Chronic interstitial pneumonia with features of organizing pneumonia in an adult horse

Abstract

A 22-y-old American Quarter Horse gelding was presented with a history of chronic progressive respiratory problems and a diffuse pulmonary nodular pattern in thoracic radiographs. The horse was euthanized, and 4 formalin-fixed samples of lung were submitted for histopathology. There were multifocal areas of marked thickening of alveolar septa as a result of proliferation of myofibroblasts embedded in fibromyxoid matrix (interpreted as “Masson bodies”), focal areas of fibrosis, and numerous papillary projections of connective tissue into bronchioles. A diagnosis of organizing pneumonia was reached. No etiology was found for this lesion. It is important to consider causes of chronic interstitial pneumonia with fibrosis in horses other than equid herpesvirus 5, such as complicated viral or bacterial pneumonia or chronic toxicoses.

Interstitial lung disease is a generic term that is used to describe damage and/or inflammation of alveolar septa.⁵ The most common presentation of interstitial lung disease is diffuse alveolar damage, which consists of damage to type I pneumocytes and/or endothelial cells. The acute (exudative) phase is characterized by edema, plasma proteins, cellular debris, surfactant, and fibrin (hyaline) membrane formation in alveoli. The subsequent proliferative phase is characterized by type II pneumocyte hyperplasia, with alveolar and interstitial damage and inflammation. If fibrin or damage persist, a chronic (fibrosing) phase may take place, with infiltration of lymphocytes, macrophages, fibroblasts, and myofibroblasts into the fibrinous exudate.^5,11,30 This chronic phase is characterized by the organization of fibrin that is invaded by fibroblasts, organized into fibrous tissue, and is incorporated into the alveolar wall and covered by epithelium, which results in thickening of the alveolar septa.^5,19 Alternatively, fibrous tissue may develop within the alveolar septa as a result of repetitive, persistent, or severe damage to epithelial or endothelial cells.⁵

Among different presentations of interstitial lung disease in humans, “organizing pneumonia” (OP) is described. OP is characterized by multifocal areas of alveolar thickening with fibromyxoid connective tissue, and extension of this tissue into bronchiolar lumens, generating lesions resembling bronchiolitis obliterans.^1,10 We describe herein a case of chronic interstitial pneumonia in a horse, with findings compatible with the cicatricial form of OP.

A 22-y-old American Quarter Horse gelding was presented to the West Coast Equine Hospital (Somis, CA) with a history of chronic progressive respiratory problems. Numerous multifocal-to-coalescing nodules in the pulmonary parenchyma were identified in radiographs of the thorax (Fig. 1). With this finding, a presumptive diagnosis of equine multinodular pulmonary fibrosis (EMPF) was reached, and the owner elected euthanasia. Four formalin-fixed pieces of lung were submitted to the California Animal Health and Food Safety Laboratory in San Bernardino for histopathology. Hematoxylin and eosin (H&E) and Masson trichrome stains were done.

Figure 1.

Lateral thoracic radiographic view of a horse with chronic interstitial pneumonia, with radiodense regions in the caudodorsal and ventral pulmonary parenchyma.

Large numbers of fusiform cells embedded in collagen (interpreted as Masson bodies), together with a mild-to-moderate inflammatory infiltrate of macrophages, lymphocytes, and plasma cells expanded the alveolar septa multifocally and obliterated ~ 80% of the examined Masson sections of lung. Interstitial fusiform cells and collagen extended into the lumens of adjacent bronchioles forming pedunculated papillary-like projections that partially or totally occupied the bronchiolar spaces and alveolar ducts (Fig. 2A, 2B), which were segmentally lined with simple low cuboidal epithelium, resembling bronchiolitis obliterans. Occasionally, fibrotic foci formed randomly arranged coalescing bundles that conferred a reticular and nodular appearance to the affected areas. There were a few random hemorrhagic foci with the presence of hemosiderophages. Interlobular septa were markedly expanded with edema and mild infiltrates of macrophages, lymphocytes, and plasma cells. The histologic findings in this horse are compatible with cicatricial OP, a pulmonary entity in humans reported in 2018.⁸

Figure 2.

