Endothelin (ET) is a peptide hormone composed of 21 amino acids which was originally found in the culture supernatant of porcine endothelial cells.　There are three subtypes of human ET, ET-1, ET-2, and ET-3, encoded by the gene family in distinct chromosomal locations.　Among these subtypes, ET-1 is produced by endothelial cells and well known as a potent vasoconstrictor.　Besides this function, ET-1 shows various functions such as cell proliferation and chemotaxis.　Additionally, ET-1 is known to have many pathophysiological roles in various diseases including cardiovascular disorders and tumors.　Based on these findings, many kinds of antagonists for blocking ET receptor-mediated cell signaling have been developed and applied to human medicine.

　Many reports indicate that ET-1 is up-regulated and mediates inflammatory reactions in the lesional tissues caused by various types of allergic diseases. For example, in human patients with asthma, ET-1 immunoreactivity was detected in bronchial epithelium and elevated ET-1 levels were found in bronchoalveolar lavage fluid and peripheral blood.　Moreover, in a mouse model of allergic lung inflammation, ET receptor antagonists were shown to be effective to prevent granulocyte infiltration into the lung.　However, it has not been clearly understood whether ET-1 has a pathophysiological role in allergic dermatitis such as atopic dermatitis and food hypersensitivity.　Therefore, a series of the present studies were carried out to understand the pathophysiological roles of ET-1, endothelin receptor A (ETA), and endothelin receptor B (ETB) in canine allergic dermatitis.

　In Chapter I, as a first step to elucidate the function of the ET system in dogs, full-length cDNAs of canine ET receptors were cloned and sequenced.　As s result, high sequence similarities of the canine ET receptors to the counterparts of mouse, rat, and human were shown.　Additionally, the expression analysis revealed that ET-1, ETA, and ETB genes were ubiquitously expressed in various organs in the dog as in the case of other mammals (Fig.1).　These results suggest that ET system has a wide range of functions in the dog as shown in the mouse, rat, and human.

　Next, in the Chapter II, plasma ET-1 concentrations and the cutaneous mRNA expression levels of ET-1, ETA, and ETB were measured in dogs with allergic dermatitis.　As the results, plasma ET-1 level was significantly lower in dogs with allergic dermatitis than healthy dogs (Fig.2), however, PPET-1 expression level was not statistically different among the non-lesional and lesional skin samples in dogs with allergic dermatitis and the skin sample from healthy dogs (Fig.3).　Meanwhile, mRNA expression levels of ET receptors were significantly lower in the lesional skins than normal skins (Fig.3).　These results indicated that ET system was down-regulated in the skin of dogs with allergic dermatitis.　Its difference from the phenomenon of the up-regulation in the allergic bronchitis in human and mouse might be due to the phase of the disease because most of the dogs with allergic dermatitis were in the chronic phase.

　Lastly, in Chapter III, to interpret the physiological role of ET system in allergic dermatitis, a study was focused on the chemotactic activity of ET-1 in the dog. Several studies have revealed the roles of ET-1 in leukocyte accumulation into the inflamed tissues.　For the chemotaxis assay, a novel assay system allowing real-time observation of the movement of the cells (KK-chamber system) was employed in this study.　I found that blood-derived neutrophils showed chemotaxis to ET-1, however, other leukocytes such as monocytes and lymphocytes did not, suggesting that ET-1-mediated chemotaxis was almost specific to neutrophils among canine leukocytes (Fig.4).　The chemotaxis of canine neutrophils was shown to be mediated by receptors other than ETA and ETB, because ET receptor antagonists for selectively blocking ETA or ETB did not eliminate the cell migration (Fig.5).　Previous studies indicated that neutrophil adhesion to vascular endothelial cells was mediated by enhancing the expression of adhesion molecules via ET receptor signaling.　These findings indicate that ET system provides a comprehensive support to the neutrophil infiltration into inflammatory site.　

　In Chapter II, I found that decreased ET-1 concentration in the plasma and decreased ET receptor expression in the lesional skin in dogs with allergic dermatitis.　Considering the chemotactic activity of ET-1 to canine neutrophils as shown in Chapter III, the down-regulation of ET system may affect the neutrophil recruitment into the lesional skin of dogs with allergic dermatitis.　Interestingly, it is known that accumulation of neutrophils does not occur in the lesional skin of dogs with allergic dermatitis, meanwhile, increase of neutrophils in the bronchial or bronchoalveolar tissues has been described in human asthmatic patients.　These findings suggest the presence of different mechanism on controlling the neutrophil infiltration between allergic dermatitis and allergic bronchitis.　This difference could be resulted from variation in the immune reaction and cytokine production from the cells between the two diseases.　The present studies revealed the association of ET system with the pathophysiology in canine allergic dermatitis and will provide a novel therapeutic approach to allergic diseases which are now one of the most problematic diseases in dogs as well as humans.

Fig.1. Reverse transcription (RT)- polymerase chain reaction (PCR) for detection of the expression of preproendothelin-1 (PPET-1), ETA, and ETB mRNAs in normal tissues and blood of dogs ( lane 1, skin; lane 2, thymus; lane 3, lung; lane 4, heart; lane 5, liver; lane 6, pancreas; lane 7, spleen; lane 8, adrenal grand; lane 9, kidney; lane 10, testis; lane 11, uterus; lane 12, bladder; lane 13, stomach; lane 14, duodenum; lane 15, colon; lane 16, mesenteric lymph node; and lane 17, blood).

Fig.2.　Plasma ET-1 concentration in dogs with allergic dermatitis in comparison to healthy dogs and dogs with congestive heart failure.　The mean values of each group are indicated by crossbars.　AD: atopic dermatitis, FH: food hypersensitivity, CHF: congestive heart failure.　 *P < 0.05 vs. healthy dogs.

