学位論文内容の要旨

博士の専攻分野の名称

博士（医 学）氏 名 小林 稔

学 位 論 文 題 名Studies on pathogenic and regulatory mechanisms of immune-related diseases.
(免疫関連疾患の発症および制御機構の解明に関する研究)

Background and Objectives Bronchial asthma, typically recognized as helper T (Th) 2 cell-mediatedairway inflammation, is pathogenically characterized by airway hyperresponsiveness (AHR),
eosinophilic airway inflammation, mucus hyperproduction in airway epithelium, and elevated serum
levels of IgE. Numerous chemicals and medicines against immunological cells have been researchedand developed for clinical use in asthma. However, patients suffering from asthma occasionallydevelop severe neutrophilia in the lung and show steroid resistance, which then results in “severeasthma”. It has been reported that neutrophil infiltration in the lung was not observed in Th2cell-associated airway inflammation models. Thus, the precise mechanisms of severe asthma are lessunderstood compared with those of Th2 cell-mediated airway inflammation. Previously, Th1- andTh2-mediated airway inflammation models were established by the adoptive cell transfer ofovalbumin (OVA)-specific Th1 or Th2 cells followed by OVA inhalation. In contrast to Th2 cells, Th1cells induce strong AHR concomitantly with neutrophilia in the lung but without mucus
hypersecretion. Therefore, the Th1 cell-mediated airway inflammation model appeared to be suitablefor characterizing the pathology of severe asthma, since little has been done to elucidate how Th1 cells
induce elevation of AHR.
Tachykinins such as substance P (SP) and neurokinin A (NKA) are located in the excitatory
non-adrenergic and non-cholinergic (NANC) nerves of the mammalian respiratory tract. Excitation ofthese nerves results in the release of tachykinins, which may be involved in the pathogenesis of airway
allergy in humans. Because those factors are produced in the airway tissues during inflammatory
responses, tachykinin receptor antagonists might become good targets for developing therapeutic
drugs for the treatment of allergic inflammation. However, the precise role of NKA/neurokinin-2
receptor (NK2R) signaling has not yet been elucidated, though SP signaling through tachykinin
neurokinin-1 receptor was demonstrated to be crucial for the induction of neutrophilia in the lung andAHR elevation. Generally, SP secreted by neurons in response to local tissue damage, is capable ofinducing and augmenting many inflammatory responses. Recent papers indicated that SP regulated
function of various leukocytes. On the other hand, NKA has known to control various vital responses
such as airway contraction, vasodilatation, and vascular permeability in human. However the functionof NKA on involvement in immune system has less defined than that of SP.
In the present work, we established a novel AHR induction model by intranasal (i.n.)
administration of IFN-γ and investigated the critical role of IFN-γ and NKA in Th1 cell-mediatedairway inflammation model. Furthermore, we confirmed the effect of NKA/NK2R signaling on
immune system.
Methods In order to elucidate the role of IFN-γ, we examined pathogenesis of the Th1 cell-dependentairway inflammation after treatment with neutralizing mAb against IFN-γ. To determine the preciserole of IFN-γ in the elevation of AHR, we directly administered i.n. IFN-γ to wild-type BALB/c miceconsecutive 3 days. At 24 h after the IFN-γ administration, pulmonary function was evaluated. To
confirm the direct effects of IFN-γ on the airway component cells such as airway smooth muscle cells(ASMCs), we prepared mouse ASMCs from airway tracheal and examined their mRNA levels with
IFN-γ stimulations. To investigate whether NK2R-mediated signaling cascade was involved in thesubsequent airway responsiveness in vitro and in vivo, we monitored calcium influx (Ca2+)i in ASMCs
after the β-methacholine chloride (Mch) stimulation with or without NK2R antagonist and evaluatedwhether NK2R antagonist inhibited AHR elevation induced by i.n. IFN-γ administration. Next,
CD11c-DTR Tg mice were i.n. injected with diphtheria toxin (DT), and then IFN-γ was further
injected into the mice to elucidate whether CD11c+ cells were related with the elevation of NKA level.
To address the effect of neuropeptide signaling on dendritic cell (DC)-mediated immune responses,
we further investigated expression levels of NKA and NK2R of GM-CSF-induced bonemarrow-derived DCs (BMDCs) in Type-1 immune condition and expression level of cytokines inNKA-stimulated DCs. In order to evaluate NK2R-mediated NKA stimulation on DC functions, wetransduced NK2R gene into DCs by retrovirus infection system and evaluated surface MHC class II
expression level after NKA stimulation. Then, we co-cultured OT-2 CD4+ T cells with Mock- orNK2R-transduced DCs in the presence of OT-2 peptides. To confirm the effect of NKA-NK2Rsignaling on antigen-specific T cell responses, we cultured spleen cells obtained from OT-2 or OT-1mice in the presence of NK2R antagonist. Finally, we administrated i.n. NK2R antagonist in Th1
cell-mediated asthma model for evaluating whether inhibition of NK2R signaling reduced
IFN--dependent AHR elevation.
Results We found here that Th1 cell-induced AHR elevation was mimicked by i.n. administration ofIFN-γ. IFN-γ directly induced NK2R expression and NKA production in the lung. Then, theIFN-γ-induced elevation of AHR was significantly inhibited by specific antagonist of NK2R in ourmodel. We confirmed that one of neurokinin producing cells was CD11c+ cells in lung and IFN-γ
stimulated BMDC produced NKA. NKA-NK2R signaling cascade activates DC-mediated immune
responses in vitro. Finally, we demonstarated that AHR was significantly inhibited by specificantagonist of NK2R in the Th1 cell-mediated airway inflammation model.
Discussion In the present study, we clearly demonstrated that NKA, one of neuropeptides which weregenerally located in the sensory nerves of mammalian respiratory tract and produced after excitationof the nerves as well as SP, would be involved in the elevation of AHR induced by severe asthma. It
might be possible to consider that severe symptoms of patients suffering from severe asthma are due
to excess activation of antigen-specific Th1 immunity and subsequent IFN--mediated augmentationof NKA/NK2R signaling, which causes the increase of Ca2+-dependent contraction of airway trachealsmooth muscle cells. Thus, our established IFN-γ-induced airway inflammation model will become auseful tool to elucidate the pathogenesis of severe asthma and to develop therapeutic drugs forIFN-γ-induced inflammatory diseases via NKA/NK2R signaling.
Conclusion Previous reports demonstrated that IFN-γ secreted by Th1 cells was the critical mediator
for the induction of AHR, however, the precise mechanism of AHR induction by IFN-γ remained
unclear. In the present study, we first established IFN-γ-mediated AHR elevation model, which wasinduced only i.n. administration of IFN-. Then, we found that IFN-γ activated NKA/NK2R signalingcascade, which was important for IFN-γ-induced AHR elevation. Furthermore, we demonstrated thatNK2R-dependent neuropeptide signaling enhanced DC function and subsequent T-cell immune
responses. Our present findings suggested that NK2R-mediated neuro-immuno cross-talk would be a
promising target for developing novel drugs in Th1 cell-mediated chronic inflammation, including
severe asthma.