Lung histopathology and immunohistochemistry from a horse with chronic interstitial pneumonia. A. Interstitial pulmonary fibrosis and the presence of a papillary-like pedunculated projection of connective tissue (*) that occupies most of the bronchiolar lumen, covered with a monolayer of epithelial cells. H&E. 200×. B. The pulmonary interstitium and the bronchiolar papillary-like pedunculated projection stain blue, indicative of collagen. Masson trichrome stain. 200×. C. Immunoreactivity for smooth muscle actin is present in areas of pulmonary fibrosis and within the intra-bronchiolar papillary-like pedunculated projection. Immunohistochemistry. Hematoxylin counterstain. 200×. D. Immunoreactivity for pancytokeratin is present in epithelial cells lining the alveolar spaces and covering the intra-bronchiolar papillary-like pedunculated projection. Immunohistochemistry. Hematoxylin counterstain. 200×.

In order to better characterize the lesion, immunohistochemistry was performed for pancytokeratin (panCK) and smooth muscle actin (SMA) on lung sections following the standard procedures of the Veterinary Teaching Hospital of the School of Veterinary Medicine, UC-Davis. Briefly, antigen retrieval was performed (Target retrieval solution S1699, pH 6 for panCK; S2368, pH 9 for SMA; Dako) in a steamer (Black & Decker) for 30 min for panCK or 20 min for SMA at 95°C, followed by a 20-min cool down. The following primary mouse antibodies were applied to tissue sections: mouse anti-panCK (Clone LU5, dilution 1:100; BioCare) and mouse anti-SMA (MU128-UC, dilution 1:300; BioGenex). Goat anti-mouse immunoglobulins (Mouse K4001; Dako) conjugated to peroxidase-labeled dextran polymer (Envision+ system-HRP; Dako) were used as a secondary reagent. All reactions were visualized with NovaRed for peroxidase (SK-4800; Vector Laboratories). Sections were counterstained with Mayer hematoxylin and coverslipped.

Alveolar connective tissue and the projections into bronchiolar lumens were densely populated with fusiform cells that were positive for SMA (Fig. 2C). Cells lining bronchi, bronchioles, and alveoli were positive to panCK (Fig. 2D). Paraffin scrolls of the lung were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University (Lansing, MI) for equid herpesvirus 5 (EHV-5; Equid gammaherpesvirus 5) PCR, with negative results. With these results, a diagnosis of chronic bronchointerstitial pneumonia with features of OP was reached, with proliferation of myofibroblasts (Masson bodies),^7,10,20 and mild multifocal lymphohistiocytic infiltrates.

OP was described in humans in 1983 and was classified as pneumonia as a result of major damage to alveolar walls. Nevertheless, when the involvement of terminal bronchioles was identified, it was described as bronchiolitis obliterans organizing pneumonia (BOOP).¹⁰ Today, it is called OP to avoid confusion.^1,10 The distinguishing histologic lesion of OP is the proliferation of fibromyxoid connective tissue containing fibroblasts and myofibroblasts (Masson bodies)^11,12 within alveolar ducts and sacs, with extension to bronchiolar lumens. These findings are similar in all cases, independent of the radiographic pattern of the cases.^3,20

The term OP has also been used as a pathologic entity that may have a different clinical significance, having been classified as primary (idiopathic or cryptogenic), secondary, and reactive.¹ The difference between primary and secondary OP is based on the determination of one or more etiologies that could have contributed to the development of the lesion.^6,22,25 Among the most common causes of secondary OP are autoimmune diseases that affect collagen, adverse inflammatory reaction to certain drugs, infections, tumors, and radiation.^1,6,24 It is believed that the involved mechanisms have in common diffuse alveolar epithelial damage.⁶ Reactive OP has minor clinical significance and accompanies other preexisting pulmonary diseases, such as neoplastic foci or granulomas in the pulmonary parenchyma.¹ Cicatricial OP has also been described, in which granulation tissue organizes to become much denser fibrous tissue but retains the pattern of OP.⁸ We speculate that our case falls under cicatricial OP, given the more organized areas of interstitial fibrosis, in addition to fibromyxoid tissue in alveolar septa and the presence of bronchiolar polyps.

