Pulmonary Langerhans cell histiocytosis in cats and a literature review of feline histiocytic diseases

Abstract

Objectives

The aim of this study was to report the clinical, radiographic and pathological features of pulmonary Langerhans cell histiocytosis in four cats, and carry out a literature review of feline histiocytic diseases.

Methods

Necropsy reports archived at the Department of Veterinary Pathology of the Federal University of Rio Grande do Sul were reviewed. The clinical information was then obtained from the clinical records at the Veterinary Hospital. Routine samples had been collected during necropsy, fixed in 10% formalin, routinely processed for histology, and stained with hematoxylin and eosin. Samples of lung were submitted for bacterial and fungal culture. Tissue sections of lung underwent immunohistochemical testing for vimentin, pancytokeratin, CD18, CD3, CD79αcy, E-cadherin and Iba1.

Results

This disease affected mixed breed cats aged 7–14 years. Clinical signs consisted of severe mixed inspiratory and expiratory restrictive dyspnea, lethargy and anorexia. Thoracic radiographs revealed different lesion profiles, predominantly of an interstitial and alveolar pattern. Grossly, the lungs were diffusely firm and did not collapse. The pleural surface was bright and irregular due to multifocal-to-coalescent, well-demarcated, white, firm nodules that also extended into and obliterated the pulmonary parenchyma. Histological changes were characterized by poorly demarcated infiltration with histiocytic cells arranged in cohesive groups within the alveolar, bronchiolar and bronchial spaces. Histiocytic cells had intense cytoplasmic immunolabeling for vimentin and Iba1, and robust membrane immunolabeling with CD18 and E-cadherin; these cells were negative for CD3, CD79αcy and pancytokeratin in all cases.

Conclusions and relevance

This article confirms that pulmonary Langerhans cell histiocytosis is a rare disease that occurs in middle-aged to older cats and causes widespread involvement of the pulmonary parenchyma, inducing acute or chronic, progressive respiratory disease characterized by mixed restrictive dyspnea that eventually leads to death. While a definitive clinical diagnosis is challenging, the nodular appearance of the pulmonary changes, together with the histological and immunohistochemistry findings, suffice for diagnostic confirmation of pulmonary Langerhans cell histiocytosis.

Introduction

Langerhans cells (LCs) are antigen-presenting dendritic cells (DCs) that originate in the bone marrow and are located in several organs. In the lungs, LCs colonize the bronchial epithelium.^1–3 Histiocytic diseases in cats are uncommon in comparison with dogs, and consist mainly of feline progressive histiocytosis (FPH), histiocytic sarcoma (HS) and pulmonary Langerhans cell histiocytosis (PLCH).^4,5

PLCH is a rare disease of older cats that causes progressive respiratory insufficiency secondary to pulmonary parenchymal infiltration with LCs that express CD18 and E-cadherin.^6–8 In humans, PLCH is an uncommon condition of young adults that is frequently associated with tobacco smoke.^3,9 It is believed to be a reactive process because regression occurs in many cases after treatment and smoking cessation.^10,11 In cats, PLCH is thought to represent a neoplastic process because of cellular morphological characteristics and extra-pulmonary lesions. ⁷ Here we report the clinical, radiographic and pathological features of feline PLCH in four cats, bringing new aspects of a poorly documented disease, and a literature review of feline histiocytic diseases.

Materials and methods

Feline necropsy reports archived at the Department of Veterinary Pathology of the Federal University of Rio Grande do Sul (UFRGS) were reviewed, and suspected PLCH cases were selected. The clinical information was then obtained from the clinical records at the Veterinary Hospital of the UFRGS. Routine samples of multiple organs had been collected during necropsy, fixed in 10% formalin, routinely processed for histology, and stained with hematoxylin and eosin. Samples of lung were kept refrigerated and were submitted to bacterial and fungal culture.

