Abstract
The pathophysiology of extrinsic allergic alveolitis (EAA) involves oxidative lung damage as well as interstitial and alveolar inflammation. Macrophages and mast cells are inflammatory components of EAA that produce both leukotrienes (LTs) and prostaglandin D2 (PGD2). In addition, PGD2 is also produced by the free-radical-catalysed peroxidation of arachidonic acid during oxidative stress. Urinary 8-iso prostaglandin F2α (8-isoPGF2α) and serum surfactant protein D (SP-D) are considered appropriate biomarkers of oxidative stress and interstitial lung disease activity, respectively. The present study aimed to assess the association of these biomarkers with the pathophysiology of EAA.
Two cases of acute EAA caused by the inhalation of fungi spores were reported. Eight asthmatic patients and six healthy control subjects were also enrolled in the current study.
The serum SP-D and urinary eicosanoid (LTE4, PGD2 metabolite (9α,11βPGF2), 8-isoPGF2α) concentrations markedly increased during the acute exacerbation phase. These concentrations decreased following corticosteroid therapy in the EAA patients. There was a significant correlation between serum SP-D and urinary 9α,11βPGF2 concentrations in the EAA patients.
In conclusion, although the present study proposes that serum surfactant protein-D and urinary eicosanoids are new biomarkers involved in the various immunological responses in extrinsic allergic alveolitis, further large-scale studies are needed to investigate the role of these compounds, not just as biomarkers, but also as biological potentiators of extrinsic allergic alveolitis.
Acute extrinsic allergic alveolitis (EAA) is characterised by oxidative lung damage 1. During oxidative stress, prostaglandin D2 (PGD2) is nonenzymatically produced by the free-radical-catalysed peroxidation of arachidonic acid (isoprostane pathway) 2. Briefly, isoprostanes are a unique series of PG-like compounds formed by the random oxidation of tissue phospholipids by oxygen radicals 2. Thus, isoprostanes contain racemic mixtures of E-, D-, F-type and thromboxane-type prostane rings 3, 4. The racemic D-ring isoprostane (12-isoPGD2) subsequently undergoes rapid epimerisation to racemic PGD2 2.
In contrast, alveolar macrophages and mast cells produce cysteinyl-leukotrienes (CysLTs) and cyclooxygenase-dependent PGD2 5, 6. Alveolar macrophages play a key role in acute EAA 7. There is also a persistent increase in the number of alveolar mast cells in EAA patients 8. EAA is categorised as a T-helper1-type disease and interferon (IFN)-γ plays a pivotal role in granuloma formation in EAA 7. Interestingly, mast cells, which express the Fcγ receptor I after incubation with IFN-γ, can produce PGD2 and CysLTs even in response to immunoglobulin (Ig)G stimulation 9.
Urinary leukotriene E4 (LTE4) is now considered to be the most reliable analytical parameter for monitoring the endogenous synthesis of CysLTs 10, 11. Similarly, urinary 9α,11β prostaglandin F2 (9α,11βPGF2) is a relatively stable PGD2 metabolite and an appropriate indicator of mast cell activation 5. Of the isoprostanes, 8-iso prostaglandin F2α (8-isoPGF2α) is the best-characterised isomer and urinary 8-isoPGF2α is considered the most accurate indicator of oxidant stress 2. Taking this into account, it was hypothesised that patients with acute EAA show increased concentrations of urinary eicosanoids (CysLTs, 9α,11βPGF2 and 8-isoPGF2α).
Surfactant protein D (SP-D), which is produced by alveolar type II and Clara cells 12, is an important regulatory molecule in both pulmonary surfactant homeostasis and first-line defence mechanisms against microbial or allergen challenges 13. Krebs von den Lungen-6 (KL-6) is also produced by alveolar type II cells 14. The measurements of serum SP-D and KL-6 concentrations contribute to the early diagnosis of interstitial lung disease (ILD) 12, 14. EAA is an acute ILD 7. Previous studies have demonstrated significant increases in SP-A and KL-6 concentrations in the bronchoalveolar lavage fluid (BALF) of acute EAA patients 15. Although plasma SP-D concentration is one of the most appropriate prognostic parameters of acute respiratory distress syndrome (ARDS) 16, knowledge of the serum SP-D profile of acute EAA patients is limited 17.