In dogs, the OP pattern has been identified as an idiopathic lung disease and has been associated with accidental intra-airway exposure of oleic acid.^17,18,21,23 In cats, OP is reported as idiopathic; in pigs, this pattern has been described with porcine circovirus 2 infection.^7,21 In mice, a transgenic model overexpressing CC chemokine ligand-2 under control of surfactant protein C promoter in type II pneumocytes responded to infectious challenge with Streptococcus pneumoniae with lesions characteristic of OP, with an inflammatory profile similar to that observed in humans.¹⁶

In horses, the main causes of acute interstitial pneumonia are viral infections, such as infection by influenza A virus or EHV-1 (Equid alphaherpesvirus 1).^5,19 When cases are not complicated with secondary bacterial infection, lesions are self-limiting, and the fibrotic stage is seen rarely.¹⁹ However, chronic interstitial lung diseases have been observed as sequelae to complicated bacterial bronchopneumonia or acute interstitial pneumonia, exposure to environmental contaminants such as silicates in cases of pneumoconiosis, exposure to toxic agents, and in chronic viral infections.^5,19 The most prominent chronic viral infection in the horse is EHV-5 infection, a condition known as EMPF.²⁷

Toxic plants associated with chronic interstitial pneumonia include the consumption of Eupatorium adenophorum or Ageratina adenophora. A. adenophora is commonly known as “sticky snakeroot” or “crofton weed” in the United States; E. adenophorum is limited to Australia.^4,5 Lesions include diffuse pulmonary edema, emphysema, proliferation of alveolar epithelial cells, and interstitial fibrosis. Lesions can be present multifocally or diffusely in the pulmonary parenchyma.^4,5 The pathogenesis of this intoxication involves the toxin 9-oxo-10,11-dehydroagerophone. The site of action depends on the affected species, which in horses is the lungs.⁴ Our horse was from Ventura County, which is between Santa Barbara and Los Angeles counties, both of which report the presence of A. adenophora (Invasive plant atlas of the United States, https://www.invasiveplantatlas.org/subject.html?sub=4543). Despite this horse being a likely candidate for exposure to this specific plant, there is no way to corroborate exposure. The toxic species of the genus Crotalaria do not occur in the geographic region in which this horse resided, making Crotalaria spp. an unlikely source of toxicosis; no sections of liver were submitted for histopathology, so exposure remains speculative.

EMPF is expressed clinically in adult horses as dyspnea, exercise intolerance, progressive weight loss, and fever.^2,27 Thoracic radiographs have demonstrated an interstitial-to-multinodular pulmonary pattern, which corresponds to white-to-gray coalescing pulmonary nodules of up 10 cm diameter.² Nodules are distributed throughout the pulmonary parenchyma with predominance along the dorsal aspect.^27,30 Histologically, the nodules are well delimited from the normal parenchyma and are composed of marked interstitial expansion of mature collagen and a mononuclear cellular infiltrate. In addition, there is marked hyperplasia of type II pneumocytes, presence of neutrophils and macrophages in alveolar spaces, and occasional intranuclear eosinophilic inclusion bodies in macrophages.^14,27,28,30 The causative agent for EMPF supported with the most scientific evidence is EHV-5.^14,26-28,30 There is a historic association between gammaherpesviruses and pulmonary fibrosis in animals and humans.^13,26

The presentation of multifocal fibrosis in cases of chronic interstitial pneumonia in horses is not always synonymous with EMPF. The morphologic diagnosis in our case (diffuse chronic interstitial pneumonia with Masson bodies) is consistent with OP, which is supported by the features revealed with H&E staining: 1) cellular infiltrates in the pulmonary parenchyma composed of fusiform cells and collagen fibers with mononuclear cell inflammation, 2) Masson bodies obliterating alveolar lumens in affected areas, and 3) the well-demarcated areas of hypercellularity that are separate from the unaffected pulmonary parenchyma.

