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Published ahead of print on September 11, 2003, doi:10.1165/rcmb.2003-0262OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 449-458, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0262OC

Surfactant Protein B in Type II Pneumocytes and Intra-Alveolar Surfactant Forms of Human Lungs

Frank Brasch, Georg Johnen, Alexandra Winn-Brasch, Susan H. Guttentag, Andreas Schmiedl, Nadine Kapp, Yasuhiro Suzuki, Klaus M. Müller, Joachim Richter, Samuel Hawgood and Matthias Ochs

Division of Electron Microscopy, Department of Anatomy, University of Göttingen, Göttingen; Institute of Pathology, University Hospital "Bergmannsheil," Bochum; Biochemical Pharmacology, Faculty of Science, Department of Biology, University of Konstanz, Konstanz, Germany; Department of Pediatrics and Cardiovascular Research Institute, University of California San Francisco, San Francisco, California; Division of Neonatology, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and Department of Ultrastructural Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Address correspondence to: Frank Brasch, M.D., Institute of Pathology, University Hospital "Bergmannsheil", Bürkle-de-la-Camp Platz 1, D-44789 Bochum, Germany. E-mail: Frank.E.Brasch{at}ruhr-uni-bochum.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein B (SP-B) is synthesized by type II pneumocytes as a proprotein (proSP-B) that is proteolytically processed to an 8-kD protein. In human type II pneumocytes, we identified not only proSP-B, processing intermediates of proSP-B, and mature SP-B, but also fragments of the N-terminal propeptide. By means of immunoelectron microscopy, proSP-B and processing intermediates were localized in the endoplasmic reticulum, Golgi vesicles, and few multivesicular bodies in type II pneumocytes in human lungs. A colocalization of fragments of the N-terminal propeptide and mature SP-B was found in multivesicular, composite, and some lamellar bodies. Mature SP-B was localized over the projection core of lamellar bodies and core-like structures in tubular myelin figures. In line with immunoelectron microscopy and Western blot analysis of human type II pneumocytes, a fragment of the N-terminal propeptide was also detected in isolated rat lamellar bodies. In conclusion, our data indicate that the processing of proSP-B occurs between the Golgi complex and multivesicular bodies and provide evidence that a fragment of the N-terminal propeptide and mature SP-B are transported together to the lamellar bodies. In human lungs, mature SP-B is involved in the structural organization of lamellar bodies and tubular myelin by the formation of core particles.

Abbreviations: bronchoalveolar lavage, BAL • composite bodies, cb • recombinant C-terminal propeptide of proSP-B, CproSP-B • immunoelectron microscopy, immuno-EM • lamellar bodies, lb • multivesicular bodies, mvb • recombinant N-terminal propeptide of proSP-B, NproSP-B • surfactant protein B, SP-B


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant is a complex and highly surface-active material composed of ~ 90% lipids and ~ 10% proteins, including the surfactant-associated proteins A, B, C, and D (for review see Ref. 1). Surfactant components are synthesized and secreted by type II pneumocytes of the alveolar epithelium. After the secretion of the characteristic surfactant storing lamellar bodies into the alveolar space, the contents undergo a structural transformation into tubular myelin (2) from which the surface film might be generated. This film covers the alveolar surface at the air–liquid interface and prevents alveolar collapse at low lung volumes by reducing alveolar surface tension in a surface area–dependent manner. Hydrophobic surfactant protein B (SP-B) is an important component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure (3, 4).

