Cathepsin H and napsin A are active in the alveoli and increased in alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a group of rare diseases with disturbed homeostasis of alveolar surfactant. While 90% of the primary adult forms are caused by granulocyte-macrophage colony-stimulating factor autoantibodies, the underlying cause of the juvenile form remains unknown. In order to distinguish primary from secondary effects in the pathogenesis of these two forms, the present authors studied the surfactant protein processing proteases napsin A and cathepsin H. In total, 16 controls, 20 patients with juvenile PAP and 13 adults with idiopathic PAP were enrolled. Amounts and activities of the proteases in the bronchoalveolar lavage fluid (BALF) were determined by immunoblotting and specific substrate cleavage. Both proteases were present and active in BALF from controls and increased in juvenile and adult PAP patients. The amount of active cathepsin H in relation to total cathepsin H was increased in PAP patients compared with controls. Cystatin C, the physiological inhibitor of cathepsin H in the alveolar space, was not increased to the same degree as cathepsin H, resulting in an imbalance of inhibitor to protease in the alveolar space. A general defect in napsin A or cathepsin H expression or activity was not the specific cause for abnormal surfactant accumulation in juvenile pulmonary alveolar proteinosis.

Elevated gelatinase activity in pulmonary alveolar proteinosis: role of macrophage-colony stimulating factor.

Pulmonary alveolar proteinosis (PAP) is an anti-granulocyte macrophage-colony stimulating factor (GM-CSF) autoimmune disease resulting in the accumulation of phospholipids in the alveoli. GM-CSF knockout (KO) mice exhibit a strikingly similar lung pathology to patients with PAP. The lack of functionally active GM-CSF correlates with highly elevated concentrations of M-CSF in the lungs of PAP patients and GM-CSF KO mice. M-CSF has been associated with alternative macrophage activation, and in models of pulmonary fibrosis, M-CSF also contributes to tissue resorption and fibrosis. Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been implicated in extracellular matrix degradation in animal models of fibrosis and asthma. We show for the first time that the lungs of PAP patients contain highly elevated levels of MMP-2 and MMP-9. PAP broncholaveolar lavage (BAL) cells but not bronchial epithelial cells expressed increased MMP-2 and MMP-9 mRNA relative to healthy controls. Both MMPs were detectable as pro and active proteins by gelatin zymography; and by fluorometric global assay, PAP-MMP activity was elevated. BAL cells/fluids from GM-CSF KO mice also demonstrated significantly elevated MMP-2 and MMP-9 gene expression, protein, and activity. Finally, PAP patients undergoing GM-CSF therapy exhibited significantly reduced MMPs and M-CSF. These data suggest that in the absence of GM-CSF, excess M-CSF in PAP may redirect alveolar macrophage activation, thus potentially contributing to elevated MMP expression in the lung.

Perflubron enhances adenovirus-mediated gene expression in lungs of transgenic mice with chronic alveolar filling.

Perfluorochemical (PFC) liquids have both low surface tension and a high capacity to dissolve O2 and CO2, and have been shown to improve gas exchange and lung compliance in animal models of lung injury. We have previously demonstrated that perflubron and other PFC liquids enhance transgene expression in lungs of spontaneously breathing normal rodents after intratracheal instillation of either adenoviral or liposomal vectors followed by a single instillation of PFC liquid. We reasoned that PFC liquids may also be useful for enhancing transgene expression in abnormal lungs. GM-CSF knockout mice develop chronic accumulation of surfactant lipids and proteinaceous material in alveolar spaces and serve as a useful model of chronic alveolar filling. Intratracheal instillation of the adenoviral vector Adlac-Z resulted in patchy in situ distribution of beta-Gal activity, predominantly in larger proximal airways. In contrast, in mice instilled with Adlac-Z followed by instillation of a single dose of perflubron (10 ml/kg body weight), increased expression was observed in distal airway and alveolar epithelial cells. In particular, expression was observed in epithelial cells of debris-filled alveoli. Spectrophotometric measure of quantitative beta-Gal activity in lung homogenates demonstrated increased activity in lungs of mice receiving Adlac-Z plus perflubron compared with lungs of animals receiving Adlac-Z alone. These studies demonstrate that use of perflubron enhances transgene expression in lungs of animals with a chronic alveolar filling process. This approach may be applicable for gene delivery in diseases marked by chronic airway or alveolar filling such as cystic fibrosis.

[A case of pulmonary alveolar proteinosis with elevated serum angiotensin-converting enzyme activity].

