important for the pathogeny of clinical conditions, such as postoperative ileus, peritonitis and encapsulating peritoneal sclerosis. Here we have examined the inflammatory effect of lipopolysaccharide (LPS) and the anti-inflammatory effect of nicotinic acetylcholine receptor stimulation in rat intestinal mesothelial cells. LPS upregulated mRNA expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS). The α7, α9 and α10 subunits of nicotinic acetylcholine receptor were detected in intestinal mesothelial cells. Nicotine (10 nM) significantly inhibited LPS-induced mRNA expression of IL-1ß and iNOS, but not TNF-α and MCP-1. In addition, the α7 nicotinic acetylcholine receptor selective agonist, PNU-282987 (10 nM), significantly inhibited LPS-induced mRNA expression of IL-1ß but not TNF-α, iNOS and MCP-1. Finally, we found that enteric nerves adhered to intestinal mesothelial cells located under the ileal serosa. In conclusion, intestinal mesothelial cells react to LPS to induce the production of nitric oxide from iNOS. The anti-inflammatory action of intestinal mesothelial cells expressing α7nAChR may be mediated via their connectivity with enteric nerves.

Expression and Function of the Cholinergic System in Immune Cells.

T and B cells express most cholinergic system components-e.g., acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase, and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Using ChATBAC-eGFP transgenic mice, ChAT expression has been confirmed in T and B cells, dendritic cells, and macrophages. Moreover, T cell activation via T-cell receptor/CD3-mediated pathways upregulates ChAT mRNA expression and ACh synthesis, suggesting that this lymphocytic cholinergic system contributes to the regulation of immune function. Immune cells express all five mAChRs (M1-M5). Combined M1/M5 mAChR-deficient (M1/M5-KO) mice produce less antigen-specific antibody than wild-type (WT) mice. Furthermore, spleen cells in M1/M5-KO mice produce less tumor necrosis factor (TNF)-α and interleukin (IL)-6, suggesting M1/M5 mAChRs are involved in regulating pro-inflammatory cytokine and antibody production. Immune cells also frequently express the α2, α5, α6, α7, α9, and α10 nAChR subunits. α7 nAChR-deficient (α7-KO) mice produce more antigen-specific antibody than WT mice, and spleen cells from α7-KO mice produce more TNF-α and IL-6 than WT cells. This suggests that α7 nAChRs are involved in regulating cytokine production and thus modulate antibody production. Evidence also indicates that nicotine modulates immune responses by altering cytokine production and that α7 nAChR signaling contributes to immunomodulation through modification of T cell differentiation. Together, these findings suggest the involvement of both mAChRs and nAChRs in the regulation of immune function. The observation that vagus nerve stimulation protects mice from lethal endotoxin shock led to the notion of a cholinergic anti-inflammatory reflex pathway, and the spleen is an essential component of this anti-inflammatory reflex. Because the spleen lacks direct vagus innervation, it has been postulated that ACh synthesized by a subset of CD4+ T cells relays vagal nerve signals to α7 nAChRs on splenic macrophages, which downregulates TNF-α synthesis and release, thereby modulating inflammatory responses. However, because the spleen is innervated solely by the noradrenergic splenic nerve, confirmation of an anti-inflammatory reflex pathway involving the spleen requires several more hypotheses to be addressed. We will review and discuss these issues in the context of the cholinergic system in immune cells.

Physiological functions of the cholinergic system in immune cells.

T and B cells, macrophages and dendritic cells (DCs) all express most of the components necessary for a functional cholinergic system. This includes choline acetyltransferase (ChAT), muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively) and acetylcholinesterase (AChE). Immunological activation of T cells up-regulates cholinergic activity, including ChAT and AChE expression. Moreover, toll-like receptor agonists induce ChAT expression in DCs and macrophages, suggesting cholinergic involvement in the regulation of immune function. Immune cells express all five M1-M5 mAChR subtypes and several nAChR subtypes, including α7. Modulation of antigen-specific antibody and pro-inflammatory cytokine production in M1/M5 mAChR gene-knockout (KO) and α7 nAChR-KO mice further support the idea of a non-neuronal cholinergic system contributing to the regulation of immune function. Evidence also suggests that α7 nAChRs are involved in suppressing DC and macrophage activity, leading to suppression of T cell differentiation into effector T cells. These findings suggest the possibility that immune function could be modulated by manipulating immune cell cholinergic activity using specific agonists and antagonists. Therefore, a fuller understanding of the immune cell cholinergic system should be useful for the development of drugs and therapeutic strategies for the treatment of inflammation-related diseases and cancers.

Nitric oxide-induced oxidative stress impairs pacemaker function of murine interstitial cells of Cajal during inflammation.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

PGD2 deficiency exacerbates food antigen-induced mast cell hyperplasia.

Prostaglandin D2 (PGD2) is a major prostanoid secreted mainly by mast cells. Although PGD2 has been identified as a modulator of allergic inflammation, its precise role remains unclear. Here we investigate the role of PGD2 in food allergy. Oral administration of ovalbumin induces allergic responses in sensitized wild-type (WT) mice. Systemic gene deficiency of haematopoietic PGD synthase (H-PGDS(-/-)) exacerbates all of the manifestations accompanying severe mast cell hyperplasia in the intestine. Morphological studies show that c-kit/FcɛRI-positive WT mast cells strongly express H-PGDS. Transplantation of H-PGDS(-/-) mast cells also aggravates ovalbumin-induced mast cell hyperplasia and allergic symptoms in mast cell null mice. H-PGDS deficiency accelerates the production of SDF-1α and the activity of MMP-9 in the antigen-stimulated intestine. SDF-1α receptor blockade or MMP-9 inhibition relieves the exacerbated mast cell hyperplasia and manifestations observed in H-PGDS(-/-). Thus, PGD2 deficiency results in food antigen-induced mast cell hyperplasia.

