Idiopathic pneumonia syndrome is a rare complication following hematopoietic stem cell transplantation (HSCT), defined by diffuse lung injury with no identified etiology, with an incidence of 2–12% . It accounts for approximately 12% of paediatric post-HSCT pulmonary complications and most patients rapidly succumb to the disease . Idiopathic pneumonia syndrome is a diagnosis of exclusion; bronchoalveolar lavage and ideally lung biopsy should be performed to rule out bacterial, viral, fungal, or non-infectious causes. Clinical presentation is variable but includes respiratory distress, non-productive cough and hypoxemia . Currently, no effective treatment exists, although disease severity is reduced in patients receiving cyclosporine and intravenous immunoglobulin as graft- vs. host disease (GvHD) prophylaxis .
The pathogenesis is multi-factorial, with toxic bystander-effects of conditioning, GvHD and occult infection implicated as risk factors . In murine models, endothelial cell activation and injury is a factor in the development of idiopathic pneumonia syndrome , and can be demonstrated histologically by endothelial leak of fibrin, endothelial apoptosis, veno-occlusive disease, venous embolization, and fibrin deposition and evidence of pulmonary hypertension, including pulmonary vascular intimal hyper-proliferation [3,4,5,6].
Endothelial cell activation is classified as Type I (immediate) or Type II (delayed), which causes de novo synthesis of proteins including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which are expressed on cell membranes . The production of endothelial nitric oxide synthase 3 (eNOS) is up-regulated during endothelial cell injury, most commonly due to sheer stress, which causes the release of nitric oxide in order to dilate blood vessels [7, 8].
Historically, few lung biopsies have been performed on our post-HSCT patients with idiopathic pneumonia syndrome. This is because the procedure is inherently risky on small sick patients on mechanical ventilation support. Furthermore, interpretation of the histological pattern has been challenging, often with no underlying disease process described. We retrospectively examined lung biopsies of patients with unexplained respiratory symptoms and signs in our primary immunodeficiency paediatric HSCT cohort, specifically looking for evidence of endothelial cell activation and injury.
Primary immunodeficiency paediatric patients transplanted between January 2000 and July 2015 in whom post-HSCT lung biopsies had been performed were identified from our departmental primary immunodeficiency and pathology databases. Medical notes were reviewed and tissue samples re-examined for evidence of endothelial cell activation and injury.
Formalin fixed and paraffin embedded tissue sections were stained using hematoxylin and eosin according to standard protocols. Additional material was stained using Ventana Benchmark autostainers with UltraView detection: ICAM-1 (ab109361), VCAM-1 (ab134047), and eNOS (ab76198) (Abcam, Cambridge, UK). The antibody dilutions used were: ICAM 1:400, VCAM 1:250 and eNOS 1:250 .
Of 313 paediatric patients with primary immunodeficiency who underwent HSCT, 9, with severe combined immunodeficiency and transplanted within the first year of life, who developed idiopathic pneumonia syndrome had lung biopsies performed, of whom eight had biopsy material available. Six received pre-HSCT conditioning chemotherapy and seven received GvHD prophylaxis (Table S1). All presented with respiratory distress requiring supplemental oxygen. The median time of symptom onset was post-transplant day +11 (range day −10 to day +36). Eight required mechanical ventilation. Two patients received defibrotide to treat clinical veno-occlusive disease (patients 3, 8). Mortality was 88% (Table S2). Four patients had ground glass appearances on chest radiography or computerised tomography, one had significant left sided consolidation, two had non-specific consolidation, and one demonstrated bilateral upper zone collapse of both lower lobes on the chest radiograph (Table S3). In patient 4, Haemophilus influenzae type B was isolated from bronchoalveolar lavage fluid on D + 33, but not on bronchoalveolar lavage fluid on D + 44, after antibiotic treatment. One had Rhinovirus identified from bronchoalveolar lavage but had no reactive inflammatory cells, and no histopathological evidence of viral interstitial lung disease. No other microorganisms were identified from other patients.