Our case differs from EMPF because it lacked several histologic features that are common in EMPF: 1) in the hypercellular areas in EMPF, there is dense and predominantly neutrophilic infiltrate in alveolar spaces, 2) the interstitial connective tissue does not extend into the bronchiolar lumen forming Masson bodies, 3) there usually are intrahistiocytic eosinophilic nuclear inclusion bodies, 4) affected areas are well demarcated with diffuse and severe thickening of alveolar septa, and 5) there is marked hyperplasia of type II pneumocytes.

Myofibroblasts have a very important role in progressive pulmonary fibrosis because of their continuous production of extracellular matrix and cytokines.²⁹ The presence of myofibroblasts and connective tissue obliterating the alveolar spaces is a feature that defines OP of humans.³ The extension of this tissue into bronchiolar lumens is a common finding in these cases.^3,6 The presence of myofibroblasts has also been detected in cases of EMPF, in both natural and experimental infections.²⁸ Therefore, the detection of myofibroblasts with immunohistochemistry is not a feature that can be used to differentiate these entities. The main difference lies on the dominant distribution of myofibroblasts and connective tissue. In EMPF, nodules with myofibroblasts and collagen are located predominantly in the pulmonary interstitium, diffusely thickening the alveolar septa,²⁸ whereas in cases of OP, the nodules with myofibroblasts and connective tissue extend and obliterate alveolar lumens (Masson bodies), frequently extending into the lumens of bronchioles.^1,3,10

The term bronchiolitis obliterans (BO) has different interpretations in human medicine and veterinary medicine. In general, the term BO has been used by human pathologists to refer to the presence of polypoid projections of granulation or fibrous tissue into the lumen of bronchioles, with narrowing and obliteration of lumens.^9,13 In human medicine, luminal granulation tissue polyps extending from the adjacent parenchyma with a restrictive effect, which are not associated with a fibrosing lung disease, are regarded as part of OP (previously known as BOOP).^1,3,10 The presence of dense granulation tissue between the bronchiolar epithelium and muscular layer, without extending into the adjacent parenchyma, is now regarded as constrictive bronchiolitis (previously known as “obliterative bronchiolitis”).⁹ In constrictive bronchiolitis, granulation tissue is much denser (frequently collagenized) than seen in OP, and typically becomes re-epithelialized^9,10; fibrosis of the bronchiolar wall causes external compression and reduces the diameter of the lumen, a lesion not commonly identified in animals.⁵ In addition, a BO syndrome, with proliferation of fibromyxoid granulation tissue, is specifically described in humans as a result of chronic rejection in lung-transplant individuals.¹⁵

In veterinary medicine, the term BO is used to describe fibrous polyps occluding bronchiolar lumens and is commonly identified in lesions of chronic bronchopneumonia in cattle, as a consequence of viral-, bacterial-. or neutrophil-mediated epithelial damage,^5,13 and is occasionally used to describe acute lesions in which neutrophils occlude the airflow.⁵ The term BOOP has been used as a common microscopic finding in animals with unresolved bronchopneumonia, in which there is alveolar fibrosis and organized exudate that becomes attached in the bronchiolar lumen,¹³ similar to the description of organizing bacterial pneumonia (classified as secondary OP or BOOP) in humans.¹⁰

Although the radiographic findings in this horse were highly suggestive of EMPF, histologic features differed substantially, and there was no molecular evidence of EHV-5 infection. These findings highlight the importance of following up such cases with an autopsy and histopathology, having in mind other infectious and non-infectious causes of chronic interstitial pneumonia in horses.