Although it is clearly established that surfactant protein A (SP-A) is secreted by type II pneumocytes as a component of pulmonary surfactant, its secretion pathway as well as its subcellular localization in the human lung are uncertain. We therefore studied the intracellular and intra-alveolar localization of SP-A in eight adult human lungs by immunohistochemistry and immunoelectron microscopy. Only type II pneumocytes could be identified as SP-A positive cells within the parenchymal region. SP-A was localized mainly in small vesicles and multivesicular bodies close to the apical plasma membrane. Only few lamellar bodies were weakly labeled at their outer membranes. Stereologic analysis showed this weak signal to be due to specific labeling. In the alveolar space, lamellar body-like surfactant forms in close proximity to tubular myelin were labeled for SP-A at their periphery. The strongest SP-A labeling was found over tubular myelin figures. Labeling for SP-A was also found in close association with the surface film and unilamellar vesicles. Our results support the hypothesis that, in the human lung, SP-A is mainly secreted into the alveolar space via an alternative pathway that largely bypasses the lamellar bodies. After secretion, the outer membranes of unwinding lamellar bodies become enriched with SP-A when tubular myelin formation is initiated. SP-A may also be involved in the transition of tubular myelin into the surface film.
The pulmonary surfactant system prevents alveolar atelectasis by reducing alveolar surface tension in a surface area– dependent manner. In addition, surfactant may be an active component of the pulmonary host defense system. Surfactant consists of ∼ 90% lipids and 10% proteins, including the surfactant apoproteins A, B, C, and D (1, 2). Surfactant, which covers the alveolar surface at the air–liquid interface, is synthesized, stored, secreted, and to a large extent recycled by type II pneumocytes of the alveolar epithelium. Besides its biochemical complexity, surfactant is also morphologically heterogeneous (1). An intracellular surfactant compartment, consisting of characteristic storage organelles termed lamellar bodies, and an intra-alveolar surfactant compartment, can be distinguished. Intra-alveolar surfactant is present in different morphologic forms which, according to contemporary models, correspond to different stages in surfactant metabolism (1). After secretion of lamellar bodies into the alveolar lumen, they undergo a major structural transformation into tubular myelin figures (3) from which the surface film is generated. “Spent” surfactant is found in the hypophase as unilamellar vesicles and can be taken back into type II pneumocytes. Intra-alveolar surfactant material can be harvested by bronchioalveolar lavage. After differential centrifugation, surface active large aggregates, ultrastructurally largely corresponding to tubular myelin figures, and converted inactive small aggregates, ultrastructurally largely corresponding to small unilamellar vesicles, can be distinguished (4).
According to its weight, the most abundant surfactant apoprotein in the alveoli is the hydrophilic surfactant protein A (SP-A). SP-A may have roles in surfactant homeostasis, structure, and surface activity, as well as its host defense functions (for review, see Refs. 2, 5–7). The essential role of SP-A for the formation and structural integrity of tubular myelin in vivo has recently been demonstrated in SP-A–deficient mice where tubular myelin is absent (8, 9). In conjunction with SP-B and SP-C, SP-A enhances the rate of adsorption of phospholipids into the surface film at the air–liquid interface in vitro (10). However, the surfactant-specific functions of SP-A might become relevant only under conditions when the respiratory system is stressed (6, 11). High SP-A levels enhance the resistance of surfactant to protein inhibition in vivo (12). This protective effect has also been shown in mice that overexpress SP-A (13), whereas the absence of SP-A results in a higher susceptibility to protein inhibition in SP-A–deficient mice (9). SP-A also inhibits surfactant secretion and stimulates the reuptake of surfactant lipids by type II pneumocytes in vitro. In addition, SP-A may have important functions in the innate host defense system of the lung (2, 11). SP-A is also present in nonalveolar pulmonary sites, as well as in other organs (7). Altered intra-alveolar SP-A levels contribute to abnormal surfactant function in a variety of lung diseases (4, 14).
Although it is clearly established that SP-A is secreted by type II pneumocytes as a component of pulmonary surfactant, its secretion pathway and its subcellular localization in the human lung are still under discussion (see Refs. 2, 6, 15). This may in part be due to the fact that the microstructural organization of the phospholipid-rich surfactant is extremely difficult to preserve for immunocytochemical analysis (5). However, methodologic improvements in immunoelectron microscopy in combination with stereologic methods now allow for good ultrastructural preservation, while maintaining antigenicity for immunolocalization studies as well as quantitative analysis to test for labeling specificity (16, 17). The aim of the present study was therefore to analyze the intracellular and intra-alveolar localization of SP-A in the parenchymal region of the adult human lung by state-of-the-art immunoelectron microscopy and stereology.