SP-B is synthesized as a proprotein in type II pneumocytes. En route from its site of synthesis to the lamellar bodies, the processing to mature SP-B involves the cleavage of the signal peptide, glycosylation of the C-terminus, followed by cleavage of the N-terminal and C-terminal propeptides (5). Previously, it has been shown that the N-terminal propeptide is required for targeting mature SP-B to lamellar bodies in type II pneumocytes (6, 7). Mature SP-B interacts with phospholipids and contributes to the rapid insertion of secreted surfactant phospholipids into the surface film (reviewed in Ref. 8). Although the processing of proSP-B has been studied by immunofluorescence and immunoelectron microscopy (9, 10), the ultrastructural localization of proSP-B, processing intermediates of proSP-B, the N-terminal propeptide, and mature SP-B in human lungs is not well characterized. This may, in part, be due to the fact that it is extremely difficult to preserve the different surfactant forms for immunocytochemical analysis. However, methodological improvements in immunoelectron microscopy (immuno-EM) now allow for good ultrastructural preservation of cellular compartments, lamellar bodies, and tubular myelin, as well as the surface film to study the distribution of the surfactant proteins (11). The aim of the present study was therefore to study the distribution of proSP-B, processing intermediates, the N-terminal propeptide, and mature SP-B in type II pneumocytes as well as to characterize the localization of mature SP-B in pulmonary surfactant forms of human lungs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human Lungs
For the present study, we used eight non-transplanted single human lungs obtained from adult donors. Left and right donor lungs were separated shortly before transplantation. One donor lung was used for transplantation and the contralateral donor lung was fixed at the time of transplantation as soon as the clinical procedure allowed. Fixation was performed by instillation of the fixative via the airways to ensure rapid and uniform fixation as described in detail below. All donor lungs were carefully examined by two board-certified pathologists (K.-M.M., F.B.) by light and electron microscopy. Donor lungs were examined only if they could not be made available for another suitable recipient by the Eurotransplant Foundation Center, Leiden, The Netherlands. Each analysis was done under the blanket agreement for pathology material to exclude a pulmonary disease of the donor. Institutional approval was given for the use of human donor material. As previously reported in detail, none of the nontransplanted donor lungs used for this article showed pathologic changes, and transplanted patients had a favorable outcome (12, 13).

Cloning of proSP-B
Using a full-length human SP-B cDNA mutated to contain an EcoR1 site at position 24 (taking the initiating methionine as position 1) and an Xho site at position 381 as a template, primers spanning residues 24–196 and 282–381 of the preproprotein were used to PCR amplify NproSP-B and CproSP-B, respectively. Both PCR products and the full EcoR1-Xho fragment encoding the full-length proprotein (residues 24–381) were cloned, sequence verified, and ligated into pET-23b vector (Novagen, Madison, WI). pET-23b provided translation initiation at the N-terminus and a six-histidine tail for purification at the C-terminus of the expressed protein.

Expression and Purification of the Recombinant C- and N-Terminal Propeptide of proSP-B
Escherichia coli BL21(DE3)pLysS transformed with recombinant N-terminal propeptide of proSP-B (NproSP-B) and recombinant C-terminal propeptide of proSP-B (CproSP-B) containing pET-23b plasmids were grown in LB broth containing 50 µg/ml ampicillin for 8–12 h to an OD600 of 0.6. IPTG was added to the culture to a final concentration of 0.4 mM to induce expression. After 3 h the broth was centrifuged and the bacterial pellet was lysed by sonication on ice in 20 mM Tris buffer, pH 7.4. The released inclusion granules were purified by centrifugation and washing in Tris buffer and then solubilized in 20 mM Tris, 6 M urea, 50 mM DTT buffer, pH 7.4. Following dilution 1:10 in 20 mM Tris, 6 M urea, 0.5 M NaCl, 3 mM reduced glutathione, 0.3 mM oxidized glutathione buffer, pH 7.4, the soluble inclusion granule contents were dialyzed twice against 10 vols of the same buffer containing 2 M urea, then against 10 vols of 20 mM Tris, 5 mM imidazole, 0.5 M NaCl binding buffer (pH 7.9). After centrifugation, the supernatant was applied to a Ni-NTA agarose column (Novagen). The column was washed and eluted using the supplier's protocols. The eluate containing protein was dialyzed against sodium phosphate buffer, pH 7, and stored in aliquots at -20°C.