Non-neuronal cholinergic system in regulation of immune function with a focus on α7 nAChRs.

In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animal's body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4(+) T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including α7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M(1)/M(5) mAChR signaling up-regulates IgG(1) and pro-inflammatory cytokine production, while α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric α7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205(+) DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating α7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.

Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus.

BACKGROUND AND PURPOSE: Post-operative ileus (POI) is induced by intestinal inflammation. Here, we aimed to clarify the effects of 5-HT3 receptor antagonists against POI. EXPERIMENTAL APPROACH: We administered three 5-HT3 receptor antagonists, ondansetron, tropisetron and palonosetron, to a mouse model of POI induced by surgical intestinal manipulation (IM). Immunohistochemistry, intestinal transit, inflammatory mediator mRNA expression and 5-HT content were measured. In some experiments, 5-HT3 A receptor null mice were used. KEY RESULTS: Three 5-HT3 receptor antagonists reduced IM-induced infiltration of inflammatory CD68-positive macrophages and myeloperoxidase-stained neutrophils. Ondansetron exhibited no anti-inflammatory actions in 5-HT3 A receptor null mice. Ondansetron inhibited expression of the chemokine CCL2, IL-1ß, IL-6, TNF-α and iNOS mRNAs up-regulated by IM, and also ameliorated the delayed gastrointestinal transit. Peritoneal macrophages, but not most infiltrating monocyte-derived macrophages, expressed 5-HT3 receptors. IM stimulation increased the 5-HT content of peritoneal lavage fluid, which up-regulated mRNA expression of proinflammatory cytokines in peritoneal macrophages. Immunohistochemical localization of 5-HT3 receptors suggests that ondansetron suppressed expression of these mRNAs in activated peritoneal macrophages, adhering to the serosal region of the inflamed intestinal wall. CONCLUSION AND IMPLICATIONS: 5-HT3 receptor antagonists were anti-inflammatory, mainly targeting peritoneal macrophages expressing these receptors. They also restored the delayed gastrointestinal transit by IM. 5-HT3 receptor antagonists should be therapeutically useful agents against POI.

SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205(+) dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity.

Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including α7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous α7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205(+) dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10 µg/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific α7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5 µg/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at α7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses.

Characterization of anoikis-resistant cells in mouse colonic epithelium.

Anoikis is a form of apoptosis triggered by inadequate or inappropriate cell matrix contacts. Anoikis resistance has been utilized to isolate adult stem or progenitor cell populations from various tissues. The aim of this study was to characterize the stem or progenitor cell markers expressed in anoikis-resistant cells isolated from mouse colonic epithelium. Mouse colonic epithelial cells were isolated by Ca(2+)-depletion, and anoikis-resistant cells were obtained by culturing the cells in ultra-low attachment dishes. Flow cytometry analysis showed that anoikis-resistant cells are positive for the epithelial cell marker CD326, but negative for the hematopoietic cell marker CD45, eliminating the possibility of hematopoietic cell contamination. The majority of anoikis-resistant cells were also positive for the stem or progenitor cell markers CD133 and DCLK1 by immunofluorescent analysis. Reverse transcriptase-polymerase chain reaction analysis revealed that anoikis-resistant cells express +4 position cell marker Hopx, but did not express the other reported stem or progenitor cell markers, Lgr5, Musashi1, Bmi1, mTert and Olfm4. CD133 and DCLK1 double positive cells were observed both apical and basal crypts in mouse proximal colonic tissues. Together, anoikis-resistant cells in mouse colon epithelium were shown to be positive for CD133, DCLK1, Hopx and CD326, but negative for CD45, Lgr5, Musashi1, Bmi1, mTert and Olfm4. This study has shown a possible approach to isolating stem cells from the intestinal epithelium.

Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function.

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M(1) /M(5) mAChR signaling upregulates IgG(1) and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP-1, also appears to be involved in modulating normal T cell function.

Suppressive effects of a pyrazole derivative of curcumin on airway inflammation and remodeling.

To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 significantly suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin or dexamethasone (DEX). CNB001 significantly inhibited the decrease of E-cadherin mRNA expression and increase of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-ß1 and TNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-ß1, CNB001 significantly downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also reported to be related to airway remodeling. Whereas DEX alone significantly increased the active SERPINE1 level, the combination of DEX and CNB001 significantly suppressed active SERPINE1. In addition, CNB001 significantly suppressed neutrophil infiltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage fluid of the murine asthma model, which was not observed in the case of DEX. In conclusion, the curcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway inflammation and remodeling.

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.

Mediatophore regulates acetylcholine release from T cells.

Immunological stimulation of T cells by phytohemagglutinin (PHA) enhances the synthesis and release of acetylcholine (ACh), suggesting a role for the lymphocytic cholinergic system in the regulation of immune function. In the present study, we used two human leukemic T cell lines as models to investigate whether mediatophore, a homooligomer of a 16-kDa subunit homologous to the proteolipid subunit c of vacuolar H(+)-ATPase (V-ATPase), is involved in mediating ACh release from T cells. Immunohistochemical analysis revealed the presence of mediatophore in the cytoplasm and on the plasma membrane of both T cell lines. Mediatophore gene expression was up-regulated by immunological T cell activation by PHA. Transfection of anti-mediatophore small interference RNA down-regulated mediatophore gene expression and significantly reduced ACh release. These results suggest that T cells express mediatophore, which then plays a key role in mediating ACh release, and that mediatophore expression is regulated by immunological stimulation.

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system.

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.