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+.     

Structural characterization of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A soluble glycoprotein of Mr = 80,000 has been isolated from lung lavage of patients with alveolar proteinosis and found to contain 5 residues of hydroxyproline, 91 residues of glycine, 3 residues of methionine, 3.8 molecules of sialic acid, 6 molecules of mannose, 5.9 molecules of galactose, 1 molecule of fucose, and 9.1 molecules of glucosamine. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted in four peptides with molecular weights of 36,000, 27,000, 12,000, and 5,000. The chemical compositions of the CNBr peptides indicated the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contained carbohydrate. Limited trypsin digestion of the glycoprotein of Mr = 80,000 resulted in four peptides with molecular weights of 62,000, 36,000, 26,000 and 18,000, the latter being the NH2-terminal peptide of the native glycoprotein molecule. The peptide of Mr = 26,000 was found to be the COOH-terminal peptide.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.     

Structural relationships of the major glycoproteins from human alveolar proteinosis surfactant

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.     

Elemental analysis of lung tissue particles and intracellular iron content of alveolar macrophages in pulmonary alveolar proteinosis

Background

Pulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images.

Results

The elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining.

Conclusion

The present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.     

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.     

Characterization of collagenous and non-collagenous peptides of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A glycoprotein of Mr 62 000 was isolated from lung lavage material of patients with alveolar proteinosis. The glycoprotein was found to contain (per molecule) 72 residues of glycine, 5 residues of hydroxyproline, 3 molecules of sialic acid, 4.9 molecules of mannose, 4.0 molecules of galactose, 0.9 molecule of fucose and 7.0 molecules of N-acetylglucosamine. Limited pepsin digestion of the glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine, and two of which contained all the carbohydrate present in the glycoprotein of Mr 62 000. The three peptides containing hydroxyproline and with high content of glycine contained a repeating -Gly-X-Y-sequence in the peptide chain. Partial amino acid-sequence analyses on the peptides derived from the digestion of the alveolar glycoprotein with various proteolytic enzymes indicate that this glycoprotein is characterized by the presence of alternating collagenous and non-collagenous regions in the same polypeptide chain.     

Pulmonary impedance and alveolar instability during injurious ventilation in rats.

The mechanical derangements in the acutely injured lung have long been ascribed, in large part, to altered mechanical function at the alveolar level. This has not been directly demonstrated, however, so we investigated the issue in a rat model of overinflation injury. After thoracotomy, rats were mechanically ventilated with either 1) high tidal volume (Vt) or 2) low Vt with periodic deep inflations (DIs). Forced oscillations were used to measure pulmonary impedance every minute, from which elastance (H) and hysteresivity (eta) were derived. Subpleural alveoli were imaged every 15 min using in vivo video microscopy. Cross-sectional areas of individual alveoli were measured at peak inspiration and end exhalation, and the percent change was used as an index of alveolar instability (%I-EDelta). Low Vt never led to an increase in %I-EDelta but did result in progressive atelectasis that coincided with an increase in H but not eta. DI reversed atelectasis due to low Vt, returning H to baseline. %I-EDelta, H, and eta all began to rise by 30 min of high Vt and were not reduced by DI. We conclude that simultaneous increases in both H and eta are reflective of lung injury in the form of alveolar instability, whereas an isolated and reversible increase in H during low Vt reflects merely derecruitment of alveoli.     

Iron homeostasis and oxidative stress in idiopathic pulmonary alveolar proteinosis: a case-control study

Background

Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.

Methods

Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.

Results

Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.

Conclusion

Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.     

Characterization of a high-molecular-weight glycoprotein isolated from the pulmonary secretions of patients with alveolar proteinosis

A high-molecular-weight glycoprotein was isolated, purified and partially characterized from the insoluble pulmonary secretions accumulating in lungs of patients suffering from pulmonary alveolar proteinosis. On electrophoresis in 5% polyacrylamide gel in the presence of sodium dodecyl sulphate and 2-mercaptoethanol, the purified protein gave one major band as detected by Coomassie Blue as well as with periodic acid/Schiff staining. An apparent mol.wt. of 250000 was estimated for this glycoprotein. Amino acid analysis showed that it contains hydroxyproline, and relatively high amounts of glycine, glutamic acid, aspartic acid and leucine. It contains approx. 6% hexose, 3% sialic acid and 2% glucosamine. The neutral sugars are galactose, mannose and fucose. An antiserum prepared in rabbits against this high-molecular-weight glycoprotein cross-reacted with two smaller glycoproteins (mol.wts. 62000 and 36000) isolated from the same pulmonary secretions of these patients. A complementary observation was also made when this large alveolar glycoprotein cross-reacted with an antiserum prepared in rabbits against the smaller glycoprotein (mol.wt. 36000). It appears that this high-molecular-weight glycoprotein may be the precursor of the two smaller glycoproteins present in the same diseased pulmonary secretions.     

Ultrastructural, histochemical, and freeze-fracture evaluation of multilamellated structures in human pulmonary alveolar proteinosis

Ultrastructural, histochemical, and freeze-fracture studies of material recovered by bronchoalveolar lavage from patients with pulmonary alveolar proteinosis revealed four types (A, B, C, and D) of multilamellated structures (MS). Type A, the major component, consisted of concentric, trilaminar structures which were composed of two electron-dense layers and a central lucent layer (5.7-7.5 nm in overall width) alternating with wider (25-30 nm) electron-lucent intervening layers. Type B MS were formed by concentric lamellae with a 5-5.3-nm periodicity. Type C MS were composed of wavy, electron-dense lamellae with a 4-4.5-nm periodicity. Type D MS were conglomerated masses of intricately arranged double or triple electron-dense layers (7.5-13.5 nm wide) alternating with wider (30-40-nm) electron-lucent layers. The electron-dense lamellae of type A, type C, and type D MS were stained with ruthenium red, the Thiéry method, and concanavalin A, indicating the presence of carbohydrate components. Freeze-fracture studies revealed smooth inner and outer surfaces in type A MS, with the fracture planes passing through the central parts of the trilaminar structures; the intervening layers contained 10-nm particles, which probably are proteins. Type B MS had smooth surfaces, and type C MS had slightly particulate surfaces; while type D MS showed tubular or polygonal structures, 350 nm wide, with rows of particles 7-8 nm in diameter. It is concluded that type A and type D MS contain proteins and carbohydrates, probably in the form of glycoproteins, as well as phospholipids, and are related to tubular myelin. Type B and type C MS are considered to contain mainly phospholipids; type C MS are also considered to contain carbohydrates and to be related to lamellar bodies of type II alveolar epithelial cells.