In eight cases of single-lung transplantation, the contralateral human donor lung was used for microscopic analysis, provided it could not be matched to another suitable recipient by The Eurotransplant Foundation Centre, Leiden, The Netherlands (see Ref. 18 for details). Two of the lungs were fixed for immunohistochemistry by intrabronchial instillation of 4% buffered formaldehyde solution. Several samples from different areas were embedded in paraffin. For immunoelectron microscopy, six single human lungs were fixed by instillation of a mixture of 4% formaldehyde (prepared from freshly depolymerized paraformaldehyde) and 0.1% glutaraldehyde in 0.2 M Hepes buffer at a pressure of 25 cm water column. Systematic uniform random samples representative of the whole organ were infiltrated with 2.3 M sucrose in PBS for 1 h, frozen in liquid nitrogen, transferred to a freeze substitution unit (Reichert AFS; Leica, Vienna, Austria), freeze substituted in 0.5% uranyl acetate in methanol at –90°C for at least 36 h, and embedded in Lowicryl HM20 (Polysciences, Eppelheim, Germany) at –45°C (see Ref. 19 for details).
Immunohistochemistry was performed using the alkaline phosphatase method. Serial sections of 4 μm thickness were mounted on poly-l-lysine slides, dried overnight, dewaxed by xylene, rehydrated in a graded series of ethanol, and finally washed in Tris buffer. An automated staining device (TechMate 500; Dako, Glostrup, Denmark) was used for the subsequent labeling procedure for SP-A and Clara cell 10 kD protein (CC10; Dr. G. Suske, Institute of Molecular Biology and Tumor Research, University of Marburg, Germany). Immunostaining was performed with the APAAP kit (Dako) according to the specifications of the manufacturer. Fast Red was used as substrate for alkaline phosphatase. Sections were finally counterstained with Mayer's haematoxylin. For electron microscopic analysis, immunolabeling of ultrathin sections of 70 nm thickness was performed with primary antibodies against SP-A and the N-terminal fragment of the precursor form of surfactant protein B (proSP-B) using protein A-gold (Department of Cell Biology, University of Utrecht, Netherlands; dilution 1:65) or gold-coupled secondary antibodies (British BioCell; Cardiff, UK; dilution 1:20) with a gold particle diameter of 5 or 10 nm for detection. Ultrathin sections were either labeled for SP-A only with a monoclonal (PE 10; Dako; IgG concentration 500 μg/ml; dilution 1:40) or polyclonal (Laboratory Dr. S. Hawgood; dilution 1:40; see Ref. 20 for details) primary antibody, or double labeling for SP-A (monoclonal) and proSP-B (Laboratory Dr. S. Hawgood; dilution 1:20) was performed. Both the monoclonal and the polyclonal antibody against SP-A yielded similar results. Control experiments were performed by omission of the primary antibody. Specificity of the antibodies against SP-A was tested by Western blotting with human bronchoalveolar lavage fluid (Figure 1), which was performed according to the standard protocol of the manufacturer (Novex/Invitrogen, Carlsbad, CA). Examination of labeled ultrathin sections was conducted using an EM 900 (LEO, Oberkochen, Germany) at an accelerating voltage of 50 kV.
Three of the human lungs were used for quantitative analysis of the intracellular distribution of SP-A labeling. A recently established parameter, relative specific labeling index (RSLI), was determined to test for preferential labeling of different cell compartments (17). Following the rules of systematic uniform random sampling for design-based stereological analysis (19), ultrathin sections from four blocks of each of the three lungs were examined. For each type II pneumocyte completely visible on the section, the numbers of gold particles labeling for SP-A in different cellular compartments (nucleus, lamellar bodies, and remaining cytoplasm including vesicles) were counted. Due to the dependence of the effective resolution of gold labeling on the size of the underlying particles (21), we chose not to subdivide the rather large compartment cytoplasm/vesicles to avoid uncertainties and misinterpretations in the allocation of the gold particles. A total of 123 type II pneumocyte profiles were analyzed. The total number of gold particles counted over type II pneumocyte profiles was 1,876, thus the mean number of gold particles counted per cell profile was 15. The observed distribution of gold particles was compared with an expected distribution which would occur if the gold particles were scattered randomly over the cells, according to the relative volume of the cellular compartments the gold particles were associated with. The relative volume fractions of the cell compartments (Vv [comp/cell]) were estimated by point counting in an earlier study (18). The expected number of gold particles for each compartment (Ngold expected) was determined according to the formula: Ngold expected = 1876 × Vv (comp/cell). The RSLI was determined for each compartment as the observed value divided by the expected value: RSLI = Ngold observed/Ngold expected. An RSLI of 1 would therefore indicate random labeling while values higher than 1 would indicate nonrandom (preferential or specific) labeling. Statistical testing for nonrandomness of labeling was performed by χ2-analysis.