A 45-year-old man with diffuse infiltrates on the chest X-ray film was admitted to the hospital. At first, sarcoidosis was suspected due to the elevated serum angiotensin-converting enzyme (ACE) activity. Transbronchial lung biopsy and bronchoalveolar lavage, however, did not lead to that diagnosis. Pulmonary alveolar proteinosis (PAP) was diagnosed after an open lung biopsy. The diffuse infiltrates on his chest X-ray film diminished spontaneously. Two years later, diffuse infiltrates increased again with an elevated serum ACE activity. In this case, it seemed that the severity of PAP was associated with serum ACE activity. We review the literature on the relationship between serum ACE activity and dysfunction of alveolar macrophages in PAP.

Serum and lavage lactate dehydrogenase isoenzymes in pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by the accumulation of lipoproteinaceous material in the alveolar space. Serum lactate dehydrogenase (LDH) has been noted to be elevated in patients with PAP in previous studies. We sought to extend this observation in a series of patients with PAP by looking at total serum LDH concentrations and LDH isoenzyme fractions measured before and after whole lung lavage. Total LDH and LDH isoenzymes were also determined in the lavage effluent. Total serum LDH was elevated before lavage in 10 of 16 patients. Prelavage serum LDH and prelavage alveolar-arterial O2 gradient showed a significant correlation (r = 0.62, p less than 0.05). A decrease in serum LDH was found after lavage in all patients in whom postlavage data was available (paired t test, p less than 0.01, n = 11), although the magnitude of this decrease varied considerably. The isoenzyme pattern before lavage was isomorphic, and this pattern was unchanged after whole lung lavage. This was in marked contrast to the LDH isoenzyme pattern observed in the lavage effluent, which showed a lower percent LDH1 and LDH2 and a higher percent LDH3, LDH4, and LDH5 when compared with the corresponding prelavage isoenzyme percentages for serum (unpaired t test, p less than 0.001). There was no correlation between the total serum LDH concentration and the total lavage LDH concentration. These data confirm that elevated serum LDH is a common finding in PAP. Furthermore, the LDH elevation found consistently in the alveolar fluid points to this as the source of the serum LDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of human pulmonary alveolar epithelial cells revealed by peroxisome changes in pulmonary proteinosis.

Regenerating areas of human lungs in pulmonary fibrosis were observed electron microscopically, and peroxidatic activity of catalase in lung peroxisomes were demonstrated cytochemically. Proliferation of Type II cells was prominent there, and some of the cells extended their cytoplasms to cover the denuded basement membrane. Unusual intermediate cells between Type II and Type I cells were observed. The extension of cytoplasmic processes with new generation of pinocytotic vesicles strongly suggested a Type I cell profile. However, catalase-positive peroxisomes were found in these cells simultaneously. From these results it was concluded that Type I cells may originate from Type II cells in human lungs as they do in experimental animals.

Extracellular alkaline phosphatase from alveolar secretions of patients with pulmonary alveolar proteinosis.

Alkaline phosphatases in alveolar secretions from the lungs of patients with pulmonary alveolar proteinosis have been studied. A soluble form of alkaline phosphatase was isolated from the secretions and characterized. The extracellular enzyme had a pH optimum at 9.95; was stimulated by Mg2+, Ni2+, and Mn2+; was inhibited by Zn2+, Be2+, Cu2+, and low concentrations (8 mM) of L-homoarginine and imidazole; and was heat-stable at 55 degrees C. The soluble phosphatase existed primarily as a high molecular weight complex (excluded from Sepharose 4B) and could be dispersed into low molecular weight forms (205 000--285 000) by treatment with n-butanol. Following butanol treatment, the thermostability of the enzyme was markedly decreased but the kinetic properties such as the Km values, activation energies, and responses to various inhibitors were unchanged. The alkaline phosphatase may originate from unusual type 2 cells present in the alveoli of patients with pulmonary alveolar proteinosis.

PUlmonary alveolar proteinosis: shunt fraction and lactic acid dehydrogenase concentration as aids to diagnosis.

The shunt fraction breathing 100 per cent O2 and serum lactic acid dehydrogenase concentration were evaluated as 2 easily obtainable, rapid laboratory procedures that might aid in the early diagnosis of pulmonary alveolar proteinosis. The mean +/- SE shunt fraction was 20 +/- 1.2 per cent in patients with alveolar proteinosis compared to 8.9 +/- 0.5 per cent in other groups of patients with diffuse lung disease (P less than 0.001). The lactic acid dehydrogenase concentration was increased in all patients with alveolar proteinosis and helped to separate further the 2 groups. Thus, when a patient presents with the findings of chronic diffuse lung disease, the lactic acid dehydrogenase concentration and the shunt fraction can be of value in suggesting the diagnosis of pulmonary alveolar proteinosis.

Phospholipase A in pulmonary secretions of patients with alveolar proteinosis.

This report presents evidence for the presence of phospholipase A2 (EC 3.1.1.4) activity in the insoluble pulmonary secretions of patients with alveolar proteinosis. The enzyme activity has a pH optimum between 7.5 and 8.5 and is stimulated by deoxycholate and Ca2+.