Fig.3.　Relative expression of PPET-1 (a), ETA (b), and ETB (c) mRNAs in the lesional and non-lesional skin samples from dogs with allergic dermatitis and normal skin samples from healthy dogs.　The mean values of each group are indicated by crossbars.　*P < 0.05 vs. normal skin.　(**)P =0.001 vs. normal skin.

Fig.4. Chemotaxis of canine neutrophils to ET-1.　The number of migrated cells were counted by KK-chamber system in the presence of a serially diluted ET-1.　The average values from the results in 3 dogs are plotted.

Fig.5. Effects of ET receptor antagonists on the chemotaxis of canine neutrophils to ET-1. Ratios of the migrated cell number in a presence of 4 kinds of ET receptor antagonists (BQ-123, BQ-610, BQ-788, BQ-701-1) to that without treatment of the antagonists are shown.　The average values of the results in 3 dogs are indicated.

　本研究は、犬においてエンドセリン-1(ET-1)とアレルギー性皮膚炎の関連を明らかにすることを目的として、一連の解析を行ったものである。

第一章:犬のエンドセリン受容体のクローニングおよび正常組織での発現解析

　犬のエンドセリン受容体(ET受容体)の2つのサブタイプであるETAおよびETBの遺伝子クローニングを行い、ET-1の前駆体であるプレプロエンドセリン-1(PPET-1)、ETA、およびETBの正常組織における発現を逆転写ポリメラーゼ連鎖反応(RT-PCR)によって解析した。クローニングした犬のET受容体遺伝子の塩基配列は、他の哺乳類の相同遺伝子との間で高い相同性を示し、ヒト、マウスおよびラットとのアミノ酸残基の相同性はそれぞれ、ETAの場合には94.1、92.0、および 91.6 %、ETBの場合には97.3、88.3、および87.4 %であった。RT-PCRの結果においては、PPET-1、ETA、およびETBが多くの犬正常組織(皮膚、胸腺、肺、膵臓、脾臓、副腎、腎臓、精巣、子宮、膀胱、胃、十二指腸、結腸、およびリンパ節)にて発現していることが確認された。以上より、ET-1は犬でも他の哺乳類と同様に多様な機能を持ち得ることが示唆された。

第二章:犬のアレルギー性皮膚炎における血漿中ET-1濃度およびET-1とET受容体の病変部での発現

　アレルギー性皮膚炎と診断された犬19頭において、その血漿中ET-1濃度を酵素免疫測定法(ELISA)にて測定し、さらに病変部でのPPET-1、ETA、ETB mRNA発現量を定量的ポリメラーゼ連鎖反応(定量PCR)によって相対定量した。血漿中ET-1濃度は、アレルギー性皮膚炎症例犬では0.55〜1.31(平均値0.92)pg/ml、正常犬では0.22〜1.77(平均値0.66)pg/mlであり、症例群の平均値が正常犬のそれよりも有意に低い結果となった(p <0.05)。一方、定量PCRの結果、PPET-1 mRNAの発現量において正常皮膚サンプル群と病変部皮膚サンプル群との間で有意な差は認められなかった。また、ETAおよびETB mRNAの発現量は病変部皮膚サンプル群において、正常皮膚サンプル群よりも有意に低いことが明らかになった(p <0.005)。喘息の場合、病変部ではET-1の発現が増強し、その組織中ET受容体の発現量および分布は健常と比較して変化がないことが報告され、さらにET-1がその病態形成に重要であることが判明している。しかしながら、本章の結果より、アレルギー性皮膚炎においてはET-1の作用が明らかに低下していることが示され、これが喘息との間の病態の違いを反映していると考えられた。

第三章:ET-1の犬の好中球に対する遊走能の解析

　犬の白血球のET-1に対する遊走能およびそれを媒介するET受容体サブタイプの解析をKK-チャンバー法による遊走実験を用いて行った。正常犬3頭より採取した末梢血から比重遠心法により末梢血単核球を分取し、また沈殿法により好中球を分取した。遊走実験の結果、好中球においてET-1に対する強い遊走能が認められ、その遊走細胞数はET-1の濃度依存的に増加した。また、ETB特異的アゴニストに対しても濃度依存的な遊走反応が認められた。しかしながら、ETAおよびETBのそれぞれに特異的な競合阻害物質であるアンタゴニストで処理した好中球および無処置の好中球を用いて遊走実験を行った結果、両者のET-1に対する遊走能に有意な差を認めなかった。このことから、ET-1に対する好中球の遊走がETAおよびETB以外の未知の受容体を介して起っている可能性が示唆された。ヒトの喘息の病変部には重度の好中球浸潤が見られる一方で、アレルギー性皮膚炎の病変部皮膚では好中球の浸潤がほとんどないことが一般的に認められている。本章の結果により好中球の浸潤にET-1が重要であることが示唆されたことから、第二章で認められたアレルギー性皮膚炎の病変部におけるET-1およびET受容体の発現低下は、本疾患と喘息の間での病態の違い、特に好中球浸潤の程度の差異と関連しているものと考えられた。

　今回の一連の研究は、ET-1、ETA、およびETBの発現と、ET-1の遊走因子としての機能を解析することにより、犬におけるアレルギー性皮膚炎の病態とET-1との関連を明らかにしたものであり、他のアレルギー疾患と本疾患との病態生理学的な差異を説明する新しい知見を提示し得たものと考える。

　本申請論文を審査した結果、審査委員一同は、博士(獣医学)の学位を授与するに値すると判断した。