Among the cells of the parenchymal region, type II pneumocytes were the only ones that were positively stained for SP-A. Within the bronchiolar epithelium, nonciliated cells positive for CC10 showed no signal for SP-A on subsequent paraffin sections (Figures 2A and 2B). Within the alveolar region, immunohistochemistry for SP-A showed a weak labeling of type II pneumocytes and a strong positive staining at the alveolar epithelial surface (Figure 2C). The tracheal epithelium and tracheal submucosal glands, which have been shown to contain SP-A and its mRNA in the human fetal and newborn lung (22), were not examined in the present study.
Ultrastructurally, the phospholipid-rich lamellar bodies within type II pneumocytes, as well as intra-alveolar surfactant subtypes, were well preserved. The strong staining on the alveolar epithelial surface that was observed by immunohistochemistry (Figure 2C) corresponded to a signal over intra-alveolar surfactant subtypes at the electron microscopic level (Figure 2D). In the alveolar space, some lamellar body–like surfactant forms in close proximity to tubular myelin were labeled for SP-A at their periphery (Figures 2D and 2E). Other lamellar body–like forms with no association with tubular myelin were devoid of any labeling. The strongest labeling for SP-A was found over the lattice structures of tubular myelin figures (Figures 2D and 2E). Labeling for SP-A was also found in close association with the surface film and unilamellar vesicles (Figure 2F). Within the phagolysosomes of alveolar macrophages, SP-A was found in tubular myelin-like structures, but not in lamellar body–like forms (Figure 3).
Within type II pneumocytes, SP-A was localized mainly in small vesicles and multivesicular bodies close to the apical plasma membrane (Figure 4A). Only a few of the multivesicular bodies showed colocalization of SP-A and proSP-B (Figure 4C), while most of them were either positive for proSP-B (Figure 4D) or SP-A. The vast majority of SP-A labeling showed no colocalization with proSP-B. Only some of the lamellar bodies were weakly labeled for SP-A at their outer membranes (Figure 4B). When comparing the labeled structures, a gradient of increasing SP-A labeling from intracellular lamellar bodies (Figure 4B) via intra-alveolar lamellar body–like forms to tubular myelin (Figures 2D and 2E) was seen.
Stereologic analysis of the intracellular SP-A labeling distribution revealed that, per single cell profile, an average of only two gold particles were counted over lamellar bodies (Table 1). However, taking the relative volumes of the cell compartments into consideration, estimation of the relative specific labeling index and statistical testing by χ2-analysis showed that this labeling pattern is due to a highly significant nonrandom labeling. Hence, stereologic analysis helped to perceive a weak signal as specific labeling.
|Compartment||Ngold observed||VV (comp/cell)||Ngold expected||RSLI|
|Lamellar bodies||248 (13.2%)||9.8%||184||1.35|
|χ2: P < 0.001|
The genetics, structure, processing, properties, and functions of SP-A have been extensively reviewed recently (5-7, 11, 23). The aim of the present study was to determine the intracellular and intra-alveolar localization of SP-A in the parenchymal region of the adult human lung (see Refs. 2, 6, 15). Aldehyde fixation, freeze substitution, low temperature embedding in Lowicryl HM20 resin and colloidal gold labeling, as performed in the present study, is ideally suited for quantitative immunoelectron microscopy (16, 21). The lungs examined in this study were obtained during clinical single-lung transplantation where the contralateral donor lung could not be made available to a suitable recipient. While one lung was transplanted, the contralateral donor lung was fixed by airway instillation to ensure rapid and uniform fixation of the whole organ for subsequent morphologic investigation. Previous conventional electron microscopic examination of human lungs obtained in this way showed excellent ultrastructural preservation of type II pneumocytes (18). Samples taken from these lungs according to the rules of systematic uniform random sampling can be expected to be representative of the whole organ, therefore avoiding sampling bias that may be seen in autopsy or biopsy specimens (18, 19). Hence, the material investigated in the present study can be regarded as normal healthy adult human lung.