For fungal culture, lung samples were seeded in Sabouraud glucose agar with chloramphenicol and cycloheximide followed by incubation at 26°C for 7 days. For bacterial culture, a sample of lung was inoculated in 5% sheep blood Mueller Hinton agar and in MacConkey agar. The sample was aerobically incubated at 37°C for 72 h. Tissue sections of lung underwent testing by immunohistochemistry (IHC) for vimentin (mouse monoclonal, 1 in 200 overnight [Zymed]), pancytokeratin (human monoclonal, 1 in 80 overnight [Dako]), CD18 (mouse monoclonal, 1 in 10 dilution for 60 mins [Peter Moore, University of California, Davis]), CD3 (rabbit polyclonal, ready-to-use [Dako]), CD79αcy (mouse monoclonal, 1 in 100 dilution overnight [Dako]), E-cadherin (mouse monoclonal, 1 in 500 dilution for 60 mins [BD Biosciences]) and ionized calcium binding adapter molecule 1 (Iba1) (rabbit polyclonal antiserum, 1 in 8000 dilution for 60 mins [Wako]). Positive controls were included simultaneously, which were previously tested (small intestine [vimentin], haired skin [pancytokeratin and E-cadherin], and lymph node [CD18, CD3, CD79αcy and Iba1]).

Results

Clinical and radiology findings

Case 1 was an 8-year-old female spayed mixed breed cat that had a 1 week history of lethargy, anorexia, weight loss and respiratory distress. Physical examination revealed mixed inspiratory and expiratory restrictive dyspnea. Blood work and serum biochemistry were normal. The cat was serologically negative for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Thoracic radiographs revealed a mixed predominantly alveolar lung pattern, characterized by disseminated, nodular opacification in all lung lobes (Figure 1a). Treatment consisted of fluid therapy with sodium lactate Ringer’s solution, aminophylline (5 mg/kg IV q12h), amoxicillin with potassium clavulanate (22 mg/kg SC q12h), tramadol hydrochloride (2 mg/kg SC q12h), fluticasone propionate in the aerosol suspension (one inhalation spray q12h), cobamamide with cyproheptadine (1000 µg/animal PO q12h and 2 mg/animal PO q12h, respectively), oxygen therapy and nebulization with saline solution three times daily. The cat remained intensely dyspneic and died 4 days later.

Figure 1

Radiographic and gross features of feline pulmonary Langerhans cell histiocytosis. (a) Thoracic radiograph, case 1. Opacification of pulmonary fields of mixed pattern, predominantly diffuse alveolar infiltration. (b) Thoracic radiograph, case 2. Nodules dispersed within the pulmonary parenchyma, structured and diffuse interstitial pattern. (c) Thoracic radiograph, case 3. Diffuse increase of the radiopacity of mixed interstitial and alveolar pulmonary pattern. (d) Gross pulmonary changes, case 1. Multifocal-to-coalescent white nodules involve all pulmonary lobes. (e) Gross pulmonary changes, case 2. Multifocal-to-coalescent white nodules exhibiting indurated areas. (f) Gross pulmonary changes, case 3. Multifocal-to-coalescent white nodules in all lung lobes

Case 2 was a 14-year-old male castrated mixed breed cat with a 24 week history of lethargy, weight loss and respiratory distress. Physical examination revealed mixed inspiratory and expiratory restrictive dyspnea. Blood work and serum biochemistry were normal. The cat was serologically negative for FIV and FeLV. Second-hand exposure to tobacco smoke was reported by the owner. Thoracic radiographs revealed multifocal-to-coalescing nodules in the lungs, with a diffuse and structured interstitial pattern (Figure 1b) consistent with neoplasia. No treatment was conducted and the cat died 1 month later.

