Corresponding author: J. Johansson, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, the Biomedical Center, P.O. Box 575, SE-751 23 Uppsala, Sweden; e-mail: email@example.com.
Background: Canine pulmonary fibrosis (CPF) occurs most commonly in West Highland White Terriers. The differing incidences of CPF among dog breeds suggest that genetic factors contribute to its pathophysiology. Pulmonary fibrosis in humans is associated with mutations in the gene coding for lung surfactant protein C (SP-C) (SFTPC).
Hypothesis/Objectives: To investigate the histopathologic changes and SP-C composition and genetic structure in dogs with CPF.
Animals: Five dogs with PF, 2 dogs with other lung diseases, and 3 healthy dogs.
Methods: Lung tissue from dogs with clinically suspected CPF and 5 control cases was analyzed histopathologically. Bronchoalveolar lavage fluid (BALF) collected postmortem from 3 terriers with histopathologically confirmed pulmonary fibrosis and the 5 controls were analyzed by Western blots, and the exons of SFTPC were sequenced for 2 dogs with PF and 1 dog with other lung disease.
Results: SP-C could not be detected in BALF of 1 dog with PF, although SP-B was present. A mutation was detected in SFTPC exon 5 of this dog. From 2 dogs with PF and in all 5 control dogs SP-B and SP-C were detected in BALF.
Conclusions: Taken together, the results indicate that canine and human lung fibrosis share histopathologic features and that analysis of SP-C and its gene in a larger set of dogs with PF is warranted.
Interstitial lung disease (ILD) in humans includes a variety of chronic lung disorders of different etiologies, the majority of unknown origin.1 Any fibrosing lung disease without known cause was formerly named idiopathic pulmonary fibrosis (IPF). Owing to the narrow definition of IPF and the unclear resemblance of the disease between dogs and humans, we prefer to use the more general term canine pulmonary fibrosis (CPF) when referring to the idiopathic disease studied herein.
Chronic interstitial pulmonary fibrosis has long been observed in dogs.2 The existence of a specific clinical entity with chronic pulmonary fibrosis in a certain breed of terrier dog, West Highland White Terrier, suggests that the condition might be analogous to IPF in humans.3
Lung surfactant is a mixture of mainly phospholipids and 4 specific surfactant proteins (SP), SP-A, SP-B, SP-C, and SP-D, which is necessary for reducing the surface tension at the alveolar air-liquid interface, thereby preventing alveolar collapse at low lung volumes. SP-A and SP-D are large water soluble proteins that are implicated in lung host defense.4 SP-B and SP-C, on the other hand, are integrated with phospholipids and affect the surface properties of surfactant, as they are secreted with phospholipid lamellar bodies (the intracellular storage form of surfactant) into the alveolar space. Both SP-B and SP-C are required for preventing small airway collapse at end expiration.5
Recently familial forms of ILD in humans have been associated with different mutations in the SP-C gene (SFTPC).6 SP-C is a very hydrophobic protein, with a highly conserved sequence between species, including humans and dogs.7 SP-C is important for normal surfactant function and is exclusively produced in type II alveolar cells of the lung.8 The mutations found in association with ILD are mostly localized to the C-terminal domain of the SP-C precursor (proSP-C), and they can result in retention of the protein in the endoplasmic reticulum, misfolding into cytotoxic aggregates and lack of detectable SP-C in the alveoli.9,10 Herein we investigated pulmonary fibrosis in dogs with respect to lung histopathology, SP-B and SP-C occurrence in bronchoalveolar lavage fluid (BALF), and SFTPC sequence with the aim to compare canine and human pulmonary fibrosis.
Materials and Methods
The study was performed with permission of the Swedish animal ethical committee (no. C 192/4) and the Swedish animal welfare agency (no. 30-2407/04).