The present study aims to assess the association of these biomarkers with the pathophysiology of EAA.
METHODS
Case reports
Case 1
A 61-yr-old, nonsmoking female suffered from summer-type EAA caused by Trichosporon asahii, the most prevalent cause of EAA in Japan 18. Cell counts revealed that 58.9% of total BALF cells were lymphocytes and the CD4/CD8 ratio of lymphocyte surface markers was 0.6. In addition, transbronchial lung biopsy specimens exhibited lymphocytic alveolitis with granulomas. The subject was diagnosed positive for precipitin to T. asahii by double immunodiffusion analysis. The positive findings were confirmed during a provocation test, following which the patient was allowed to return home.
Case 2
A 48-yr-old, nonsmoking female suffered from occupational EAA caused by Aspergillus niger, predominantly isolated from house dust in her workplace (a linen room). In addition to being strongly positive for precipitins to both house dust and A. niger extract, determined by double immunodiffusion analysis, the patient was also positive for a serum-specific IgG antibody against A. niger (10.8 mg·dL−1). High-resolution computed tomography of the patient's chest revealed supportive radiographic findings 7. The results from both cases are shown in table 1⇓.
Serum Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) concentrations on admission
Both EAA patients fulfilled the American-European Consensus Conference criteria for ARDS (table 1⇑) 19. Intensive corticosteroid treatment (i.v. administration of 1,000 mg·day−1 methylprednisolone for 3 days, followed by oral administration of 0.5 mg·kg−1 prednisone) was tapered over the 7-week period, resulting in gradual improvements of both clinical symptoms and radiographic findings.
Control subjects
Eight (six female) stable asthmatic patients (mean age (range) 58 (33–73) yrs) were enrolled as diseased control subjects. Six (three female) healthy control subjects (44 (29–58) yrs) were also enrolled for comparative analysis of urinary eicosanoid data. All the subjects were nonsmokers. Permission to conduct the study was obtained from the Ethics Committee of the National Sagamihara Hospital (Japan) and all participating subjects gave informed consent.
Measurements
Serum and spot urine samples were collected between 09:00–11:00 h. In the case of subjects with acute EAA, urine samples were collected on admission and following therapy. The samples were analysed for KL-6, SP-D and eicosanoid concentrations by methods previously described 10, 12, 17, 20. Double immunodiffusion analysis of precipitating antibodies against 18 different fungal species was performed according to the Ouchterlony method. A specific IgG antibody against A. niger was performed utilising the liquid-phase immunoassay AlaSTAT microplate system (Diagnostic Products Corporation, Los Angeles, USA).
Data analysis
Serum and urinary data were expressed as mean and median, respectively. Relationships were analysed using Spearman's rank correlation test. A p-value <0.05 was regarded as statistically significant.
RESULTS
Serum KL-6 and SP-D concentrations were markedly higher in the acute EAA patients than in the eight asthmatic patients (table 1⇑). As shown in figure 1⇓, urinary eicosanoid concentrations in the acute EAA patients (LTE4: 420 and 185 pg·mg−1-creatinine; 9α,11βPGF2: 658 and 382 pg·mg−1-creatinine; 8-isoPGF2α: 393 and 537 pg·mg−1-creatinine) were markedly higher than in the healthy control subjects (LTE4: 45 pg·mg−1-creatinine; 9α,11βPGF2: 43 pg·mg−1-creatinine; 8-isoPGF2α: 187 pg·mg−1-creatinine). Median values of urinary LTE4, 9α,11βPGF2 and 8-isoPGF2 concentrations in the asthmatic patients were 55, 79 and 235 pg·mg−1-creatinine, respectively. The serum SP-D, KL-6 and urinary eicosanoid concentrations decreased following corticosteroid therapy in the EAA patients (figs 1⇓ and 2⇓). There was a significant correlation between serum SP-D and urinary 9α,11βPGF2 concentrations in the EAA patients (p<0.05, rs = 1; fig. 2⇓).
a) Urinary leukotriene E4 (LTE4), b) 9α,11β prostaglandin F2 (9α,11βPGF2), and c) 8-iso prostaglandin F2α (8-isoPGF2α) concentrations in the extrinsic allergic alveolitis patients. BA: asthmatic patients; HC: healthy control subjects. Horizontal bars indicate median values.