By immunohistochemistry, type II pneumocytes showed a weak cytoplasmic labeling for SP-A. This finding is in agreement with results from late fetal and neonatal human lungs (22, 24). For a more detailed quantitative analysis of the staining pattern in human type II pneumocytes, immunoelectron microscopy using colloidal gold was performed in the present study. In contrast to the intra-alveolar labeling pattern, the intracellular distribution of SP-A was not unambiguously interpretable on a merely qualitative basis. Intracellularly, SP-A was found mainly in small cytoplasmic vesicles and multivesicular bodies of type II pneumocytes, but only to a minor extent in the periphery of some lamellar bodies. We therefore performed a quantitative stereologic analysis to see if the signal over lamellar bodies is due to specific or background labeling. Although only two gold particles per single cell profile were counted over lamellar bodies, the estimation of the relative specific labeling index clearly showed this weak signal to be due to a nonrandom distribution of gold particles, i.e., due to specific labeling.
The weak peripheral labeling of lamellar bodies observed in the present study is in principal agreement with observations in the rat lung (25, 26) and macroscopically normal adult human lung tissue obtained from surgical specimens removed for malignant tumor (27). Others have reported a weak but more even distribution of SP-A labeling over the lamellar bodies in the newborn human lung (28), on occasional lamellar bodies in the rat lung (29). Several possible reasons might account for these discrepancies. There are species differences in the ultrastructural organization of lamellar bodies between humans and rodents (30) that might be responsible for a different localization of surfactant proteins. Such differences might also be present during different developmental stages. Furthermore, differences in the fixation and embedding protocols lead to different degrees of ultrastructural preservation of the phospholipid-rich lamellae, which may influence where and how much SP-A can be detected. Because the preservation of lamellar bodies for immunoelectron microscopic studies can be regarded as a compromise between retaining ultrastructure and maintaining antigenicity, it is not impossible that a better ultrastructural preservation might lead to some degree of inhibition of antigen-antibody reactions in the tightly spaced lamellae of well preserved lamellar bodies (31).
In 1973, Gil and Reiss formulated the hypothesis that some proteins present in tubular myelin are added after secretion of lamellar bodies, and suggested that these proteins may originate in an intracellular site other than the lamellar bodies (32). In the present study, most of the SP-A in type II pneumocytes was not located within lamellar bodies. Our results therefore support the concept that SP-A secretion is at least in part independent of the regulated secretion of lamellar bodies (33). This was based on biochemical data (34-40) and supported by immunoelectron microscopic data in the rat (29) and newborn human lung (28), showing that intra-alveolar surfactant is enriched in SP-A when compared with lamellar bodies. In contrast to its high content in intra-alveolar surfactant, SP-A might account for only around 1% of total lamellar body protein (34, 35), and only around 4–8% of total lung SP-A might be present in lamellar bodies (35, 39). The stereologic data from the current study fit very well with estimations based on biochemical data that ∼ 10% of intracellular SP-A are secreted together with lamellar bodies, whereas 90% are secreted by other means (36). Other biochemical studies revealed higher amounts of SP-A in lamellar bodies, thus favoring the hypothesis of cosecretion of SP-A and surfactant phospholipids (41-44). In vitro measurements of protein levels in isolated lamellar bodies, however, are hampered by the fact that, during the isolation procedure, this fraction might be contaminated by other organelles of similar density or that some components, due to alterations of the microstructural integrity of lamellar bodies, might have been lost (45). It is also possible that secondary association of SP-A and lamellar bodies might happen during and after cell disruption (42). These methodologic problems might explain the discrepancies in SP-A levels reported from lamellar body–enriched fractions isolated from lung tissue homogenates. Furthermore, the present study, demonstrating SP-A–positive small vesicles and multivesicular bodies close to the apical plasma membrane by a direct approach, provides possible morphologic evidence for a mainly separate secretion of SP-A in the adult human lung.
In type II pneumocytes, multivesicular bodies are involved in the post-translational assembly of surfactant components into lamellar bodies as well as in reuptake of surfactant material and subsequent recycling and/or degradation, thus representing the junction point between the biosynthetic and endocytotic pathway (46, 47). In agreement with the present findings, an SP-A–positive subpopulation of multivesicular bodies and small vesicles located at the apical cell border has been described in type II pneumocytes of the rat lung (25). In the current study, we found SP-A to be colocalized with proSP-B in multivesicular bodies only to a minor extent, whereas the vast majority of SP-A labeling was not colocalized with proSP-B. Because proSP-B is routed directly to lamellar bodies via multivesicular bodies (48), the present results might be interpreted in two different ways: either (i) a subpopulation of SP-A–positive multivesicular bodies, mainly distinct from proSP-B positive multivesicular bodies, exists that is involved in post-translational transport of de novo synthesized SP-A from the Golgi apparatus; or (ii) the small vesicles and multivesicular bodies containing SP-A belong, at least in part, to the endosomal/lysosomal system involved in reuptake of secreted surfactant components. In the latter case, the current findings would support the concept that newly synthesized SP-A enters the intra-alveolar compartment before entering the lamellar bodies (38, 40).
