幼猫增生性坏死性耳炎（PNOE）：首次证实为T细胞介导的细...

王帆

Proliferative and necrotising otitis in a kitten: first demonstration of T-cell-mediated apoptosis

幼猫增生性坏死性耳炎：首次证实为T细胞介导的细胞调亡

翻译：唐翔

Otitis externa in cats is relatively uncommon. This report describes a case of a rare, visually distinctive, proliferative and necrotising otitis in a three-month-old Persian kitten. The cat had proliferative, erythematous and necrotic tissue covering most of the proximal pinnae and vertical ear canals. On histopathological examination, the most striking feature was the existence of scattered apoptotic appearing keratinocytes within severely hyperplastic epithelium. For the first time, immunohistochemistry was used to show a closed association between CD3+ T cells and caspase-3 stained keratinocytes, consistent with a keratinocyte apoptosis by epidermal infiltrating T cells. Treatment was initiated using topical tacrolimus twice daily and an ear cleanser once daily. A marked improvement was observed after 10 days of treatment and the lesions completely resolved over a period of three weeks. The origin of T cells directed against keratinocytes is currently unknown.

猫外耳炎相对少见。本报告描述了一例3月龄的波斯幼猫罕见的，视觉上独特的增生坏死性耳炎。猫耳廓近端和垂直耳道大部分有增生、红斑和坏死组织覆盖。在组织病理学检查中，最显著的特征是在严重增生的上皮内存在散在的凋亡的角质形成细胞。免疫组织化学首次显示CD3 + T细胞和caspase-3染色的角质形成细胞之间存在密切的关联，与表皮浸润T细胞引起的角质形成细胞凋亡一致。开始使用外用他克莫司每日两次和耳部清洁剂每日一次进行治疗。治疗10天后观察到明显改善，病变在3周内完全消退。目前尚不清楚直接针对角质形成细胞的T细胞的来源。

INTRODUCTION

引言

Otitis externa in cats is relatively uncommon when compared with the dog and is most often associated with ear mite infestation, inflammatory polyps, ear canal neoplasia and underlying allergic conditions. A unique proliferative and necrotising otitis has been recently described in kittens (Gross and others 2005; Mauldin and others 2007). Herein, the authors document a similar otitis in a kitten, in which the histopathological features, of immunohistochemical staining and response to treatment are consistent with a keratinocyte apoptosis by epidermal-infiltrating T cells.

与犬相比，猫的外耳炎相对不常见，并且通常与耳螨感染、炎性息肉、耳道肿瘤和潜在的过敏性疾病相关。最近在幼猫中描述了一种独特的增生性坏死性耳炎。在此，作者记录了幼猫的类似耳炎，其中组织病理学特征、免疫组织化学染色结果和治疗反应与表皮浸润T细胞引起的角质形成细胞凋亡一致。

CASE HISTORY

病史

A three-month-old female Persian kitten was presented for evaluation of unusual proliferative lesions in both ear canals. The cat lived indoor with several others cats, and none presented dermatologic signs. Lesions were first noticed by the owner 15 days before the consultation and progressed very rapidly. The cat was treated only with a topical ear cleanser.

一只3月龄的雌性波斯猫被就诊原因是双侧耳道不寻常的增生性病变。这只猫与其他几只猫一起住在室内，其他猫没有出现皮肤症状。咨询前15天畜主首次注意到病变，进展非常迅速。猫仅接受局部洗耳剂治疗。

On examination, the cat was in good health. The only physical abnormalities found related to both ear canals (Fig 1). The cat had proliferative, erythematous, verrucous and necrotic tissue covering most of the proximal concave pinna and vertical ear canal of both ears. The tissue was friable and bled easily. Gentle manipulation of the tissue exposed underlying erosions and ulcers. Friable material from the plaques and a thick exudate with a foul odour occluded the ear canals. Several dark brown crusts that were trapped in the hair coat were observed in the preauricular region of the face. Otoscopic examination was not possible. Lesions had developed very rapidly and had coalesced. The cat appeared indifferent to the lesions.