A significant correlation between serum surfactant protein D (SP-D) and urinary 9α,11β prostaglandin F2 (9α,11βPGF2) concentration was found in the extrinsic allergic alveolitis patients. rs = 1; p<0.05.
DISCUSSION
The present authors demonstrated, for the first time, that acute EAA is characterised by eicosanoid overproduction. Although increased urinary LTE4 concentration has been reported in patients with ARDS 21, to the current authors' knowledge this is the first report demonstrating a PGD2 overproduction in patients with ARDS or even acute EAA. The CysLT and PGD2 overproduction may well be associated with the increased cyclooxygenase activity of both alveolar macrophages 6 and mast cells 5. However, the higher urinary 9α,11βPGF2 concentrations in EAA patients are mainly considered to be a reflection of mast cell activation 5, 22. Interestingly, mast cells activated by aggregated IgG, following IFN-γ-induced upregulation of Fcγ receptor I, can produce both PGD2 and CysLTs 9, which is consistent with the pathophysiology of EAA.
8-isoPGF2α is an accurate biomarker of oxidative stress in vivo 2. The present study demonstrated that urinary 8-isoPGF2α concentration is increased in acute EAA patients, suggesting a central role for oxidant stress in the pathogenesis of acute EAA. The isoprostane pathway also contributes to the PGD2 overproduction in acute EAA 2, although the current approach was unable to ascertain its relative contribution to PGD2 production. It has recently been discovered that isoprostanes containing D- or E-type prostane rings are excreted into the urine as conjugates with N-acetyl cysteine sulfoxide, suggesting that these metabolites may be used as biomarkers to estimate whole-body production of D- or E-type isoprostanes 23. Future experiments using this methodology will hopefully provide even more answers.
Increased 8-isoPGF2α concentrations have been reported in the breath condensate of patients with ARDS 24, ILD 25 and asthma 26. Recently, Wood et al. 27 demonstrated that despite high variability, sputum 8-isoPGF2α concentrations were significantly increased in patients with severe persistent asthma. The present study also demonstrated similar findings in that the two asthmatic patients with extremely high urinary 8-isoPGF2α concentrations (458 and 525 pg·mg−1-creatinine, respectively) were characterised by severe persistent asthma and hypereosinophilia (11.2 and 17.2%, respectively).
These eicosanoids are also known to possess various other biological activities, such as being potent constrictors of pulmonary vascular smooth muscle and causing plasma exudation 4–6. Although the full extent of the biological activity of the eicosanoids in acute EAA remains to be determined, the various components of the eicosanoid metabolic pathways may become therapeutic targets in acute EAA.
Consistent with a previous case report by Tanaka et al. 17, serum SP-D concentrations in the acute EAA patients were markedly increased. In contrast, serum SP-D concentrations in the asthmatic patients were low, which is in accordance with data by Koopmans et al. 28 Interestingly, the serum SP-D concentrations subsequently decreased and showed a significant correlation with urinary 9α,11βPGF2 concentrations in the EAA patients. Serum SP-D is a biomarker of ILD activity 12 and SP-D plays a protective role in pulmonary inflammation 13. PGD2 and its metabolite, 15-d-PGJ2, have the potential to serve as downregulators of lung injury induced by bleomycin 29. Taken together, these findings suggest that both SP-D and PGD2 appear to be important regulatory factors in the pathophysiology of EAA.
In conclusion, the present study proposes that serum surfactant protein D and urinary eicosanoids are new biomarkers involved in various immunological responses in extrinsic allergic alveolitis. Further large-scale studies are needed to investigate the role of these compounds, not just as biomarkers, but also as potentiators of extrinsic allergic alveolitis.
- Received September 10, 2004.
- Accepted August 15, 2005.
- © ERS Journals Ltd
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