Stereologic analysis of our material revealed that at least some SP-A is present in the periphery of lamellar bodies of the adult human lung. This finding is in good agreement with earlier morphometric observations in the rat lung (26). The fact that only a few lamellar bodies were labeled might be explained either by the fact that a subpopulation of lamellar bodies exists that contains SP-A, or as the result of the probability of random sectioning through the distinct, small peripheral labeled areas of the lamellar bodies (49). Whether this signal is, at least in part, due to reuptake of intra-alveolar SP-A and subsequent intracellular transfer to lamellar bodies via coated pits and vesicles, cytoplasmic vacuoles, and multivesicular bodies, as discussed by several authors (25, 31, 33, 49-52), remains to be further investigated. This idea is further supported by the finding that internalized purified SP-A is also located mainly at the periphery and in multivesicular structures of the lamellar bodies of cultured rat type II pneumocytes (49).
The intra-alveolar metabolism of surfactant includes transformation of secreted lamellar bodies into tubular myelin figures, as shown by Williams in 1977 (3). In her study, Williams also demonstrated the presence of particles close to the four corners of the tubular myelin lattice and on transitional membranes extending from secreted lamellar bodies to tubular myelin, whereas they were lacking on the membranes of secreted lamellar bodies. These electron dense particles are now thought to represent SP-A (1), because this location at the corners of tubular myelin is in agreement with results from immunoelectron microscopic studies in the rat lung (29). The current study demonstrates a gradient of increased SP-A labeling from intracellular lamellar bodies via intra-alveolar lamellar body– like forms to tubular myelin. The present results therefore indicate that, in the human lung, SP-A is mostly added to intra-alveolar surfactant during the transformation of secreted lamellar bodies into tubular myelin figures. Compared with the internal lamellae, the outer membranes of unwinding lamellar bodies that are in close association with tubular myelin are enriched with SP-A. A possible mechanism for the interaction of SP-A with surfactant lipids during tubular myelin formation, where outer membranes of lamellar bodies are thought to act as targets for separately secreted SP-A, has been proposed recently (53). Our observations on the peripheral labeling of freshly secreted lamellar bodies, which provide further ultrastructural evidence for this model in the adult human lung, are also in line with earlier immunoelectron microscopic data obtained in the rat (26) and newborn human lung (28). Although the amount of lipid-free SP-A in the hypophase has been estimated to be only around 1–3% (31, 54), this might still be sufficient to initiate transformation of freshly secreted lamellar bodies, because lamellar body–like forms only account for around 1–2% of the total intra-alveolar surfactant volume (55).
The strong presence of SP-A over the lattice structures of tubular myelin figures has already been demonstrated in the rat lung (25, 26, 29, 56, 57) and neonatal (28) and adult human lung (27). Furthermore, the current findings have demonstrated the presence of SP-A in close association with the surface film. This localization fits with the concept that SP-A might facilitate the incorporation of lipids from the surface-associated surfactant reservoir into the surface film (58). In this regard, it would be interesting to know whether SP-A is really localized within the surface film or just nearby. However, the resolution of the indirect labeling technique used in the present study does not allow a definitive answer. Resolution is determined by the size of the detection complex and its point of attachment to the primary antibody and has been estimated to be ∼ 15–18 nm in one direction (21, 31). This makes it impossible to decide whether a gold particle localizes an antigen exactly within a membrane or just underneath it.
In addition, the present study has also demonstrated labeling for SP-A over unilamellar vesicles, which largely correspond to inactive small surfactant aggregates. This is in agreement with the distribution of instilled SP-A in the rat lung (31). The presence of considerable amounts of SP-A, but not SP-B, in small aggregates after conversion in vitro has been shown biochemically (59). Wright and colleagues (60), on the other hand, reported only small amounts of SP-A in an intra-alveolar surfactant subfraction enriched in small vesicles. Methodologic differences and difficulties related to the isolation and subfractionation of intra-alveolar surfactant subtypes most likely account for these quantitative differences. However, it is also possible that several subpopulations of small vesicles that differ in SP-A content might exist in the alveolar space. Further studies addressing this question and its implications for surfactant reuptake are necessary.
Our finding that no SP-A signal could be detected over nonciliated bronchiolar epithelial cells that were positive for CC10 confirms previous results that no SP-A mRNA was detectable in bronchiolar epithelial cells in human lungs by in situ hybridization (61). This points out the importance of possible species differences in surfactant protein distribution. Species differences in the expression of SP-A between humans and rodents have already been emphasized (11, 61). Inconsistencies in the detection of SP-A in nonciliated airway epithelial cells might at least in part be due to the fact that this cell population is very heterogeneous, thus displaying both inter- and intraspecies variations (62). Although, in the present study, no SP-A presence could be demonstrated in CC10-positive nonciliated bronchiolar epithelial cells of adult human donor lungs, the situation might be different in fetal and neonatal human lungs (22) as well as in macroscopically normal human lung material obtained from surgical resections and in several disease states (Brasch and coworkers, unpublished observations; 11, 27, 63).
