Bronchopulmonary fluids in pulmonary alveolar proteinosis

The alveoli of patients with pulmonary alveolar proteinosis (P.A.P.) become filled with a proteinaceous material, the origin of which is uncertain. The purpose of this study was to examine bronchopulmonary washings from four adults and one child with this disease who had total lung lavage for treatment of the hypoxemia caused by the disease process. In each instance a liter or more of lavage fluid was concentrated approximately four hundred fold by ultrafiltration for protein analysis. Quantitative determinations of the proteins in this fluid and in serum were performed in quadruplicate by the Lowry method, radial diffusion, or radioimmunoassay. There was a selective increase of IgG in washings from all five patients with P.A.P., and there was a lesser increase of IgA, suggesting either a local production or a mechanism for concentrating these immunoglobulins. Albumin usually comprised a similar proportion of the total protein in serum and washings, but alpha-2 macroglobulin comprised less than half that in serum in each instance, suggesting that equal transudation of large and small proteins did not occur. There was considerable variation in the relative proportion of IgM, IgD, and IgE in washings compared to serum, suggesting that each of these immunoglobulins is metabolized (synthesized, transported, or catabolized) by an independent mechanism in the lung. Secretory piece was demonstrated in the bronchial washings of each patient, including controls, as were two soluble nonserum proteins. Fibrinogen was identified in the washings of the three patients with P.A.P. Large amounts of insoluble surfactant-like material were also present in the washings of each of the patients with P.A.P.     

The molecular basis of pulmonary alveolar proteinosis

Pulmonary alveolar proteinosis (PAP) comprises a heterogenous group of diseases characterized by abnormal surfactant accumulation resulting in respiratory insufficiency, and defects in alveolar macrophage- and neutrophil-mediated host defense. Basic, clinical and translational research over the past two decades have raised PAP from obscurity, identifying the molecular pathogenesis in over 90% of cases as a spectrum of diseases involving the disruption of GM-CSF signaling. Autoimmune PAP represents the vast majority of cases and is caused by neutralizing GM-CSF autoantibodies. Genetic mutations that disrupt GM-CSF receptor signaling comprise a rare form of hereditary PAP. In both autoimmune and hereditary PAP, loss of GM-CSF signaling blocks the terminal differentiation of alveolar macrophages in the lungs impairing the ability of alveolar macrophages to catabolize surfactant and to perform many host defense functions. Secondary PAP occurs in a variety of clinical diseases that presumedly cause the syndrome by reducing the numbers or functions of alveolar macrophages, thereby impairing alveolar macrophage-mediated pulmonary surfactant clearance. A similar phenotype occurs in mice deficient in the production of GM-CSF or GM-CSF receptors. PAP and related research has uncovered a critical and emerging role for GM-CSF in the regulation of pulmonary surfactant homeostasis, lung host defense, and systemic immunity.     

肺泡蛋白沉积症的病理学特点与诊断

目的探讨肺泡蛋白沉积症（PAP）的病理学特点及其诊断方法。方法对39例PAP进行了常规的光镜观察,并用淀粉酶消化后过碘酸雪夫染色（D—PAS）、淀粉酶消化后黏液卡红（D-黏卡）、奥新兰（AB）进行组织化学染色。结果肺活检及尸检肺组织的病理改变有如下特点：（1）肺泡腔内及部分小支气管腔内充满嗜伊红性细颗粒状蛋白性物质,并有针状裂隙。（2）蛋白性物质中杂有多少不等的退变的肺泡上皮细胞及脱落的肺泡上皮细胞。（3）肺泡Ⅱ型上皮细胞增生。（4）肺泡间隔毛细血管充血,间质炎症不明显。（5）支气管壁炎症不明显。（6）周围肺组织代偿性肺气肿。支气管肺泡灌洗液（BALF）石蜡包埋切片显微镜下可见片状嗜伊红性细颗粒状蛋白性物质,其中可见针状裂隙。组织化学染色结果肺泡腔内或BALF中嗜伊红性细颗粒状蛋白性物质D—PAS阳性（呈红色）,D-黏卡阴性,AB弱阳性（淡蓝色）或阴性。肺活检及BALF取得标本是诊断PAP的重要方法和途径。结论典型的PAP肺泡腔内或BALF中出现均质嗜伊红性细颗粒状蛋白性物质并有针状裂隙,蛋白性物质D—PAS阳性,D-黏卡阴性具有诊断意义。     

Pulmonary alveolar proteinosis mimicking idiopathic pulmonary fibrosis

Pulmonary alveolar proteinosis (PAP) is a rare disorder of unknown etiology. While most biopsies from patients with PAP disclose a prominent positive periodic acid-Schiff, diastase-resistant intra-alveolar exudate, and minimal interstitial thickening, interstitial fibrosis and honeycombing can be the predominant histologic findings. We recently identified two patients with PAP who initially were thought to have idiopathic pulmonary fibrosis, but whose biopsies ultimately disclosed PAP with considerable interstitial fibrosis. Because the clinical and radiographic presentation of PAP can overlap with other interstitial lung disorders, it should be considered in the differential diagnosis of unexplained idiopathic interstitial pneumonias in young patients.     