Five dogs with clinical signs of chronic progressive lung disease and with histopathologic diagnosis of CPF (CPF1–CPF5; 4 West Highland White Terriers and 1 Tibetan Terrier, 1 male, 4 female, age 11–12 years) and 5 controls (C1–C5; West Highland White Terrier, Rhodesian Ridgeback, Labrador Retriever, Tibetan Spaniel, Norwich Terrier, 2 males, 3 females, age 7–14 years) were included in the study. Three of the control dogs (C1–C3) had no signs or history of respiratory disease, according to case history, physical examination, and blood analysis and were euthanized because of intervertebral disc prolaps (C1), malignant melanoma (C2), or osteoarthritis (C3). The other 2 control dogs (C4 and C5) had diseases that affect the lungs, 1 with myxomatous mitral valve degeneration and pulmonary edema (C4) and 1 with acute arterial pulmonary thrombosis (C5). All 7 dogs with CPF or signs of dyspnea (C4 and C5) were treated pharmacologically, most of them with a combination of bronchodilators, systemic steroids and occasionally with antibiotics, furosemide, or both. Histopathologic examination of postmortem specimens of the lungs was performed on all 10 dogs. BALF was available from 8 out of 10 dogs, the exceptions being dogs CPF3 and CPF4, who were retrospectively collected cases. SP-C gene analysis was performed for 2 of the dogs with clinical and histopathologically verified CPF (CPF1 and 2), and for 1 control with pulmonary thrombosis (C5).
The 7 dogs with signs of respiratory disease were diagnosed by thoracic radiographs and/or histopathology at the University Animal Hospital, Swedish University of Agricultural Sciences. A hematologic profile consisting of CBC and serum biochemistry profile in combination with urine analysis were performed for diagnostic purposes and were within reference ranges unless stated otherwise. All blood samples were first analyzed automatically in a Cell-Dyn 3500a and subsequently blood smears were manually checked under a light microscope. Radiographs of the thorax were performed on all CPF cases (CPF1–CPF5) and 1 control (C4) and assessed by board certified radiologists. No thorax radiographs were taken on dog C5 since it was euthanized on arrival to the hospital. When appropriate, additional screening with blood gas analysis, cardiac and abdominal ultrasonography, and ECG was performed.
Postmortem tissue specimens from lung were fixated in neutral-buffered 10% formalin, embedded in paraffin, sectioned, and placed on glass slides. The sections were stained with hematoxylin and eosin, van Gieson stain, and when applicable also with Masson's Trichrome and periodic acid Schiff (PAS).
BALF was sampled within 24 hours postmortem by instilling 200–300 mL of isotone, sterile sodium chloride via an endotracheal tube and after aspiration. The BALF was analyzed for the presence of SP-B and SP-C, and for phospholipid contents by phosphorous analysis. No cytology was performed on the BALF samples, because of possible postmortem changes.
For analysis of phosphorous contents, BALF samples were centrifuged at 15,800 ×g for 2 hours at 4 °C in order to spin down surfactant phospholipid vesicles and the pellets were dissolved in 50 mM Tris, pH 7, by ultrasonication for 1 hour. The concentration of phospholipids in BAL samples was calculated from the phosphorous contents determined by lipid digestion in 70% perchloric acid.11
For analysis of SP-C and SP-B, equal surfactant amounts (50 μg of phospholipids) were resolved by SDS-PAGE in 16.5% acrylamide gels and electrophoretically transferred to a PVDF membrane in 20 mM Tris, 150 mM glycine, 0.05% SDS, and 20% methanol. After the transfer, the membrane was blocked for 1 hour in 5% skimmed milk and washed for 10 minutes with 0.1% PBS-Tween and 3 × for 10 minutes with PBS. Immunodetection of SP-C was made with rabbit antiserum against recombinant SP-C12,b at a titer of 1 : 100. For detection of SP-B an anti-human SP-B rabbit antibody (provided by Prof. Timothy E. Weaver, Cincinnati, USA), which cross-reacts with SP-B from all species analyzed was used at a titer of 1 : 15,000. This was followed by incubation with anti-rabbit immunoglobulin conjugated with horseradish peroxidase at a titer of 1 : 5,000. An enhanced chemiluminescence kitc was used to visualize the bands of SP-C and SP-B. Previous analysis of calf surfactant showed that no degradation of SP-C could be detected in BALF collected 24 hours postmortem.13