Antisera
Polyclonal antibodies were raised in rabbits against human SP-A, sheep SP-B, and recombinant human NproSP-B and CproSP-B, using standard protocols. IgG was purified from the serum by protein-A affinity chromatography (Pierce, Helsingborg, Sweden). A monoclonal antibody against human SP-B was generated by Y.S. Polyclonal rabbit antisera against cow or human SP-B were kindly provided by Dr. J. Whitsett (Cincinatti, OH) or purchased from Chemicon (Temecula, CA). The polyclonal antiserum against the N-flanking propeptide epitope Q186-Q200 (anti–NFlankSP-B) of proSP-B was characterized earlier (10, 14, 15).

Human Bronchoalveolar Lavage
In the present study, Western blot data from bronchoalveolar lavage (BAL) fluids from six adult humans (kindly provided by Dr. F. Bühling, University of Magdeburg, Germany) with no pathologic alterations are presented. BAL was routinely performed for diagnostic purpose during fiberoptic bronchoscopy. No patients were lavaged with more saline than used on those for whom no research use of the BAL were contemplated. Institutional approval was given for the use of human BAL fluids, and patients gave informed consent for the use of clinically unused material.

Isolated Human Type II Pneumocytes
Isolated type II pneumocytes were prepared as previously described (16). Briefly, human fetal lung from second trimester therapeutic abortions (14–23 wk estimated gestational age) were obtained under protocols approved by the Committee for Human Research, Children's Hospital of Philadelphia. Fetal lung parenchyma was dissected free of large airways, chopped into 1 mm3 explants, and cultured in Waymouth media on a rocking platform. After overnight culture as explants, epithelial cells were isolated by digestion with trypsin, collagenase, and DNase followed by panning on plastic culture dishes to remove adherent fibroblasts. Cells were then cultured for up to 7 d in 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine to induce type 2 cell differentiation. Cells were frozen at –70°C for protein analysis.

Rat Lungs
Eight male Wistar rats were obtained from the Zentralinstitut für Versuchstierforschung in Hannover, Germany. All animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85–92, revised 1985).

Isolation of Rat Lamellar Bodies and Rat BAL
Because it was not possible to purifiy enough lamellar bodies from isolated human type II pneumocytes, and because nontransplanted human donor lungs were completely fixed by instillation for assessment of potential pathologic changes, lamellar bodies were isolated from rat lungs for Western analysis. Lamellar bodies were isolated from four rat lung homogenates by upward flotation on a discontinuous sucrose gradient using a modification of the method of Duck-Chong (17). Cell-free fluid from the first pass of BAL was used for Western blot analysis without prior reduction of the samples.

Fixation, Sampling, and Processing for Immunohistochemistry and Immunoelectron Microscopy
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 (immuno-EM), six single human and four rat lungs were fixed by instillation or perfusion of a mixture of 4% formaldehyde (prepared from freshly depolymerized paraformaldehyde) and 0.1% glutaraldehyde in 0.2 M Hepes buffer. 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 (11).

Labeling Procedure
Immunohistochemistry was performed using the alkaline phosphatase method. Sections of 4 µm thickness were mounted on poly-L-lysine slides, dried overnight, dewaxed with 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 labeling procedure. 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 microscopical analysis, ultrathin sections of 70 nm thickness from five blocks of each lung were prepared. Immunolabeling of ultrathin sections was performed with primary antibodies against SP-B using gold-coupled secondary antibodies (British BioCell, Cardiff, UK) with a gold particle diameter of 10 nm for detection. Ultrathin sections of human lung specimens were labeled for SP-B either with a monoclonal antibody or polyclonal antisera. The monoclonal antibody and the polyclonal antisera against SP-B yielded identical results in human lungs. Ultrathin sections of rat lung specimens were labeled for SP-B with a polyclonal antiserum.

The double labeling for CproSP-B and SP-B, NproSP-B and SP-B, as well as SP-A and SP-B was performed as described above with the following exceptions: the ultrathin sections of human lung specimens were transferred to the primary polyclonal antiserum (anti–CproSP-B, anti–NproSP-B, or anti–SP-A) and the monoclonal antibody against SP-B diluted in blocking buffer for 60 min. Immunoreactivity was visualized by incubation with secondary 5 nm anti-rabbit and 15 nm anti-mouse gold-coupled antibodies diluted in blocking buffer.