查体：猫身体健康。发现的唯一异常与双侧耳道有关（图1）。猫有增生性、红斑性、疣状和坏死组织覆盖双耳近端耳廓凹面和大部分垂直耳道。组织易碎，容易出血。轻柔操作暴露出下方糜烂和溃疡组织。来自斑块的易碎物质和具有恶臭的厚渗出物堵塞了耳道。在面部耳前区观察到数处深棕色结痂，嵌入被毛中。无法进行耳镜检查。病变发展非常迅速，并已融合。猫对病变表现无不适感。

Based on the history and the visually distinctive clinical features, a presumptive diagnosis of proliferative and necrotising otitis was made. Ear cytology showed entire neutrophils and macrophages intermingled with some acantholytic cells and numerous exclusively extracellular cocci and rods. Bacterial culture from the ear canal revealed bacteria: Pasteurella multocida, Staphylococcus epidermidis and a Bacillus species. Cytological and bacteriological findings were consistent with bacterial colonisation rather than infection.

根据病史和视觉上独特的临床特征，推定诊断为增生性坏死性耳炎。耳部细胞学检查整个显示为中性粒细胞和巨噬细胞与一些棘层松解细胞和大量仅细胞外球菌和杆菌混合。耳道细菌培养显示细菌：多杀巴斯德菌、表皮葡萄球菌和芽孢杆菌属。细胞学和细菌学结果与细菌定殖一致，而不是感染。

Two 6-mm skin punch biopsy specimens were obtained from each ear under general anaesthesia. The samples were fixed in 10% neutral buffered formalin, routinely processed, sectioned at 5 µm and stained with haematoxylin and eosin.

在全身麻醉下从每只耳朵采集两个6 mm皮肤活检样本。将样本固定在10%中性缓冲福尔马林中，常规处理，以5µm切片，并用苏木精和伊红染色。

Histopathologic examination revealed a parakeratotic hyperkeratosis with intermingled neutrophilic crusts. The epidermis was acanthotic and spongiotic. There was a marked papillomatous hyperplasia of the epidermis and of the outer root sheath of hair follicles (Fig 2). Numerous individually shrunken and hypereosinophilic keratinocytes with pyknosis ( apoptotic cells) were observed at all levels of the epidermis and outer root sheath of hair follicles (Fig 3). Apoptosis became focally confluent, leading to superficial necrosis. A marked lymphocytic exocytosis was present in all levels of the epidermis and, most often, lymphocytes closely surrounded the affected keratinocytes (satellitosis). The superficial dermis was oedematous and the vessels were dilated. The dermis had perivascular to diffuse inflammatory infiltrate, which consisted mainly of lymphocytes, and small numbers of mastocytes, macrophages, plasma cells and neutrophils.

组织病理学检查显示角化不全性角化过度伴混杂的中性粒细胞性结痂。表皮棘层肥厚，海绵状。表皮和毛囊外根鞘有明显的乳头状增生（图2）。在表皮和毛囊外根鞘的各层均观察到许多单独皱缩和嗜酸性过多的角质形成细胞伴固缩（凋亡细胞）（图3）。细胞凋亡变得局灶性融合，导致浅表坏死。表皮的各层平均存在明显的淋巴细胞胞吐作用，最常见的是，淋巴细胞紧密包围受累的角质形成细胞（卫星现象）。真皮浅层水肿，血管扩张。真皮出现血管周围至弥漫性炎性浸润，主要包括淋巴细胞以及少量肥大细胞、巨噬细胞、浆细胞和中性粒细胞。