Case 3 was a 7-year-old female spayed mixed-breed cat with an 8 week history of lethargy, hyporexia, hypodipsia, weight loss and respiratory distress. The cat had received ampicillin and fluticasone via aerosol for 20 days, with no clinical improvement seen. On physical examination, the cat had a fever (40^oC), serous nasal discharge, and mixed inspiratory and expiratory restrictive dyspnea. Blood work and serum biochemistry were normal. The cat was serologically negative for FIV and FeLV. Thoracic radiographs revealed a diffuse increase in radiopacity in an interstitial and alveolar pattern (Figure 1c). Treatment consisted of ampicillin (5 mg/kg IV q24h), acetylcysteine (70 mg/kg IV q8h), ampicillin (20 mg/kg IV q8h), salbutamol sulfate aerosol suspension (one inhalation spray q12h), cobamamide (1000 µg/animal PO q12h) and cyproheptadine (2 mg/animal PO q12h), and oxygen therapy and nebulization with saline solution three times a day. Fine-needle aspiration of the lung was performed under anesthesia with meperidine (5 mg/kg IM) and propofol (1 mg/kg IV dose dependent). After clipping of hair and aseptic preparation with chlorhexidine 2%, a 25 G × 7 mm needle attached to a 10 ml syringe was inserted in to the dorsal intercostal space. Aspiration was performed in pulmonary fields guided by radiography. Personal protective equipment such as gloves, leaded aprons, eyeglasses and thyroid shields were worn. The samples were stained with a Romanowsky stain (Diff-Quick) and visualized by light microscopy. The cytological evaluation was inconclusive owing to low cellularity (formed by a few red blood cells, occasional macrophages in phagocytic activity and rare neutrophils). Clinical signs did not regress and the cat was euthanized.

Case 4 was a 10-year-old male castrated mixed breed cat with a 4 week history of lethargy, hyporexia, hypodipsia, weight loss and respiratory distress. Physical examination revealed mixed restrictive dyspnea. Thoracic radiographs demonstrated mixed diffuse pulmonary radiopacity with interstitial and alveolar patterns. The patient was subjected to oxygen therapy, but died less than 24 h later.

Pathological findings

All cats were submitted to a full necropsy. Grossly, the lungs were diffusely firm and did not collapse. The pleural surface was bright and irregular owing to multifocal-to-coalescent, well-demarcated, white, firm nodules that also extended into and obliterated the pulmonary parenchyma (Figure 1d–f). Similar nodules were also present in the sternal and tracheobronchial lymph nodes, spleen and liver in case 1 and in the kidneys in case 3. Both thyroid glands were enlarged in case 2. No bacterial or fungal growth was yielded on lung samples.

Histologically, pulmonary lesions in all cats were characterized by poorly demarcated infiltration with histiocytic cells arranged in cohesive groups within the alveolar, bronchiolar and bronchial spaces (Figure 2a,b). These histiocytic cells were round-to-slightly elongated and had indistinct borders and a moderate amount of pale, eosinophilic, homogeneous-to-finely-vacuolated cytoplasm. The nuclei were round-to-oval, occasionally eccentric and reniform, and had hyperchromatic chromatin with inconspicuous nucleoli (Figure 2c). There was moderate anisocytosis and anisokaryosis, and one mitosis in each × 400 field. Other findings included marked hypertrophy of the pulmonary septal and arteriolar smooth muscle, hyperplasia of type II pneumocytes, proliferation of fibrous connective tissue, alveolar edema, hemorrhages and scattered inflammatory infiltrates composed of lymphocytes, plasma cells and occasional macrophages. Multiple areas of parenchymal pulmonary necrosis were also seen in case 2. Infiltrates of histiocytic cells in the liver, spleen and sternal and tracheobronchial lymph nodes in case 1, thyroid and parathyroid in case 2 and kidneys in case 3, were similar to that described in the lungs.