The triple labeling for NproSP-B, CproSP-B, and SP-B was performed as described in detail elsewhere (18). Briefly, sections were labeled first with the primary polyclonal antiserum against CproSP-B and the monoclonal antibody against SP-B without having been mounted on a grid. The immunoreaction was visualized by incubation with a secondary 10 nm anti-rabbit and 15 nm anti-mouse gold-coupled antibody. Finally, sections were mounted with the labeled plane downwards on a Formvar-coated copper or nickel grid and dried overnight. The next day, the grids were labeled with the third primary antibody against NproSP-B and a secondary 5 nm anti-rabbit gold-coupled antibody. Control experiments were performed with omission of the primary antibodies or using preimmune serum, nonspecific serum, or irrelevant antibodies (i.e., anti-VEGF). Examination of labeled ultrathin sections was conducted using an EM 900 (LEO, Oberkochen, Germany) at an accelerating voltage of 50 kV. Per individual lung, a minimum of 50 type II pneumocytes (~ 10 type II pneumocytes per block) with at least five lamellar body profiles per cell and a minimum of 200 intra-alveolar unilamellar vesicle profiles were analyzed. Therefore, in the six human lungs used for immuno-EM a minimum of 1,500 lamellar body profiles and 1,200 unilamellar vesicle profiles were investigated. Because our sampling procedure followed the rules of systematic uniform random sampling our data can therefore be regarded as unbiased and, by definition, representative for the whole lung (11).

Detection of Surfactant Proteins by Western Blot Analysis
For immunoblotting, BAL fluids or extracts of type II pneumocytes were separated on 4–12% NuPage Bis-Tris polyacrylamide gels and then transferred to nitrocellulose membranes according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). After blocking (2 h in 5% fetal calf serum/0.1% Tween20/0.5% bovine serum albumin in Tris-buffered saline pH 7.5), membranes were incubated overnight with antibodies against NproSP-B, NFlankSP-B, CproSP-B, or SP-B. As secondary antibody, an anti-rabbit IgG alkaline phosphatase conjugate (substrate: NBT/BCIP) was used (Promega, Madison, WI). For the detection of proSP-B, proSP-B processing intermediates, fragments of the N-terminal propeptide, and mature SP-B, all Western blots were performed under nonreducing conditions. The specificity of the antisera against SP-A was shown previously (11).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of proSP-B, Processing Intermediates of proSP-B, and Fragments of the N-Terminal Propeptide in Isolated Human Type II Pneumocytes and Isolated Rat Lamellar Bodies
Both anti–CproSP-B (Figure 1a) and anti–NproSP-B (Figure 1b, lane 1) identified three bands at ~ 39 kD, ~ 40 kD, and ~ 42 kD in isolated type II pneumocytes corresponding to (pre)proSP-B (~ 40 kD), nonglycosylated proSP-B (~ 39 kD), and glycosylated proSP-B (~ 42 kD). The ~ 23 kD proSP-B processing intermediate was detected by anti–CproSP-B (Figure 1a) and anti–NFlankSP-B (Figure 1c), whereas only anti–NFlankSP-B identifed a ~ 9-kD proSP-B processing intermediate (Figure 1c). The ~ 23-kD and ~ 9-kD proSP-B processing intermediates were not detected by anti–NproSP-B (Figure 1b, lane 1). However, anti–NproSP-B identified ~ 18 kD, ~ 17 kD, ~ 15 kD, and ~ 5 kD fragments of the N-terminal propeptide in human type II pneumocytes (Figure 1b, lane 1). Within isolated rat lamellar bodies, anti–NproSP-B detected only a ~ 5-kD fragment of the N-terminal propeptide (Figure 1b, lane 2).



