Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model.

BACKGROUND: The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce. OBJECTIVES: To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model. METHODS: GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro. RESULTS: Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties. CONCLUSION: Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide system activation and tissue remodeling.

Neuropeptide signaling activates dendritic cell-mediated type 1 immune responses through neurokinin-2 receptor.

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function of NKA-NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuropeptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-γ stimulation significantly induced NK2R mRNA and remarkably enhanced surface protein expression levels of bone marrow-derived DCs. In addition, the DC-mediated NKA production level was significantly elevated after IFN-γ stimulation in vivo and in vitro. We found that NKA treatment induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC class II and promoted Ag-specific IL-2 production by CD4(+) T cells after NKA stimulation. Furthermore, blockade of NK2R by an antagonist significantly suppressed IFN-γ production by both CD4(+) T and CD8(+) T cells stimulated with the Ag-loaded DCs. Finally, we confirmed that stimulation with IFN-γ or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific T cell responses via activation of DC function, suggesting that the NKA-NK2R cascade would be a promising target in chronic inflammation caused by excessive type 1-dominant immunity.

IFN-γ elevates airway hyper-responsiveness via up-regulation of neurokinin A/neurokinin-2 receptor signaling in a severe asthma model.

The adoptive transfer of OVA-specific Th1 cells into WT mice followed by OVA inhalation induces a significant elevation of airway hyper-responsiveness (AHR) with neutrophilia but not mucus hypersecretion. Here, we demonstrate that the airway inflammation model, pathogenically characterized as severe asthma, was partly mimicked by i.n. administration of IFN-γ. The administration of IFN-γ instead of Th1 cells caused AHR elevation but not neutrophilia, and remarkably induced neurokinin-2 receptor (NK2R) expression along with neurokinin A (NKA) production in the lung. To evaluate whether NKA/NK2R was involved in airway inflammation, we first investigated the role of NKA/NK2R-signaling in airway smooth muscle cells (ASMCs) in vitro. NK2R mRNA expression was significantly augmented in tracheal tube-derived ASMCs of WT mice but not STAT-1(-/-) mice after stimulation with IFN-γ. In addition, methacholine-mediated Ca(2+) influx into the ASMCs was significantly reduced in the presence of NK2R antagonist. Moreover, the NK2R antagonist strongly inhibited IFN-γ-dependent AHR elevation in vivo. Thus, these results demonstrated that IFN-γ directly acts on ASMCs to elevate AHR via the NKA/NK2R-signaling cascade. Our present findings suggested that NK2R-mediated neuro-immuno crosstalk would be a promising target for developing novel drugs in Th1-cell-mediated airway inflammation, including severe asthma.

NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1(-/-) mouse ileum.

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1(-/-) mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1(-/-) mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1(-/-) mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1-knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1-associated proteins.

Influence of viral infection on the development of nasal hypersensitivity.

BACKGROUND: The underlying relationship between viral infections and allergic diseases of the upper respiratory tract has not been well clarified. METHODS: In order to clarify the relationship between viral infection and nasal hypersensitivity, mice were sensitized with ovalbumin (OVA) and then infected intranasally with respiratory syncytial virus (RSV), after which their nasal sensitivity to histamine or antigen was examined. RESULTS: Non-sensitized mice showed transient mild nasal hypersensitivity following nasal administration of histamine after intranasal RSV inoculation. In mice sensitized with OVA, RSV infection significantly exaggerated their nasal hypersensitivity to histamine and OVA. Treatment of these mice with a neurokinin (NK)-1/NK-2 receptor antagonist, but not with anti-IL-5 antibodies, reduced their hypersensitivity. The infiltration of nasal mucosa with eosinophils was temporarily associated with accelerated rate of RSV elimination in these animals. CONCLUSION: RSV infection induced transient nasal hypersensitivity. Several mechanisms, including impairment of nasal epithelial cells are thought to mediate this effect. In allergen-sensitized mice, RSV inoculation strongly enhanced nasal hypersensitivity.

Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice.