In conclusion, our results support the hypothesis that, in the human lung, SP-A is secreted into the alveolar space by type II pneumocytes mainly via an alternative pathway that largely bypasses the lamellar bodies. After secretion, the outer membranes of unwinding lamellar bodies become enriched with SP-A when tubular myelin formation is initiated. SP-A may also be involved in the transition of tubular myelin into the surface film. The present study underlines the remarkable complexity in the localization of SP-A within the different morphologic forms of intracellular and intra-alveolar surfactant. In addition, the usefulness of stereologic methods to perceive a weak signal as specific labeling in surfactant protein distribution studies has been demonstrated. In experimental studies, this quantitative approach could also be used to compare the labeling between different groups.
The authors thank S. Freese, A. Gerken and H. Hühn (Göttingen) and S. Geiger and S. Schaub-Kuhnen (Bochum) for their expert technical assistance.
|1.||Hawgood, S. 1997. Surfactant: composition, structure, and metabolism. In The Lung: Scientific Foundations, 2nd ed. R. G. Crystal, J. B. West, P. J. Barnes, and E. R. Weibel, editors. Lippincott-Raven, Philadelphia. 557–571.|
|2.||Wright J. R.Immunomodulatory functions of surfactant. Physiol. Rev.771997931962|
|3.||Williams M. C.Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs. J. Cell Biol.721977260277|
|4.||Lewis, J. F., R. J. Novick, and R. A. W. Veldhuizen. 1997. Surfactant in Lung Injury and Lung Transplantation. Springer, New York.|
|5.||Hawgood, S. 1992. The hydrophilic surfactant protein SP-A: molecular biology, structure and function. In Pulmonary Surfactant: From Molecular Biology to Clinical Practice. B. Robertson, L. M. G. Van Golde, and J. J. Batenburg, editors. Elsevier, Amsterdam. 33–54.|
|6.||McCormack F. X.Structure, processing and properties of surfactant protein A. Biochim. Biophys. Acta14081998109131|
|7.||Khubchandani K. R., Snyder J. M.Surfactant protein A (SP-A): the alveolus and beyond. FASEB J.1520015969|
|8.||Korfhagen T. R., Bruno M. D., Ross G. F., Huelsman K. M., Ikegami M., Jobe A. H., Wert S. E., Stripp B. R., Morris R. E., Glasser S. W., Bachurski C. J., Iwamoto H. S., Whitsett J. A.Altered surfactant function and structure in SP-A gene targeted mice. Proc. Natl. Acad. Sci. USA93199695949599|
|9.||Ikegami M., Korfhagen T. R., Whitsett J. A., Bruno M. D., Wert S. E., Wada K., Jobe A. H.Characteristics of surfactant from SP-A-deficient mice. Am. J. Physiol.2751998L247L254|
|10.||Hawgood S., Benson B. J., Schilling J., Damm D., Clements J. A., White R. T.Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28–36 in surfactant lipid adsorption. Proc. Natl. Acad. Sci. USA8419876670|
|11.||Mason R. J., Greene K., Voelker D. R.Surfactant protein A and surfactant protein D in health and disease. Am. J. Physiol.2751998L1L13|
|12.||Yukitake K., Brown C. L., Schlueter M. A., Clements J. A., Hawgood S.Surfactant apoprotein A modifies the inhibitory effect of plasma proteins on surfactant activity in vivo. Pediatr. Res.3719952125|
|13.||Elhalwagi B. M., Zhang M., Ikegami M., Iwamoto H. S., Morris R. E., Miller M. L., Dienger K., McCormack F. X.Normal surfactant pool sizes and inhibition-resistant surfactant from mice that overexpress surfactant protein A. Am. J. Respir. Cell Mol. Biol.211999380387|
|14.||Griese M.Pulmonary surfactant in health and human lung diseases: state of the art. Eur. Respir. J.13199914551476|
|15.||Rooney S. A., Young S. L., Mendelson C. R.Molecular and cellular processing of lung surfactant. FASEB J.81994957967|
|16.||Skepper J. N.Immunocytochemical strategies for electron microscopy: choice or compromise. J. Microsc.1992000136|
|17.||Mayhew, T. M., J. M. Lucocq, and G. Griffiths. 2002. Relative labelling index (RLI): a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on TEM thin sections. J. Microsc. (In press)|
|18.||Fehrenbach H., Schmiedl A., Wahlers T., Hirt S. W., Brasch F., Riemann D., Richter J.Morphometric characterisation of the fine structure of human type II pneumocytes. Anat. Rec.24319954962|