肺泡蛋白沉着症定量CT分析与肺功能相关性研究

目的 探讨定量CT参数与肺功能检杳指标在肺泡蛋白沉着症(PAP)全肺灌洗术前后的相关性及应用价值.方法 对20例PAP患者共37次肺CT及同期肺功能检查结果进行相关性分析.对15例20次(两肺19次,单肺1次)行全肺灌洗前后的CT资料进行对比分析.定量CT参数采用患者的最大吸气末全肺容积、全肺质量、全肺含气体积、平均肺体积、平均肺密度及全肺含气体积/全肺容积.肺功能检查指标采用用力肺活量(FVC)、第1秒用力呼吸量(FEV_1)、1秒率(FEV_1/FVC)、呼吸峰流速(PEF)、肺一氧化碳弥散量(D_LCO)以及肺一氧化碳弥散常数(D_LCO/VA),以中国成人肺功能正常参考值作为正常预计值,分别取其占正常预计值的百分比进行相关性分析.结果 (1)全肺质量与FVC%、FEV_1%具有相关性(均P<0.05);全肺含气体积与FVC%、(FEV_1/FVC)%、D_LCO%、D_LCO/VA%之间存在相关性(均P<0.05);平均肺体积、平均肺密度及全肺含气体积/全肺容积分别与FVC%、FEV_1%、(FEV_1/FVC)%、PEF%、D_LCO%、(DLCO/VA)%之间具有相关性(均P<0.05);在所有指标中以全肺含气体积/全肺容积与D_LCO%、(D_LCO/VA)%关系最密切(r=0.725、0.725,均P=0.000).(2)PAP患者经全肺灌洗术后,定最CT检查全肺质量显著减低[(1613±459)g比(1293±351)g,P=0.000],两肺的平均肺密度明显减低[(0.5178±0.15)g/ml比(0.4312±0.10)g/ml,P=0.000],平均肺体积明显增加[(1.216±0.70)ml/g比(1.573±0.61)ml/g,P=0.001],全肺含气体积/全肺容积升高(0.5110±0.14比0.6005±0.10,P=0.000).全肺灌洗前后的全肺容积与全肺含气体积呈正相关(r=0.911,P=0.000).结论 通过最大吸气末行螺旋CT扫描获得的PAP的定量CT参数与肺功能各项指标之间相关性好,因而本法是一种有效的CT肺功能成像技术.全肺灌洗术后以定量CT结果中的全肺质量、平均肺密度及平均含气肺体积改善明显.     

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

Ultrastructural, histochemical, and freeze-fracture studies of material recovered by bron-choalveolar 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 phos-pholipids, and are related to tubular myelin. Type B and type C MS are considered to contain mainly phos-pholipids; type C MS are also considered to contain carbohydrates and to be related to lamellar bodies of type II alveolar epithelial cells.     

Autoimmune pulmonary alveolar proteinosis: Clinical course and diagnostic criteria

Pulmonary alveolar proteinosis (PAP) is caused by accumulation of surfactant components in the alveoli and terminal airways. All forms of PAP are caused by insufficient surfactant clearance by alveolar macrophages. Autoimmune PAP, a rare, antibody-mediated disease, that compromises 90% of cases of PAP, is causes by IgG autoantibodies that block GM-CSF effect, a crucial step for macrophage maturation. Alveolar filling may progress to respiratory insufficiency, but the course of the disease is variable. Patients usually complain of dyspnea, mainly with exertion, and cough. Chest CT shows highly suggestive ground grass opacification crossed by heavy septal lines, leading to the typical “crazy paving” appearance. Bronchoalveolar lavage reveals “milky” fluid, containing semisolid remnants of surfactant components, packed as lamellar bodies. The surfactant appears granular and pink on PAS staining, and lung architecture is preserved. These cytological and pathological characteristics are diagnostic for PAP. In addition, a high titer of IgG anti GM-CSF autoantibodies is highly sensitive and specific for the diagnosis. The trigger for antibodies formation and their role (if any) in regulation GM-CSF activity in the normal state are unknown. Based on the specificity of these characteristics we suggest a structured framework for the diagnosis of Autoimmune PAP. Lung lavage with a large volume of saline is the standard therapy, and is effective in most patients. However, immune-modulatory therapy, by either supplying exogenous GM-CSF, or by inhibiting the CD20 + antibody forming cells, with Rituximab, is also effective in many patients. The precise role of each therapy, alone or in combination, should be systematically studied.     