BACKGROUND: A wealth of clinical observation has suggested that stress and asthma morbidity are associated. We have previously established a mouse model of stress-exacerbated allergic airway inflammation, which reflects major clinical findings. OBJECTIVE: The aim of the current study was to investigate the role of the neurokinin- (NK-)1 receptor in the mediation of stress effects in allergic airway inflammation. METHODS: BALB/c mice were systemically sensitized with ovalbumin (OVA) on assay days 1, 14, and 21 and repeatedly challenged with OVA aerosol on days 26 and 27. Sound stress was applied to the animals for 24 hours, starting with the first airway challenge. Additionally, one group of stressed and one group of nonstressed mice received the highly specific NK-1 receptor antagonist RP 67580. Bronchoalveolar lavage fluid was obtained, and cell numbers and differentiation were determined. Airway hyperreactivity was measured in vitro by electrical field stimulation of tracheal smooth-muscle elements. RESULTS: Application of stress in sensitized and challenged animals resulted in a significant increase in leukocyte number in the bronchoalveolar lavage fluid. Furthermore, stressed animals showed enhanced airway reactivity. The increase of inflammatory cells and airway reactivity was blocked by treatment of animals with the NK-1 receptor antagonist. CONCLUSION: These data indicate that the NK-1 receptor plays an important role in mediating stress effects in allergen-induced airway inflammation.

The role of neural inflammation in asthma and chronic obstructive pulmonary disease.

The tachykinins substance P and neurokinin A are found within airway nerves and immune cells. They have various effects on the airways that can contribute to the changes observed in asthma and chronic obstructive pulmonary disease. Both tachykinin NK(1) and NK(2) receptors have been involved in the bronchoconstriction and the proinflammatory changes induced by substance P and neurokinin A. Tachykinin NK(1) and NK(2) receptor antagonists have activity in various animal models of allergic asthma and chronic bronchitis. It is suggested that dual NK(1)/NK(2) and triple NK(1)/NK(2)/NK(3) tachykinin receptor antagonists have potential in the treatment of obstructive airway diseases.

Antibody-mediated gastrointestinal dysmotility in scleroderma.

BACKGROUND & AIMS: Defects in enteric excitatory neurotransmission have been proposed to underlie the gastrointestinal dysmotility associated with scleroderma (systemic sclerosis). This study investigated whether patients with scleroderma produce antibodies that inhibit M3-muscarinic or neurokinin receptor-mediated intestinal contractions, either directly or via an effect on L-type voltage-gated calcium channels (VGCCs). METHODS: Responses of mouse colon longitudinal muscle to stimulation by the muscarinic agonist carbachol (1-300 micromol/L) and neurokinin-1 and -2 receptor agonists were measured in the absence and presence of serum (2%) or immunoglobulin G (IgG) (0.3-1.0 mg/mL) from patients with scleroderma, those with other autoimmune disorders, and healthy controls. The role of L-type VGCCs in carbachol- and tachykinin-evoked contractions was assessed using nicardipine. RESULTS: M3-muscarinic receptor-mediated contractions were inhibited by Ig fractions from 7 of 9 patients with scleroderma (limited and diffuse forms), 4 of 4 patients with primary Sjögren's syndrome, and 3 of 3 patients with secondary Sjögren's syndrome. Ig fractions from healthy controls did not inhibit the M3-muscarinic receptor-mediated contractions. Inhibition by Ig was concentration-dependent; a maximum inhibition of approximately 40% occurred at 0.6 mg/mL IgG. Both M3-muscarinic and neurokinin receptor-mediated contractions were L-type VGCC dependent. Patient sera had no effect on responses to neurokinin receptor stimulation, demonstrating the lack of antibodies inhibiting L-type VGCCs. CONCLUSIONS: Functional antibodies specifically inhibiting M3-muscarinic receptor-mediated enteric cholinergic neurotransmission may provide a pathogenic mechanism for the gastrointestinal dysfunction seen in patients with scleroderma.

Spinal NK2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation.

The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK2 receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 (1.4-14 nmol) dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK2 receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation.

Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastrointestinal tract.

Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.

Endogenous tachykinins play a role in IL-1-induced neutrophil accumulation: involvement of NK-1 receptors.

Pretreatment of mice with capsaicin resulted in approximately 40% inhibition of the polymorphonuclear leucocyte (PMN) influx elicited by interleukin-1 (IL-1) injected into a murine air-pouch. This inhibition was mimicked by two selective antagonists of neurokinin-1 (NK-1) tachykinin (TK) receptors, i.e. CP-96,345 and RP-67,580, but not by the inactive enantiomer RP-68,651. A selective NK-2 antagonist, SR-48,968, was inactive. The natural peptide, substance P (SP), and a selective NK-1 agonist, (Sar9)SP, induced PMN infiltration into the murine air-pouch, whereas a selective NK-2 agonist, (beta Ala8)NK-A(4-10), was ineffective. Moreover, SP-induced PMN accumulation was prevented by co-administration of RP-67,580 and CP-96,345, but not by RP-68,651. These findings suggest that the release of an endogenous TK, possibly SP, may occur following IL-1 injection in vivo, indicating a contributory role for neuropeptides in the PMN migration elicited by this cytokine. The action of selective agonists and antagonists suggests the involvement of NK-1 receptors.