Figure 2

Histological and immunohistochemistry findings of feline pulmonary Langerhans cell histiocytosis. (a) Multifocal-to-coalescent infiltration of cells with histiocytic appearance, arranged in cohesive groups occupying bronchioles and alveolar spaces. Hematoxylin and eosin, × 200. (b) Histiocytic cells obliterate the alveolar spaces. Hematoxylin and eosin, × 400. (c) Histiocytic cells are round to slightly elongated, with indistinct borders, moderate amount of pale and eosinophilic cytoplasm, round to oval nuclei, occasionally eccentric and reniform and hyperchromatic chromatin with inconspicuous nucleoli. Hematoxylin and eosin, × 1000. (d) Histiocytic cells exhibit robust membranous immunolabeling with CD18. Immunohistochemistry, 3,3’-diaminobenzidine (DAB), × 400. (e) Histiocytic cells exhibit robust cytoplasmic immunolabeling with E-cadherin. Immunohistochemistry, permanent red, × 400. (f) Histiocytic cells exhibit intense cytoplasmic immunolabeling for ionized calcium binding adapter molecule 1. Immunohistochemistry, DAB, × 400

Histiocytic cells had intense cytoplasmic immunolabeling for vimentin, and robust membrane immunolabeling with CD18 (Figure 2d) and E-cadherin (Figure 2e), and intense cytoplasmic immunolabeling for Iba1 (Figure 2f); these cells were negative for CD3, CD79αcy, and pancytokeratin in all cases.

Literature review

Histiocytic diseases are proliferative disorders of DCs or macrophages that have been documented in humans and other animal species, especially dogs and less often cats.^4,5,6,12 DCs are part of the adaptive immune response, acting as antigen-presenting cells, ⁶ and consist of LCs and interstitial DCs. ¹ Gross and histological changes are often insufficient to confirm the diagnosis of histiocytic diseases, and IHC is a means to identify the cell of origin in such cases.^6,13 Reports of histiocytic diseases of the cat are limited to FPH, HS and PLCH. ⁴

FPH

FPH is the most common histiocytic disease in cats, ¹² occurring in older cats, ranging in age from 2 to 17 years.^6,12–14 It behaves clinically as a low-grade HS, which develops from resident interstitial DCs in the skin. ¹² It is considered a rare disease with no breed predilection and a slight predilection for females.^12,13

Clinically, early FPH is typically characterized by a solitary skin nodule, although multiple nodules, papules or plaques can also develop. Lesions are located mainly on the head, extremities or trunk, and are non-pruritic, non-painful, firm and often alopecic. As the disease progresses, cutaneous lesions tend to coalesce and eventually ulcerate. Lesions can wax and wane, but spontaneous regression has not been reported.^6,12,13 Over time, lesions can become invasive and affect internal organs. ¹² Diagnostic confirmation is based on histology and IHC profile. ¹³

Histologically, lesions consist of diffuse, poorly demarcated histiocytic cell infiltrates in the dermis and subcutaneous tissues;^6,12,13 approximately 40% of cases can display epitheliotropism.^6,12,13 During the initial stages histiocytic cells are well differentiated, but as the disease progresses, cellular pleomorphism, mitotic count and multi-nucleation occurs, and lesions more closely resemble a HS. ⁶ Cells are large and have round-to-polygonal, faintly eosinophilic cytoplasm that occasionally exhibit clear vacuoles. Nuclei are oval and have finely dispersed chromatin, with a small, discrete single nucleolus. A mild-to-moderate degree of anisocytosis and anisokaryosis is observed, and the mitotic count varies, but is usually low.^6,12 Immunohistochemically, histiocytes express CD1, CD18, major histocompatibility complex (MHC II) and Iba1, and E-cadherin negativity.^6,12,13,15

FPH has a poor long-term prognosis as it tends to respond poorly to chemotherapy, ² and treatment measures are not well defined. ⁵ Successful surgical resection depends on the number and location of nodules. ¹² The use of corticosteroids, antibiotics, interferon, nitrogen mustard, vincristine and vinblastine were administered to cats with this disease.^12,14

HS complex

HS arises from cells with the phenotypic profile of interstitial DCs.^6,12 While any tissue can be affected, ⁴ masses are more frequent in the bone marrow, lung, lymph nodes, meninges, periarticular tissues of extremities, skin, nasal cavity and spleen.^5,6,16–19 Hemophagocytic HS is an aggressive malignant neoplasm of macrophage origin that is mainly described in dogs and rarely in cats;^6,20–22 in contrast with HS, hemophagocytic HS originates from macrophages in the splenic red pulp and bone marrow. ⁴