Immunohistochemical staining was performed on formalin-fixed, paraffinembedded tissue for CD3 and active caspase-3. Briefly, formalin-fixed, paraffin-embedded tissue was sectioned at 4 µm, mounted and deparaffinised. The sections were incubated with rabbit antihuman CD3 (rabbit polyclonal antibody, N1580, Dako Corporation, diluted 1:200) and rabbit antihuman cleaved caspase-3 (rabbit monoclonal antibody, clone 9662, Cell signaling, diluted 1:400). They were stained using a standardised strepavidin-biotin immunoperoxidase technique and diaminobenzidine as the chromogen. Immunohistochemistry revealed that the infiltrating lymphocytes were predominantly CD3+T cells (Fig 4). Immunostaining for active caspase-3 showed cytoplasmic and occasional nuclear staining in most of the keratinocytes with morphology consistent with apoptosis (Fig 5).

对福尔马林固定、石蜡包埋的组织进行CD3和活性caspase-3的免疫组织化学染色。简言之，福尔马林固定、石蜡包埋的组织以4µm切片，封片并脱蜡。用兔抗人CD3（和兔抗人cleaved caspase-3（兔单克隆抗体，克隆9662，细胞信号传导，1:400稀释）孵育切片。采用标准化链霉亲和素-生物素免疫过氧化物酶技术染色，二氨基联苯胺为显色剂。免疫组织化学显示浸润淋巴细胞主要为CD3 + T细胞（图4）。活性caspase-3免疫染色显示，在大多数角质形成细胞中存在细胞质和偶见细胞核染色，其形态与细胞凋亡一致（图5）。

Based on clinical and histopathological features, a diagnosis of proliferative and necrotising otitis was made.

根据临床和组织病理学特征，诊断为增生性坏死性耳炎。

Treatment was initiated using topical 0.03% tacrolimus ointment (Protopic

0.03%; Astellas Pharma) twice daily and an ear cleanser once daily (Epi-Otic; Virbac). A marked improvement was observed after 10 days of treatment with an 80% decrease in the overall lesional area and proliferative tissue. The lesions in both ear canals completely resolved over a period of three weeks (Fig 6). At this time, otoscopic examination was normal. Treatment was continued for a further two weeks once daily, then every other day for two weeks and after which it was stopped. There was no recurrence of otitis or the proliferative plaques with a follow-up of 1.5 year.

开始局部使用0.03%他克莫司软膏每日两次和洗耳剂每日一次，清洗后使用耳药使用进行治疗。治疗10天后观察到明显改善，总体病变面积和增生组织减少80%。双侧耳道病变在3周内完全消退（图6）。此时耳镜检查正常。继续治疗2周，每日一次，然后隔日一次，持续2周，之后停药。随访1.5年无耳炎复发及增生性斑块。

DISCUSSION

讨论

A unique proliferative and necrotising otitis has been described in kittens (Gross and others 2005; Mauldin and others 2007). The disease occurs classically in young cats and is visually distinctive. The rapid appearance of a proliferative, friable and necrotic tissue covering pinnae and ear canals in a kitten, that appears indifferent to the lesions and is otherwise clinically healthy, is very suggestive of this entity.The lesions could spontaneously regress in 12 to 24 months (Gross and others 2005) but it is not always the case (Mauldin and others 2007). In our case, a favourable and rapid response was observed following the topical application of tacrolimus.

在幼猫中描述了一种独特的增生性坏死性耳炎。该病典型地发生于幼猫，视觉上具有独特性。在幼猫中迅速出现覆盖耳廓和耳道的增生、脆弱和坏死的组织，与病变无关，在其他方面临床健康，非常提示该疾病。病变可在12-24个月内自发消退，但并不总是如此。在我们的病例中，他克莫司外部给药后观察到有利且快速的反应。

Similar to the previous reports, on histopathological examination, the most striking feature is the existence of scattered apoptotic-appearing keratinocytes within severely hyperplastic epidermis and superficial follicular epithelium. Luminal folliculitis is another common finding.  Superficial and luminal pustulation and eosinophilic epidermal microabscesses have been occasionally reported but it was not observed in the present case.