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  		Figure 1. Western blot analysis of isolated human type II pneumocytes and isolated rat lamellar bodies for proSP-B, proSP-B processing intermediate, and fragments of the N-terminal propeptide. In isolated human type II pneumocytes (hT2), ~ 40 kD (pre)proSP-B, ~ 39 kD non-glycosylated proSP-B, and ~ 42 kD glycosylated proSP-B were detected by anti–CproSP-B (a) and anti–NproSP-B (b, lane 1). Whereas anti–CproSP-B (a) and anti–NFlankSP-B (c) identified a ~ 23-kD proSP-B processing intermediate, anti–NproSP-B (b) detected fragments of the N-terminal propeptide (~ 18 kD, ~ 17 kD, ~ 15 kD, and ~ 5 kD) in isolated human type II pneumocytes (hT2, lane 1) and a ~ 5-kD fragment of the N-terminal propeptide in isolated rat lamellar bodies (rLB, lane 2). Furthermore, anti–NFlankSP-B (c) identified a ~ 9-kD proSP-B processing intermediate in isolated human type II pneumocytes.

 
Subcellular Localization of proSP-B, Processing Intermediates of proSP-B, Fragments of the N-Terminal Propeptide, and Mature SP-B in Type II Pneumocytes in Human Lungs
To localize proSP-B, processing intermediates, fragments of the N-terminal propeptide, and mature SP-B in type II pneumocytes of human lungs, we used specific polyclonal (anti–NproSP-B, anti–CproSP-B, anti–SP-B) and monoclonal antibodies (anti–SP-B). At the light microscopic level, the immunohistochemical reaction product appeared as a fine cytoplasmic (anti–NproSP-B, anti–CproSP-B) or granular (anti–SP-B) red staining (not shown). We next looked at the distribution of CproSP-B and NproSP-B in freeze-substituted Lowicryl HM20 embedded human lung specimens using immunogold EM. At the ultrastructural level, the C-terminal propeptide (CproSP-B, 10 nm gold particles) was localized in the ER, Golgi vesicles (vesicles), and rarely in multivesicular bodies (mvb) (Figure 2a), but not in composite or lamellar bodies (lb). Double labeling for CproSP-B (5-nm gold particles) and SP-B (15-nm gold particles) showed that Golgi vesicles (vesicles) were labeled only for CproSP-B, whereas most mvb and all composite as well as lb contained only SP-B (Figure 2b). Surprisingly, anti–NproSP-B (10-nm gold particles) labeled not only ER and Golgi vesicles, but also many mvb, composite bodies (cb), and some lb (Figures 2c and 2d). Because anti–NproSP-B not only identified fragments of the N-terminal propeptide but also (pre)proSP-B, we performed double and triple labeling experiments for NproSP-B, CproSP-B, and SP-B to further characterize intracellular compartments. Double labeling experiments confirmed that NproSP-B (5-nm gold particles) and SP-B (15-nm gold particles) are colocalized in mvb, cb, and some lb (Figure 2e). Furthermore, triple labeling for NproSP-B (5-nm gold particles), CproSP-B (10-nm gold particles), and SP-B (15-nm gold particles) showed that anti–NproSP-B and anti–CproSP-B labeled the ER, Golgi vesicles (vesicle) and rarely mvb, whereas only anti–NproSP-B and anti–SP-B labeled mvb, cb, and some lb (Figure 2f).



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  		Figure 2. Immunolocalization of CproSP-B, NproSP-B, and SP-B in human type II pneumocytes. Immuno-EM labeling for CproSP-B (10-nm gold particles) (a) and double-labeling for CproSP-B (5-nm gold particles), and SP-B (15-nm gold particles) (b) showed that the endoplasmic reticulum and Golgi vesicles were labeled only for CproSP-B, whereas most of the mvb contained only SP-B. Surprisingly, immuno-EM labeling for NproSP-B (10-nm gold particles) (c and d), double-labeling for NproSP-B (5-nm gold particles), and mature SP-B (15-nm gold particles) (e) as well as triple-labeling for NproSP-B (5-nm gold particles), CproSP-B (10-nm gold particles), and mature SP-B (15-nm gold particles) (f) showed a colocalization of NproSP-B and CproSP-B in the endoplasmic reticulum and Golgi vesicles (f), as well as a colocalization of NproSP-B and SP-B in multivesicular, composite (arrow), and some lb (c–e). CproSP-B was rarely found in mvb (f).