Sequence Analysis of SFTPC
EDTA blood was collected and DNA was either extracted immediately or the blood sample was frozen and stored at −20°C until further analysis. Genomic DNA was prepared from the blood samples using a kit.d PCR was performed for amplification of the SP-C gene using a forward primer upstream from exon 1, 5′-tgtcccctctccctaccggc-3′ (starting at position −77) and a reverse primer downstream of exon 5, 5′-gggttccccagttccgggtc-3′ (g.2320) with reference to the 1st base of the canine SP-C gene. The PCR product was separated on a 0.8% agarose gel, and extracted by a gel purification kit.d
The amplified canine SP-C gene was used as template and sequence PCR was performed using the Big Dye Sequencing kit.e Five different primers were used; the 2 described above and forward 5′-agggtgccccgcttgtgcag-3′ (g.661) covering exon 2, 5′-aagagggctgagggtggagg-3′ (g.1173) for exon 3, and 5′-actctcccgggtgcctagcc-3′ (g.1529) for exon 4. The primers were designed from intron sequences adjacent to the respective exon. The sequence data were obtained by using a 310 Genetic Analyser.e
All dogs with CPF had mild to moderate dyspnea, tachypnea, or both with intermittent coughing. Some owners complained about exercise intolerance in the dogs. The most prominent clinical finding was distinctive diffuse inspiratory crackles and most dogs also wheezed. Because of the refractory nature of the disease, all dogs with PF progressed despite treatment, predominately with steroids and bronchodilators, and were euthanized or died between days 0 and 276 after diagnosis. Three of the control dogs (C1–C3) had no signs of respiratory disease clinically, or in the case history. The 2 other dogs (C4 and C5) had tachypnea and mild dyspnea, including intermittent wheezing.
Radiology of dogs with PF revealed moderate to severe interstitial to broncho-interstitial pattern in the lung field, but in all cases parts of the lungs showed normal air filled tissue. One dog with signs of dyspnea not related to CPF (C4) had increased opacity of the lung-field with alveolar to mixed broncho-interstitial pattern. In 2 of the dogs with histopathologically confirmed PF, a suspected right-sided cardiac enlargement could be seen (CPF4 and CPF5). In 1 dog (CPF3), the thoracic radiographs also revealed neurogenic pulmonary edema and inguinal hernia with dislocated and possibly strangulated small bowel loops, which had been caused by external trauma because of a road traffic incidence. The suggested radiologic diagnoses were pulmonary fibrosis, chronic bronchial disease or, for 1 non-CPF dog (C4), chronic pulmonary edema.
Ultrasound and ECG Findings
Both dogs that had evidence of right-sided cardiomegaly on thoracic radiographs (cor pulmonale) underwent echocardiography. On the ECG, 1 dog had sinus arrhythmia with signs of P-pulmonale (high P-wave amplitude) but mean electrical axis (MEA) in the frontal plane within reference range, whereas the other case had sinus arrhythmia with PQRS deflections and MEA within reference range.
Hematology and Biochemical Findings
Hematologic and biochemical profiles were obtained in all cases. No consistent abnormalities were observed and in general the measurements were within the reference ranges. One dog (CPF4) had moderately elevated activities of serum alkaline phosphatase (sALP) (5.8 μkat/L ref: < 5.0). The raised sALP levels were likely induced by exogenous steroids instituted because of dermatologic problems. One dog (CPF3) had a moderate leukocytosis (17.1 × 109 cells/L ref: 5.2–14.1) and mild eosinophilia (2.4 × 109 cells/L ref: 0.1–1.2).