Elevated IL-10 inhibits GM-CSF synthesis in pulmonary alveolar proteinosis

Pulmonary alceolar proteinosis (PAP) is an autoimmune lung disease characterized by accumulation of surfactant material within the lung. Autoantibodies to GM-CSF as well as high levels of IL-10 are also found in the lungs in PAP. Previous studies suggest that treatment with recombinant GM-CSF is beneficial for patients with low levels of GM-CSF antibodies. The role of IL-10 in PAP, however, is unknown and the hypothesis that IL-10 may affect PAP GM-CSF synthesis has not been addressed. The current findings show that GM-CSF secretion is significantly compromised in PAP bronchoalveolar lavage (BAL) cells compared to controls, but surprisingly, GM-CSF mRNA levels are elevated. In contrast, IL-10 protein and mRNA levels are both highly elevated in PAP. In vitro analysis of GM-CSF regulation indicates that both secretion and mRNA levels are sharply reduced by IL-10 and increased by anti-IL-10 antibody. The phenomenon of elevated GM-CSF mRNA in BAL cells appears not to be due to lack of negative feedback by GM-CSF protein. Results suggest that in PAP, GM-CSF synthesis is deficient and associated with negative regulation by IL-10. Furthermore, IL-10 gene expression becomes even more elevated in patients who do not respond to recombinant GM-CSF therapy and have high anti-GM-CSF titers. Based on these observations, we hypothesize that IL-10 may be an indicator of PAP clinical response to GM-CSF therapy.     

Hyaluronic acid in the pulmonary secretions of patients with alveolar proteinosis

Hyaluronic acid was the only glycosaminoglycan found in the pulmonary secretions of patients with alveolar proteinosis. The compound gave a hexouronate/hexosamine molar ratio of about 11. Glucosamine constituted over 98% of the hexosamines, the remaining being galactosamine. It moved as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis, and this spot disappeared after digestion with hyaluronidase. It was associated with small amounts of proteins, the major amino acids of which are aspartic acid, glutamic acid, glycine, alanine, and leucine.A preliminary report of this work was presented in abstract form at the Fourth International Symposium on Glycoconjugates, September 27 to October 1, 1977, Woodshole, Massachusetts.     

Accumulation of surfactant protein D in human pulmonary alveolar proteinosis.

Surfactant protein D (SP-D) is a collagenous calcium-dependent carbohydrate-binding protein that is structurally related to the serum mannose-binding proteins and pulmonary surfactant protein A. SP-D was initially characterized as a biosynthetic product of freshly isolated rat type II cells and first purified in chemical amounts from bronchoalveolar lavage of rats with silica-induced alveolar lipoproteinosis. The present studies describe the characterization of human SP-D isolated from therapeutic bronchoalveolar lavage of patients with pulmonary alveolar proteinosis. Human proteinosis SP-D was extracted from the 10,000 x g pellet of bronchoalveolar lavage with 100 mmol/L glucose or ethylenediamine tetraacetic acid, and specifically bound to and eluted from maltosyl-agarose. The protein cross-reacted with monospecific antibodies to rat SP-D by enzyme-linked immunosorbent assay and immunoblot and eluted near the position of rat SP-D on reverse-phase high performance liquid chromatography. When chromatographed on 4% agarose (A-15M) in the presence of ethylenediamine tetraacetic acid, the solubilized human proteinosis SP-D eluted near the void volume and earlier than rat SP-D dodecamers or human SP-D multimers in the lavage supernatant. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of proteins in the lavage pellet with antibodies to the carbohydrate-binding domain of proteinosis human SP-D demonstrated covalently cross-linked multimers of SP-D monomers (43 kd, reduced) and multimers of trimeric components stabilized by disulfide and non-disulfide bonds. These studies describe the isolation and biochemical characterization of human SP-D and demonstrate the abnormal accumulation of this protein in the air spaces of patients with alveolar proteinosis.     