|19.||Fehrenbach H., and M. Ochs. 1998. Studying lung ultrastructure. In Methods in Pulmonary Research. S. Uhlig and A. E. Taylor, editors. Birkhäuser, Basel. 429–454.|
|20.||Joe P., Wallen L. D., Chapin C. J., Lee C. H., Allen L., Han V. K. M., Dobbs L. G., Hawgood S., Kitterman J. A.Effects of mechanical factors on growth and maturation of the lung in fetal sheep. Am. J. Physiol.2721997L95L105|
|21.||Griffiths, G. 1993. Fine structure immunocytochemistry. Springer, Berlin.|
|22.||Khoor A., Gray M. E., Hull W. M., Whitsett J. A., Stahlman M. T.Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithelium in the human fetus and newborn. J. Histochem. Cytochem.41199313111319|
|23.||Floros J., Hoover R. R.Genetics of the hydrophilic surfactant proteins A and D. Biochim. Biophys. Acta14081998312322|
|24.||Morotti R. A., Cangiarella J., Gutierrez M. C., Jagirdar J., Askin F., Singh G., Profitt S. A., Wert S. E., Whitsett J. A., Greco M. A.Congenital cystic adenomatoid malformation of the lung (CCAM): evaluation of the cellular components. Hum. Pathol.301999618625|
|25.||Walker S. R., Williams M. C., Benson B.Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung. J. Histochem. Cytochem.34198611371148|
|26.||Haller E. M., Shelley S. A., Montgomery M. R., Balis J. U.Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms. J. Histochem. Cytochem.40199214911500|
|27.||Balis J. U., Paterson J. F., Paciga J. E., Haller E. M., Shelley S. A.Distribution and subcellular localization of surfactant-associated glycoproteins in human lung. Lab. Invest.521985657669|
|28.||DeMello D. E., Heyman S., Phelps D. S., Floros J.Immunogold localization of SP-A in lungs of infants dying from respiratory distress syndrome. Am. J. Pathol.142199316311640|
|29.||Voorhout W. F., Veenendaal T., Haagsman H. P., Verkleij A. J., Van Golde L. M. G., Geuze H. J.Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice. J. Histochem. Cytochem.39199113311336|
|30.||Stratton, C. J. 1984. Morphology of surfactant producing cells and of the alveolar lining layer. In Pulmonary Surfactant. B. Robertson, L. M. G. Van Golde, and J. J. Batenburg, editors. Elsevier, Amsterdam. 67–118.|
|31.||Savov J., Wright J. R., Young S. L.Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and Clara cells. Am. J. Physiol.2792000L118L126|
|32.||Gil J., Reiss O. K.Isolation and characterization of lamellar bodies and tubular myelin from rat lung homogenates. J. Cell Biol.581973152171|
|33.||Ikegami M., Jobe A. H.Surfactant protein metabolism in vivo. Biochim. Biophys. Acta14081998218225|
|34.||Froh D., Ballard P. L., Williams M. C., Gonzales J., Goerke J., Odom M. W., Gonzales L. W.Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro. Biochim. Biophys. Acta105219907889|
|35.||Oosterlaken-Dijksterhuis M. A., Van Eijk M., Van Buel B. L. M., Van Golde L. M. G., Haagsman H. P.Surfactant protein composition of lamellar bodies isolated from rat lung. Biochem. J.2741991115119|
|36.||Froh D., Gonzales L. W., Ballard P. L.Secretion of surfactant protein A and phosphatidylcholine from type II cells of human fetal lung. Am. J. Respir. Cell Mol. Biol.81993556561|
|37.||Rooney S. A., Gobran L. I., Umstead T. M., Phelps D. S.Secretion of surfactant protein A from rat type II pneumocytes. Am. J. Physiol.2651993L586L590|
|38.||Ikegami M., Ueda T., Purtell J., Woods E., Jobe A.Surfactant protein A labeling kinetics in newborn and adult rabbits. Am. J. Respir. Cell Mol. Biol.101994413418|
|39.||Doyle I. R., Barr H. A., Nicholas T. E.Distribution of surfactant protein A in rat lung. Am. J. Respir. Cell Mol. Biol.111994405415|
|40.||Osanai K., Mason R. J., Voelker D. R.Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol.191998929935|
|41.||Snyder J. M., Mendelson C. R.Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development. Biochim. Biophys. Acta9201987226236|
|42.||O'Reilly M. A., Nogee L., Whitsett J. A.Requirement of the collagenous domain for carbohydrate processing and secretion of a surfactant protein, SP-A. Biochim. Biophys. Acta9691988176184|