Blood Gas Analysis
Blood gas analysis was carried out in 1 dog (CPF2) with severe respiratory distress. It was hypoxemic with normocapnia and had an alveolar-arterial gradient >20 mmHg, indicating a significant mismatch between ventilation and perfusion.
In the 3 control dogs without signs of respiratory disease (C1–C3), no clinically important findings were recorded in the lungs. In the control dog with myxomatous mitral valve degeneration (C4), findings consistent with chronic pulmonary edema were recorded including a mild interstitial fibrosis and moderate numbers of alveolar macrophages with hemosiderin in the cytoplasm (heart failure cells) in the alveoli. In control dog C5 a thrombus was present in a large artery with areas of necrosis in adjacent lung parenchyma. No signs of interstitial fibrosis were detected in the lung.
In the 5 dogs with PF (CPF1–CPF5), consistent histopathologic findings were multifocal areas with interstitial fibrosis with thickening of the alveolar septa and varying numbers of mononuclear inflammatory cells, mainly lymphocytes (Fig 1a). The extracellular matrix stained strongly with Masson's trichrome confirming presence of collagen (Fig 1b). In case CPF5, marked centers of proliferating fibroblasts were found in addition to severe interstitial fibrosis (Fig 1d). Multifocal areas of hyperplasia and hypertrophy of type II pneumocytes were present in all 5 CPF cases. Abnormally shaped type II pneumocytes, sometimes multinucleated, were present in 3 of the dogs (CPF1, CPF4, and CPF5) (Fig 1b). Vacuolated macrophages were present in the alveoli and multinucleated alveolar macrophages were recorded in 2 dogs (CPF1 and CPF4). In 2 dogs (CPF2 and CPF3) amorphous, strongly PAS-positive material was present in the alveoli (Fig 1c). Areas with apparently normal lung tissue were present in all dogs.
SP-C and SP-B in BALF
Three CPF cases (CPF1, CPF2, and CPF5) and the 5 controls were analyzed for the presence of SP-B and SP-C in BALF by Western blots. In CPF1, no SP-C was found, although immunoreactivity for SP-B was detected (Fig 2). In the other 2 dogs with CPF (CPF2 and CPF5) and the 5 control dogs SP-B and SP-C were detected (Fig 2). SP-B was present as a monomer (9 kDa), dimer (17 kDa) or as oligomers of various sizes (data not shown). For the 2 retrospectively retrieved CPF cases CPF3 and CPF4, BALF was not obtained for SP analysis.
Sequence Analysis of SFTPC Exons
The SP-C gene was amplified and its exons sequenced for 2 CPF cases (CPF1 and CPF2) and for 1 control (C5). This revealed no mutations that influence the expression of SP-C. However, in exon 5, 2 deviations compared with the published sequence14 were found for CPF1 (position 12 G > C), for CPF2 (position 78 T > C), and for C5 (both position 12 G > C and position 78 T > C). These deflections apparently do not correlate to pulmonary disease or presence of SP-C, and may hence be considered polymorphisms.
Even though interstitial pulmonary fibrosis has long been recognized in dogs, the etiology is still unknown. Here we analyzed 5 cases of CPF for histopathologic appearance, 3 of which were also analyzed for SP-B and SP-C contents in BALF and 2 of which were also analyzed for SP-C gene sequence. In the present case series the clinical observations were almost identical to the clinical picture described in the 1st characterization of chronic pulmonary disease in West Highland White Terriers; slowly but progressively exacerbating dyspnea, sometimes accompanied by cor pulmonale.3 In that study 29 West Highland White Terriers with pulmonary fibrosis were studied, but only 4 cases were confirmed by histopathology. In the present study, 5 histopathologically confirmed cases of CPF are analyzed. This principally shows that the number of analyzed confirmed cases is small. This situation calls for larger studies on CPF cases and controls with and without respiratory disease to further elucidate the histopathology and etiology of pulmonary fibrosis.