Basophilic staining in pulmonary alveolar proteinosis. Report of three cases

Material filling the alveoli in pulmonary alveolar proteinosis has been shown to be eosinophilic by hematoxylin-eosin (HE) staining of lung tissue. No previous evidence has suggested a different result from a frozen section. We noted three cases, in 1972, 1973, and 1982, of basophilic staining of this characteristic alveolar material on unfixed, frozen sections; these cases later showed the usual eosinophilic properties when HE staining was performed at room temperature on formaldehyde solution-fixed, paraffin-embedded sections. These observations are important in that failure to recognize this possibility may initially lead the surgical pathologist away from the correct diagnosis or falsely suggest a problem in stains or staining techniques.     

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.     

Anti-GM-CSF titer predicts response to GM-CSF therapy in pulmonary alveolar proteinosis

Pulmonary alveolar proteinosis (PAP) is an idiopathic disease characterized by the accumulation of surfactant in the pulmonary airspaces. The development of a PAP-like syndrome in the GM-CSF knockout mouse and resolution of disease by local GM-CSF expression strongly implicates GM-CSF in surfactant homeostasis and disease pathogenesis. Based on murine data, GM-CSF therapy was administered to PAP patients, with a subset responding to therapy. The lack of response to GM-CSF therapy in some patients is unexplained. In adult idiopathic PAP there appears to be no intrinsic cellular defect in synthesizing or secreting GM-CSF and/or function in the GM-CSF receptor. Subsequent studies have shown the presence of circulating, neutralizing anti-GM-CSF antibodies in all adult PAP patients studied to date. Whether the anti-GM-CSF is causally related to the PAP disease and whether it should be the target of manipulation remains to be determined. The present study quantified the anti-GM-CSF levels sequentially in PAP patients receiving GM-CSF therapy. The data indicate that titers of circulating anti-GM-CSF predict response to GM-CSF therapy. In addition, we present data from a patient undergoing plasmapheresis in which anti-GM-CSF titer decreased with improvement in the lung disease. Together, these data support the hypothesis that PAP is an anti-GM-CSF autoimmune disease due to the development of antibodies, which results in the deactivation or neutralization of GM-CSF.     

A macrophage-suppressing 40-kD protein in a case of pulmonary alveolar proteinosis

Summary Pulmonary alveolar proteinosis (PAP) is a rare disease of unknown etiology. Macrophage dysfunctions are claimed to be involved in the pathogenesis. We investigated phagocytosis and oxidative metabolism of alveolar macrophages in a case of pulmonary alveolar proteinosis. These cells phagocytize normally and phagocytizable stimulants cause a normal oxidative burst. In response to the membrane signals phorbolmyristate acetate and aggregated immunoglobulin, however, no stimulated turnover of the oxidative metabolism can be observed. A 40-kD protein found in the lavage fluid mediates this macrophage-inhibiting effect. This phenomenon may contribute to the frequent opportunistic infections seen in PAP patients. It can be concluded from our data that the high frequency of infections with opportunistic species in these patients can be reduced by therapeutic bronchoalveolar lavage. By this procedure the abnormal macrophage-suppressing protein can be washed out of the lung at an early stage of the disease.Abbreviations AM alveolar macrophage - BM blood monocyte - BAL bronchoalveolar lavage - CL chemiluminescence - Ig immunoglobulin - kD kilodalton - PAP pulmonary alveolar proteinosis - PMA phorbolmyristate acetate - SRBC sheep red blood cells - Z zymosan     

Ultrastructural analysis of pulmonary alveolar proteinosis induced by methylnaphthalene in mice.

Pulmonary alveolar proteinosis was induced at a 100% incidence in B6C3F1 female mice by twice weekly painting the back skin with methylnaphthalene for 30 weeks to give a total dose of 7.14 g/kg b.wt. Semithin sections were used for defining areas of type II pneumocyte hyperplasia and hypertrophy and associated proteinosis. Ultrastructurally, alveolar spaces were found to be filled with numerous myelinoid structures resembling the lamellar bodies of type II pneumocytes. Mononucleated giant cells (balloon cells, BC) containing numerous myelinoid structures, lipid droplets and many electron dense amorphous ascicular crystals were closely associated with this extracellular membranous material. Stacks of elastic fibers stained with tannic acid and bundles of collagen fibers were loose and discontinuous in the interstitium of affected areas. The following pathogenesis is hypothesized: type II pneumocytes produce increased amounts of lamellar bodies due to their hyperplasia and hypertrophy and develop to form BC which liberate numerous myelinoid structures on their rupture. Epidermal absorption of methylnaphthalene is an efficient reliable method of induction of this internal disease.