与以前的报道相似，在组织病理学检查中，最显著的特征是在严重增生的表皮和浅表毛囊上皮内存在散在的凋亡样角质形成细胞。毛囊腔毛囊炎是另一种常见结果。表皮和毛囊腔脓疱及嗜酸性表皮微脓肿偶有报道，但在本病例中未见。

Currently, the pathogenesis of the disease is not well characterised. Gross and others (2005) suggested that brightly eosinophilic and shrunken keratinocytes could represent a dyskeratotic rather than an apoptotic process. In the current case, an immunohistochemistry was performed with antihuman activated caspase-3 antibody. Caspases(i.e. cysteinyl- aspartatecleaving proteases), proteins belonging to the cysteine protease family, are cardinal molecules in the apoptotic process. They are synthetised as inactive pro-enzymes and, when activated, can cleave a broad range of cellular targets and ultimately cause apoptosis. In human beings, detection of activated caspase-3 is an important mean of identifying apoptotic cells in tissue sections. The technique seems to be more specific that the “TUNEL” method commonly used to reveal apoptosis. It facilitates its use to differentiate between necrosis and apoptosis and to detect apoptotic cells even before morphological changes take place (Walker and Quirke 2001). Feline caspase-3, a 278-amino acid protein, is very similar to the human form sharing 87% homology (Yamazaki and others 2004). Active caspase-3 has been measured in the cat by flow cytometric detection of cells stained with antihuman activated caspase-3 antibody (Natoni and others 2006). Moreover, immunohistochemistry with this antibody has been shown to be a sensitive and specific method to identify apoptotic cells in the testes of cats (Jewgenow and others 2009). To the knowledge of the authors, skin immunohistochemistry using this antibody has never been performed in the cat. In our case, cytoplasmic and occasional nuclear staining for activated caspase-3 was observed in most of the keratinocytes with morphology consistent with apoptosis. Some of these cells were closely surrounded with CD3+ T cells, a phenomenon known as satellitosis. These observations are consistent with an induction of keratinocyte apoptosis by epidermal-infiltrating T cells. In our case, the favourable and rapid response observed following the topical application of tacrolimus, that is a calcineurin inhibitor known to inhibit T-cell-mediated keratinocytes apoptosis (Trautmann and others 2001), supports the hypothesis of T-cell aetiology.

目前，该疾病的发病机制尚未确定。Gross等(2005)认为明显的嗜酸性和皱缩角质形成细胞可能代表角化不良而不是凋亡过程。在当前病例中，用抗人活化caspase-3抗体进行免疫组织化学检查。半胱天冬酶（即半胱氨酰-天冬氨酸分离蛋白酶）是属于半胱氨酸蛋白酶家族的蛋白质，是凋亡过程中的主要分子。它们被合成为无活性的前酶，当被激活时，可以针对广泛的细胞靶标，并最终引起细胞凋亡。在人类中，检测活化的caspase-3是识别组织切片中凋亡细胞的重要手段。该技术似乎比常用于揭示细胞凋亡的“TUNEL”方法更具体。它有助于区分坏死和凋亡，甚至在发生形态学变化之前检测凋亡细胞。猫caspase-3是一种由278个氨基酸组成的蛋白，与人形式非常相似，同源性为87%。通过流式细胞术检测抗人活化caspase-3抗体染色的细胞，在猫中测定了活性caspase-3。此外，已证明该抗体的免疫组织化学是鉴别猫睾丸中凋亡细胞的灵敏和特异性方法。据作者所知，从未在猫中进行过使用该抗体的皮肤免疫组织化学。在我们的病例中，在大多数角质形成细胞中观察到活化caspase-3的细胞质和偶见细胞核染色，形态与细胞凋亡一致。其中一些细胞被CD3 + T细胞紧密包围，这种现象被称为卫星现象。这些观察结果与表皮浸润T细胞诱导角质形成细胞凋亡一致。在我们的案例中，他克莫司（一种已知抑制T细胞介导的角质形成细胞凋亡的钙调磷酸酶抑制剂）外用给药后观察到的有利且快速的反应，支持T细胞病因学假设。