|43.||Young S. L., Ho Y. S., Silbajoris R. A.Surfactant apoprotein in adult rat lung compartments is increased by dexamethasone. Am. J. Physiol.2601991L161L167|
|44.||Alcorn J. L., Mendelson C. R.Trafficking of surfactant protein A in fetal rabbit lung in organ culture. Am. J. Physiol.2641993L27L35|
|45.||Clements, J. A. 1986. Some relationships among structure, composition, and functional characteristics of lung surfactant. In Physiology of the Fetal and Neonatal Lung. D. V. Walters, L. B. Strang, and F. Geubelle, editors. MTP Press, Lancaster, UK. 169–182.|
|46.||Williams M. C.Vesicles within vesicles: what role do multivesicular bodies play in alveolar type II cells? Am. Rev. Respir. Dis.1351987744746|
|47.||Kalina M., Socher R.Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells. J. Histochem. Cytochem.381990483492|
|48.||Voorhout W. F., Veenendaal T., Haagsman H. P., Weaver T. E., Whitsett J. A., Van Golde L. M. G., Geuze H. J.Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment. Am. J. Physiol.2631992L479L486|
|49.||Kalina M., McCormack F. X., Crowley H., Voelker D. R., Mason R. J.Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro. J. Histochem. Cytochem.4119935770|
|50.||Ryan R. M., Morris R. E., Rice W. R., Ciraolo G., Whitsett J. A.Binding and uptake of pulmonary surfactant protein A (SP-A) by pulmonary type II epithelial cells. J. Histochem. Cytochem.371989429440|
|51.||Young S. L., Fram E. K., Larson E., Wright J. R.Recycling of surfactant lipid and apoprotein-A studied by electron microscopic autoradiography. Am. J. Physiol.2651993L19L26|
|52.||Stevens P. A., Wissel H., Zastrow S., Sieger D., Zimmer K. P.Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes. Am. J. Physiol.2802001L141L151|
|53.||Palaniyar N., Ridsdale R. A., Hearn S. A., Possmayer F., Harauz G.Formation of membrane lattice structures and their specific interaction with surfactant protein A. Am. J. Physiol.2761999L642L649|
|54.||Baritussio A., Alberti A., Quaglino D., Pettenazzo A., Dalzoppo D., Sartori L., Pasquali-Ronchetti I.SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant. Am. J. Physiol.2661994L436L447|
|55.||Ochs M., Nenadic I., Fehrenbach A., Albes J. M., Wahlers T., Richter J., Fehrenbach H.Ultrastructural alterations in intraalveolar surfactant subtypes after experimental ischemia and reperfusion. Am. J. Respir. Crit. Care Med.1601999718724|
|56.||Williams M. C., Benson B. J.Immunocytochemical localization and identification of the major surfactant protein in adult rat lung. J. Histochem. Cytochem.291981291305|
|57.||Coalson J. J., Winter V. T., Martin H. M., King R. J.Colloidal gold immunoultrastructural localization of rat surfactant. Am. Rev. Respir. Dis.1331986230237|
|58.||Schürch S., Qanbar R., Bachofen H., Possmayer F.The surface-associated surfactant reservoir in the alveolar lining. Biol. Neonate6719956176|
|59.||Veldhuizen R. A., Inchley K., Hearn S. A., Lewis J. F., Possmayer F.Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates. Biochem. J.2951993141147|
|60.||Wright J. R., Benson B. J., Williams M. C., Goerke J., Clements J. A.Protein composition of rabbit alveolar surfactant subfractions. Biochim. Biophys. Acta7911984320332|
|61.||Phelps D. S., Floros J.Localization of surfactant protein synthesis in human lung by in situ hybridization. Am. Rev. Respir. Dis.1371988939942|
|62.||Massaro G. D., Singh G., Mason R. J., Plopper C. G., Malkinson A. M., Gail D. B.Biology of the Clara cell. Am. J. Physiol.2661992L101L106|
|63.||Broers J. L. V., Jensen S. M., Travis W. D., Pass H., Whitsett J. A., Singh G., Katyal S. L., Gazdar A. F., Minna J. D., Linnoila R. I.Expression of surfactant associated protein-A and Clara cell 10 kilodalton mRNA in neoplastic and non-neoplastic human lung tissue as detected by in situ hybridization. Lab. Invest.661992337346|
Abbreviations: relative specific labeling index, RSLI; surfactant protein A, SP-A.
(Received in original form March 21, 2001 and in revised form August 31, 2001)