HS affects mainly adult cats,^16,23 ranging in age from 1 to 16 years.^{16–20,24–28} Although no breed predisposition is apparent, most affected cats are domestic shorthairs and this may be an effect in the general population of cats.^{16,20–24,26–28}

HS is a highly aggressive neoplasm that typically metastasizes to lymph nodes or other organs, including kidneys, liver and lungs.^6,16 The clinical signs of HS vary according to the location of the masses.^4,6 Weight loss, lethargy, anemia, thrombocytopenia, splenomegaly and lymphomegaly are common findings in cases of HS in cats.^{16,20,21,23,24,27} Nasal discharge and increased upper respiratory sounds, as well as dyspnea, are signs described in cases of HS with involvement of the respiratory system.^18,19 Seizures were observed in a cat with HS located in the nasal cavity that extended to cranial cavity, ¹⁸ and lameness in a case with involvement of periarticular tissues of the extremities. ¹⁷ There are reports of cats with mediastinal and vertebral canal histiocytic sarcoma,^25,26 with progressive hindlimb ataxia and paresis. ²⁶

It is uncertain whether tumors represent a multicentric disorder or metastatic foci from a primary neoplasm.^6,12 Differentiation between late-stage FPH and primary HS should be based on the clinical history with emphasis on the clinical course. ¹²

Gross lesions are usually white-to-tan, homogeneous masses, but diffuse organ enlargement by infiltrating neoplastic histiocytic cells can also occur.^4,6,16 Histologically, lesions are most frequently composed of sheets of large and pleomorphic cells, with multinucleated giant cells that usually have marked cytological atypia and numerous bizarre mitoses.^4,17 Nuclei are oval to reniform and have prominent nucleoli. ²⁸ Erythrophagocytosis and extramedullary hematopoiesis are frequent features in hemophagocytic HS. ⁴ In IHC, HS can express CD1a, MHC II, CD18, Iba1 and lysozyme.^{12,15–19,27,28} In hemophagocytic HS, macrophages express markers most consistent with macrophage differention.^4,20 Treatment with corticosteroids, antibiotics and vincristine has had limited success.^{16,20–23,25,26}

PLCH

PLCH is a rare disease of aged cats (10–15 years) that causes progressive respiratory insufficiency. ⁶ Clinically, cats with this disorder have tachypnea, increased respiratory effort or open-mouth breathing. ⁴

Gross findings are characterized by multinodular-to-diffuse involvement of all lung lobes.^4,5,7 Histologically, lesions consist of cohesive histiocytic infiltrates, which obliterate terminal airways and extend to pleural surfaces. These cells are moderately pleomorphic and typically stain positive for CD18, E-cadherin and vimentin,^4,5,7 which supports a LC phenotype. ⁷ Bronchodilators, diuretics and corticosteroids are typically administered to cats with PLCH, but these treatments tend to be unsuccessful. ⁷

Discussion

The diagnosis of PLCH in the current cases was suspected based on the clinical and pathological findings, and confirmed based on the IHC profile of the infiltrating histiocytic cells. Although the number of cats included in the present study is small, all cats were mixed breed and older, similar to what has been reported by other researchers. ⁷

All cats had similar clinical signs, consisting mainly of severe respiratory distress, anorexia, weight loss and lethargy, similar to other cases described in the literature.^4,7 Respiratory signs and more specifically restrictive dyspnea in cases of PLCH are caused by the widespread obliteration of alveolar spaces by the intense and characteristic histiocytic infiltrates. ⁷ The clinical course in the current cases ranged from 1 to 28 weeks, reflecting the varied acute or chronic clinical progression reported in cases of PLCH.^4,8

Thoracic radiographs in the present study revealed a mixed pulmonary pattern that was predominantly interstitial and alveolar. That is in contrast with what has been reported in the veterinary literature, where PLCH is mainly associated with a bronchointerstitial pattern.^4,7 In the present study, cytological evaluation of a pulmonary aspirate was not an effective method for diagnostic confirmation of PLCH. There are few reports of feline PLCH, with little information related to treatment. In cases of the present study, supportive therapy was initiated, but no clinical response was observed and the cats died, supporting the progressive nature of feline PLCH.