 
Ultrastructure of LB and Tubular Myelin in Human and Rat Lungs
Despite the fact that we used the same protocol for the preparation of human and rat lung specimens, the morphology of the surfactant forms varied markedly between rat and human as described in previous reports (2, 19, 20). In the human lung, the lb exposed concentrically arranged lamellae and a projection core (Figures 3b and 3c). Depending on the section plane, the projection core was located in the center or at the periphery of the lb. Whereas the central core was surrounded by concentrically arranged lamellae, the lamellae were attached to the peripheral core at an acute or right angle (Figure 3c). At high magnification, densely stacked membrane segments, randomly arranged, were found in the projection core (Figure 3c). The tubular myelin of human lung exhibited lamellae arranged in a square lattice and either dense core particles with randomly oriented stacks of densely packed disc-like membrane segments (asterisks in Figure 3e) or target-like myelin structures with concentrically arranged densely stacked membranes around an electron lucent center (number signs in Figure 3f). In rat lungs, lb did not contain a projection core (Figures 4a and 4b) and the tubular myelin figures only showed lamellae organized into a square lattice (Figure 4c) (2).



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  		Figure 3. Localization of SP-B in type II pneumocytes and intra-alveolar surfactant forms of human lungs. In type II pneumocytes, SP-B (10-nm gold particles) was found in mvb (a) and over the projection core (pc) of the lb (b–c). In the intra-alveolar surfactant, SP-B (15-nm gold particles) was found over the projection core of lb-like surfactant forms (lbl) (d and e) and in tubular myelin figures either over dense core particles (asterisks) (e) or target-like myelin structures (number signs) (f). No SP-A (5-nm gold particles) was detected in lb in the process of secretion (d). In the intra-alveolar surfactant, we found some lb-like surfactant forms (lbl*) in close proximity to tubular myelin (tm) labeled for SP-A at their periphery (e), whereas the strongest labeling for SP-A was detected over the lattice structure of tubular myelin figures (f).

 


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  		Figure 4. Localization of SP-B in type II pneumocytes and intra-alveolar surfactant forms of rat lungs. A scattered labeling for SP-B was found over lb in type II pneumocytes (a) and also over lb in process of secretion (b). In intra-alveolar tubular myelin figures (tm), SP-B was associated with the lamellae of the lattice structure (c).

 
Distribution of Mature SP-B in LB and Tubular Myelin Figures in Human Lungs
Immunohistochemistry demonstrated that anti-SP-B produced a strong granular staining in type II pneumocytes (not shown). At the ultrastructural level, mature SP-B (10 nm secondary antibody-gold complex) was found in mvb (Figure 3a) and lb in type II pneumocytes (Figures 3b and 3c). At higher magnification, a strong labeling for mature SP-B was restricted to the projection core of the lb (Figure 3c).

Immuno-EM Double Labeling for SP-B and SP-A in Human Lungs
The localization of mature SP-B (15 nm secondary antibody-gold complex) over the projection core was also found in lb in the process of secretion (Figure 3d) as well as intraalveolar lamellar body-like surfactant forms (lbl) (Figures 3d and 3e). In tubular myelin figures, SP-B was localized either over dense core particles (asterisks in Figure 3e) or target-like myelin structures (number signs in Figure 3f). No SP-A (5 nm secondary antibody-gold complex) was detected in lb in the process of secretion (Figure 3d). Furthermore, we identified some lamellar body-like surfactant forms (lbl*) in close proximity to tubular myelin (tm) labeled for SP-A at their periphery (Figure 3e), whereas others (lbl) with no association to tubular myelin figures were devoid of any labeling (Figures 3d and 3e). The strongest labeling for SP-A was detected over the lattice structure of tubular myelin figures (Figure 3f). Whereas SP-A was associated with the surface film and small unilamellar vesicles (11), no SP-B was detected over these structures (not shown). To exclude an artificial distribution of mature SP-B in the human lung produced by one antibody, three different polyclonal antisera and one monoclonal antibody were used. No differences between the polyclonal antisera and the monoclonal antibody were observed.