However, the reason for the generation of the T cells is still unknown. Appearance of cytotoxic lymphocytes could be associated with numerous factors including drug administration, infection, neoplasia, connective tissue disease and autoimmunity. A viral origin has been suspected in the previous described cases. Interestingly, calicivirus has been shown to induce apoptosis in cultured cells by activating capsase-3 (Natoni and others 2006), and papillomavirus infection is commonly responsible for a papillomatous hyperplasia of the epidermis. However, there is currently no evidence to link this syndrome to any virus. Polymerase chain reaction (Gross and others 2005) and immunohistochemical staining (Mauldin and others 2007) did not reveal evidence of herpesvirus, calicivirus or papillomavirus infection. It is, however, possible that a virus may have triggered the reaction without persistence of the antigen or detection of the antigen with immunoperoxidase staining. An example of this is the papillomavirus-associated feline Bowenoid carcinoma in situ. The welldeveloped Bowenoid lesions may have negative immunoperoxidase staining, but lesions that are throught to be precursors (viral plaques) are more likely to have positive staining for papillomavirus antigen (Wilhelm and others 2006). Possible spontaneous remission is in favour of a viral origin that could be spontaneously cleared in some cats.

然而，T细胞生成的原因仍不清楚。淋巴细胞毒性的出现可能与许多因素相关，包括给药、感染、肿瘤、结缔组织疾病和自身免疫。在先前描述的病例中怀疑为病毒来源。有趣的是，已证明杯状病毒通过激活capsase-3诱导细胞凋亡，乳头瘤病毒感染通常导致表皮乳头瘤样增生。然而，目前还没有证据将这种综合征与任何病毒联系起来。聚合酶链反应和免疫组织化学染色未发现疱疹病毒、杯状病毒或乳头瘤病毒感染的证据。然而，病毒可能在抗原不持续存在或免疫过氧化物酶染色未检测到抗原的情况下触发反应。例如乳头瘤病毒相关的猫鲍温样原位癌。发育良好的鲍温样病变可能具有阴性免疫过氧化物酶染色，但作为前体（病毒斑块）的病变更可能具有阳性乳头瘤病毒抗原染色。可能的自发缓解支持在一些猫中可自发清除的病毒来源。

In conclusion, the demonstration of a T-cell-induced keratinocyte apoptosis gives new insights about the pathogenesis of the disease. Further studies are needed to characterise the target of lymphocytes.

总之，T细胞诱导的角质形成细胞凋亡的证实为该病的发病机制提供了新的见解。需要进一步的研究来表征淋巴细胞的靶标。

FIG 1. Left ear before treatment

图1 治疗前的左耳。

FIG 2. Severe papillomatous hyperplasia of the epidermis and external root sheath of hair follicle (H&E, x 50)

图2.表皮和毛囊外根鞘重度乳头状增生(H&E，x 50)

FIG 3. Apoptotic keratinocytes (arrows) (H&E, ×200)

图3.凋亡的角质形成细胞（箭头）(H&E，×200)

FIG 4. Immunohistochemistry with CD3. Note the numerous CD3+ T cell within severely hyperplastic epidermis (×100)

图4. CD3免疫组织化学。注意重度增生的表皮内有大量CD3 + T细胞(×100)

FIG 5. Immunohistochemistry with anticaspase 3 antibody. Note the cytoplasmic and nuclear staining for activated caspase-3 in most of the keratinocytes with a morphology consistent with apoptosis (×100)

图5.抗caspase-3抗体的免疫组织化学。注意大多数角质形成细胞中活化caspase-3的细胞质和细胞核染色，其形态与细胞凋亡一致(×100)

FIG 6. Left ear after 20 days of treatment

图6 治疗20天后的左耳。

笨笨

仁心仁术

Urumqi冬