A histopathologic pattern of usual interstitial pneumonia (UIP) is essential for the diagnosis of IPF in humans.15 The histologic key features of UIP are destruction of the lung architecture with fibrosis and honeycombing, with the most severe lesions present in the subpleural parenchyma.15 Mild interstitial inflammation with lymphocytes and plasma cells, hyperplasia of type II pneumocytes and scattered fibroblastic foci are also present.15 The present study shows that human IPF associated with SFTPC mutations and CPF share several similar histopathologic features, such as diffuse alveolar septal widening by fibrosis, type II pneumocyte hyperplasia, mild interstitial inflammation, vacuolated macrophages in the alveoli, and centers of proliferating fibroblasts. In 2 of the canine cases with PF, prominent PAS-positive material was found in the alveoli, which are features related to alveolar lipo-proteinosis. This has likewise been reported in humans with ILD.16,17 In all the dogs with PF, parts of the lung were normal, according to radiographic and histopathologic examination. This is also true for humans with IPF.16,18 No reliable conclusions can be drawn at this point because only a small number of cases are investigated, but these crude histopathologic similarities warrant the further investigation of dogs with PF in possible development of a model for human IPF/ILD.
In a retrospective study, 6 cases of West Highland White Terriers with pulmonary fibrosis were investigated by light microscopy, ultrastructure analysis, and immunohistochemistry and then compared with human IPF.19 The study suggested that chronic pulmonary disease of West Highland White Terriers is a result of aberrant collagen regulation and thus different to IPF. This might be true; however, the fibrosis could be considered as an end-stage situation of inflammatory response, with still unknown etiology. One of the differences between human and dog lung fibrosis claimed19 is the absence of fibroblast foci in the latter. Herein we, however, detected centers of proliferating fibroblasts (Fig 1d) similar to published histologic pictures of human fibroblast foci.20
Because lung fibrosis is mainly found in terrier breeds and especially frequently reported in West Highland White Terriers,3,21 there is a suspicion that the susceptibility to develop PF has genetic components. With the recent completion of the dog genome sequence,14 we are in an excellent position to perform genetic mapping in predisposed breeds in parallel with clinical studies. Using the dog as a model may lead to further understanding of this complex disease, considering the high degree of conservation between canine and human DNA and protein sequences.14
The dog with pulmonary fibrosis without detectable SP-C and mutation in the SFTPC exons was a Tibetan Terrier. Further studies are required to address the question whether breed related differences are of relevance in the pathogenesis of CPF and whether different mutations can give rise to apparently similar disease phenotypes, analogous to the situation in humans. However, in this dog with PF and without detected SP-C, the SP-B content was normal. It is not likely that SP-C is selectively degraded by the ongoing inflammatory process because of secondary pneumonia. Human cases with ILD and SP-C deficiency in BALF without mutations in the coding sequence of SFTPC have been reported.22,23 The subjects also had reduced, but detectable, levels of SP-A and SP-B in BALF.23 The lack of alveolar SP-C in these cases was not caused by SP-B abnormalities, and remains unexplained. It might be speculated that cases with SP-C deficiency in humans and dogs can be caused by a mutation outside of the coding sequence, for instance a regulatory region, or in any gene important for the processing of proSP-C.
In summary, this study analyses SPs in association with pulmonary fibrosis in dogs. It also describes a robust method for detection of SP-B and SP-C in BALF, which could be used for screening of a larger number of dogs. The present histopathologically confirmed case of CPF without detectable SP-C in the alveoli further calls for additional studies of SPs in dogs with pulmonary fibrosis.
aAbbot Diagnostics, IL
bAltana AG, Konstanz, Germany
cAmersham Biosciences, Uppsala, Sweden
dQiagen, VWR International, Stockholm, Sweden
eABI prism, Stockholm, Sweden
We are grateful to the Swedish West Highland White Terrier club and private dog owners for help in retrieving cases, and to Prof. Tim Weaver for generous gifts of antibodies. This study was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning and AGRIA Pet Insurance Research Foundation.