On review of previously examined pre-mortem biopsy samples, none had histo-pathological signs of viral, bacterial or fungal infection. Five had evidence of widespread capillary leak of fibrin in alveolar spaces. No patients showed evidence of active veno-occlusive disease in lung tissue. Four showed arterial myo-intimal thickening (a form of severe remodeling possibly related to previous injury), indicative of pulmonary hypertension, two of which had alveolar capillary fibrin leak. Two had obliterative bronchiolitis. One had fibrin deposition, intra-epithelial T cells and atypical mildly reactive pneumocytes. At post-mortem, one patient sample had intra-alveolar fibrin, veno-occlusive disease, pulmonary hypertension, intra-epithelial T cells, and obliterative bronchiolitis. Taken together, these specimens show pathological evidence of endothelial cell damage (Fig. 1).
Seven paraffin-embedded samples were available for additional staining. The photomicrographs of ICAM-1, VCAM-1, and eNOS are representative of all cases. The details of the expression pattern (strength W/S; extent 0–3) of these immunostains are tabulated (Table 1). All demonstrated ICAM-1 positive staining in pneumocytes and/or vascular endothelial cells. Three were also positive for VCAM-1 and five samples were positive for eNOS staining in vascular endothelium (Table 1, Figure S1). Cases 1, 5 and 7 stained positive for all three antibodies, case 2 was positive for ICAM and VCAM without eNOS and cases 3 and 8 positive for ICAM and eNOS without VCAM. There were no cases where eNOS was expressed without either ICAM-1 or VCAM-1. Conversely, ICAM-1 was expressed in all cases where eNOS was positive, which shows that there was no non-specific staining of eNOS. Normal control samples where not available from patients without pneumonitis, or with infective pneumonia/pneumonitis. However, a control sample from a patient with Omenn Syndrome and interstitial pneumonitis was negative for these stains (Figure S2).
Our patients with clinical and radiological evidence consistent with idiopathic pneumonia syndrome, all showed underlying disease processes which can be interpreted as associated with endothelial cell activation injury. In particular, we show histological evidence of type II endothelial cell activation, with display of ICAM-1 and/or VCAM-1, and endothelial injury, with endothelial upregulation of eNOS. This, together with histological findings of intra-alveolar fibrin and pulmonary hypertension suggests that endothelial cell activation injury may be a causative factor underlying idiopathic pneumonia syndrome in these patients.
Possible factors initiating endothelial cell activation injury include previous infection, conditioning agents or the inflammation associated with primary immunodeficiency syndromes or may be caused by other unidentified cellular or inflammatory responses . Due to the invasive nature of lung biopsies, it is not possible to fully determine the pathophysiological sequence of events prior to symptoms of idiopathic pneumonia syndrome arising. Control samples from un-affected patients, or even those with infection-related pneumonia are also unavailable, although we were able to compare our results with lung tissue taken from a patient with inflammatory pneumonitis, who did not have evidence of endothelial cell activation injury. Further work with murine models would enhance our understanding of cellular events prior to the development of idiopathic pneumonia syndrome. Defibrotide modulates endothelial cell activation and is often effective in treating veno-occlusive disease and thrombotic microangiopathy, both diseases also due to endothelial cell activation [3, 9], although two patients in this series had received this for symptoms of veno-occlusive disease.
We have shown that endothelial cell activation injury may underlie the pathology of idiopathic pneumonia syndrome in a non-experimental setting. Further investigation, particularly in animal or tissue models, using indicators of advanced direct endothelial cell damage such as markers of apoptosis (e.g., cleaved caspase-3 or TUNEL) may be able to dissect this process further. Defibrotide may prove to be useful in the treatment of patients with idiopathic pneumonia syndrome, although stratifying which patients will benefit from this treatment requires further study.
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We acknowledge support for this work by the MRC/ESPRC Newcastle Molecular Pathology Node.
TA collected the data, performed the analysis and interpretation of the data and the writing of the manuscript. JS collected the data and contributed to manuscript writing, MAS, CO’B, AC, MT, MB contributed equally to the conceptualization of the research, manuscript writing and critical review, AS performed the analysis and interpretation of the data, manuscript writing and critical review and ARG designed and conceptualized the research project, analysis and interpretation of the data, manuscript writing and critical review at every level of the research stages.
Conflict of interest
The authors declare that they have no conflict of interest.
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Altmann, T., Slack, J., Slatter, M. et al. Endothelial cell damage in idiopathic pneumonia syndrome. Bone Marrow Transplant 53, 515–518 (2018). https://doi.org/10.1038/s41409-017-0042-z