Gross lesions in cases of PLCH are characterized by infiltrative nodules involving all pulmonary lobes.^5,7 Initially, multifocal nodules correspond histologically to involvement of the peri-bronchial parenchyma but tend to coalesce and involve the pleural surface overtime,^4,5,7 which can explain the variation in radiographic patterns in this study. As described in other cases, minor involvement of lymph nodes, spleen, liver, thyroid and parathyroid gland, and kidney were observed in all the current cases. Histologically, infiltrating histiocytic cells were cohesive, had marked cellular and nuclear pleomorphism, and obliterated the peri-bronchial parenchyma, alveolar septa and alveolar spaces.^4,7 Pulmonary smooth muscle hypertrophy, fibrosis, and scattered lymphocytes and plasma cells were also frequent, as previously reported. ⁷

PLCH in humans has been associated with tobacco smoke, especially in young adults.^9,29 Tobacco smoke supposedly causes damage to the bronchial epithelium, which prompts the release of peptides that stimulate alveolar macrophages to secrete cytokines that activate resident antigen-presenting LCs.^3,29 Genetic and other factors, such as prior treatment with chemotherapeutic agents, may also be involved in the pathogenesis of PLCH.^3,29 The cats in the present study had no history of other lung diseases. Interestingly, one cat (case 2) had been reportedly exposed to tobacco smoke, but the significance of this finding remains elusive. PLCH in humans is classified as a reactive disorder.^10,11 While the morphological features of the histiocytic infiltrates and the presence of extrapulmonary lesions of PLCH may favor a neoplastic rather than a reactive process in cats, molecular studies are lacking and the true underlying mechanism of this disorder remains widely unknown.^4,7

Diagnostic confirmation in cases of PLCH relies on the IHC profile of histiocytic cells.^4,7 In the present study, the cells were strongly positive for vimentin, CD18, Iba1 and E-cadherin, indicative of a LC phenotype.^4,6,7 Iba1 expression in neoplastic cells confirms the histiocytic origin of the tumor.^5,15 The leukointegrin CD18 is strongly expressed on all leukocytes, and has been used as a histiocytic marker, but this is dependent upon exclusion of lymphocyte differentiation ⁵ , as demonstrated in the present cases where the cells were negative for CD3 and CD79αcy. LCs are the only histiocytic cells that express the adhesion molecule E-cadherin, which confirms the diagnosis of PLCH in association with the IHC labeling for Iba1 and CD18.^4,7 FPH is the most frequent histiocytic disease of cats. Lesions are commonly limited to the skin, but visceral involvement has been reported; however, histiocytic cells in cases of FPH do not express E-cadherin, as they do not have a LC phenotype.^12,30

PLCH may share clinical, radiographic, and pathological findings with other pulmonary diseases, such as infectious processes (fungal and bacterial pneumonia), feline asthma and neoplasms. ⁸ No evidence of infection was detected in the present study; in addition, the absence of pancytokeratin in immunolabeling by infiltrating cells excludes the possibility of epithelial neoplasia. ⁸

Conclusions

This article confirms that PLCH occurs in middle-aged to older cats and causes widespread involvement of the pulmonary parenchyma, inducing acute or chronic, progressive respiratory disease characterized by mixed restrictive dyspnea that eventually leads to death. Thoracic radiographs reveal different lung pattern lesion profiles, including a mixed, predominantly interstitial and alveolar pattern. No significant changes in blood counts and biochemical tests were identified. While a definitive clinical diagnosis is challenging, the nodular appearance of the pulmonary changes, together with the histological findings and the use of antibodies specific in IHC for histiocytic lineage, such as CD18 and Iba1, in addition to E-cadherin for LC, is sufficient for diagnostic confirmation of PLCH.