Distribution of Mature SP-B in LB and Tubular Myelin Figures in Rat Lungs
In the rat lung, a moderate to strong labeling for mature SP-B (10 nm secondary antibody-gold complex) was found over lamellar bodies in type II pneumocytes and also over lamellar bodies in the process of secretion (Figures 4a and 4b). In intra-alveolar tubular myelin figures, mature SP-B was associated with the lamellae of the lattice structure (Figure 4c).

Identification of SP-B in Human Type II Pneumocytes, Isolated Rat LB and Human as well as Rat BAL Fluid
In isolated human type II pneumocytes (T2), monoclonal anti–SP-B identified monomeric (~ 8 kD) and dimeric (~ 18 kD) SP-B (Figure 5a, lane 1). However, monoclonal anti–SP-B detected only dimeric (~ 18 kD) SP-B in human BAL fluids (Figure 5a, lane 2). No differences in specificities were observed for mono- and polyclonal anti–SP-B (Figure 5a, lane 2, and Figure 5b). The polyclonal antisera against SP-B also identified mature dimeric SP-B in rat BAL fluids, but the band was detected at ~ 17 kD and was not as sharp as in the human BAL (Figure 5b, lane 2). In isolated rat lamellar bodies, monomeric (~ 8 kD) and dimeric SP-B (~ 17 kD) were found (Figure 5b, lane 3).



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  		Figure 5. Western blot analysis of isolated human type II pneumocytes, human BAL, rat BAL, and isolated rat lb for SP-B. The monoclonal antibody (a, lane 2) as well as polyclonal antisera against SP-B (b, lane 1) detected mature dimeric ~ 18-kD SP-B in human BAL (hBAL) fluids. In the rat BAL (rBAL) (b, lane 2) and isolated rat lb (rLB) (b, lane 3), the polyclonal antisera also identified mature dimeric SP-B, but the band was detected at ~ 17 kD and was not as sharp as in the human BAL. Monomeric SP-B (~ 8 kD) was only found in isolated human type II pneumocytes (hT2) (a, lane 1) and isolated rat lb (b, lane 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hydrophobic surfactant protein B (SP-B) is an important factor in surfactant homeostasis and hereditary SP-B deficiency leads to fatal neonatal respiratory disease (4). Mature SP-B is a 79 amino acid homodimer of ~ 18 kD that is synthesized as a (pre)proprotein of ~ 40 kD (8, 21). The post-translational modification and processing of the primary translation product involves cleavage of the signal peptide (~40 kD {Rightarrow} ~39 kD), the glycosylation of the C-terminus (~39 kD {Rightarrow} ~42 kD) followed by cleavage of the N-terminal and C-terminal propeptide (5, 22). The post-translational processing of ~ 42 kD glycosylated proSP-B is at least a three-step process with two distinct cleavages of the N-terminal propeptide (~ 42 kD proSP-B {Rightarrow} ~ 23 kD processing intermediate and ~ 9 kD processing intermediate {Rightarrow} ~ 8 kD mature SP-B) and one of the C-terminal propeptide (~ 23 kD processing intermediate {Rightarrow} ~ 9 kD processing intermediate) (10). Autoradiographic, immuno-EM, and immunofluorescence studies demonstrated that proSP-B is processed to mature SP-B en route from the Golgi complex via the multivesicular and composite bodies to the lamellar bodies in type II pneumocytes (Figure 6) (23, 24).



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  		Figure 6. Processing, trafficking, and distribution of SP-B in type II pneumocytes and intra-alveolar surfactant forms in human lungs. En route from its site of synthesis to the lb the post-translational processing of proSP-B to mature SP-B is at least a three-step process with two distinct cleavages of the N-terminal propeptide and one of the C-terminal propeptide (adapted from A. Korimilli and coworkers [10]). The processing of proSP-B to mature SP-B occurs between Golgi vesicles and mvb. The colocalization of fragments of the N-terminal propeptide (ocher dots) and mature SP-B (red dots) in multivesicular, composite, and some lamellar bodies and the identification of a ~ 5-kD fragment of the N-terminal propeptide in lb provide evidence for the concept that the N-terminal propeptide of proSP-B is involved in the transport of mature SP-B to lb (6, 7). In human lungs, mature SP-B is involved in the structural organization of lb by the formation of a projection core. Mature dimeric SP-B is secreted via the lb in the intra-alveolar space (54), whereas SP-A (green dots) largely bypasses the lb (11, 55, 56). After secretion, the outer membranes of unwinding lb become enriched with SP-A when tubular myelin formation is initiated (11). SP-A may also be involved in the transition of tubular myelin into the surface film (11).

 
The proteolytic cleavage of the major part of N-terminal propeptide (~ 42 kD {Rightarrow} ~ 23 kD) is the first processing step of proSP-B, but the fate of the cleaved N-terminal propeptide in type II pneumocytes is not clear (10, 25, 26). However, biochemical data clearly indicated that the N-terminal propeptide is required for targeting SP-B to lamellar bodies (6, 7). Futhermore, incomplete processing of proSP-B and proSP-C in babies with mutations in the N-terminal propeptide region of proSP-B might be due to the inability to route proSP-B and proSP-C to the proper cellular compartments (27). As shown by immuno-EM, our antiserum against the N-terminal propeptide of proSP-B labeled not only the ER and Golgi vesicles, but also multivesicular, composite, and some lamellar bodies. Furthermore, anti–NproSP-B identified not only proSP-B but also ~ 18-kD, ~ 17-kD, ~ 15-kD, and ~ 5-kD fragments of the N-terminal propeptide in isolated human type II pneumocytes. Using only anti–NproSP-B, it is not possible to distinguish between proSP-B and fragments of the N-terminal propeptide in subcellular compartments. However, proSP-B was also detected by anti–CproSP-B, whereas fragments of the N-terminal propeptide or mature SP-B were identified only by anti–NproSP-B or anti–SP-B, respectively. Therefore, we performed an immuno-EM double labeling for NproSP-B and mature SP-B as well as triple labeling for CproSP-B, NproSP-B, and mature SP-B. In line with previous immunofluorescence and immuno-EM studies (9, 10), our immuno-EM data indicate that the cleavage of the C-terminal propeptide occurs between the Golgi complex and multivesicular bodies and that mature SP-B is generated in multivesicular bodies (Figure 6). Because multivesicular, composite, and some lamellar bodies showed a colocalization of NproSP-B and mature SP-B, immuno-EM provides morphologic evidence that mature SP-B and fragments of the N-terminal propeptide are transported together to the lamellar bodies. To corroborate the transport of fragments of the N-terminal propeptide to the lamellar bodies, we isolated these organelles from rat lungs. In line with immuno-EM and Western blot analysis of isolated human type II pneumocytes, a ~ 5-kD fragment of the N-terminal propeptide was also detectable in isolated rat lamellar bodies. Furthermore, a similar fragment of the N-terminal propeptide (N129-L181) was generated from recombinant proSP-B by an aspartic protease present in lamellar bodies (F. Brasch and F. Bühling, unpublished observation). In conclusion, our data provide evidence for the concept that the N-terminal propeptide of proSP-B is necessary for the transport of SP-B to lamellar bodies in type II pneumocytes (6, 7) (Figure 6).

The structural preservation of the lipid-rich surfactant forms and maintenance of antigenicity are a prerequisite for the ultrastructural localization of mature SP-B in the intracellular and intra-alveolar surfactant. Using immuno-EM standard protocols, the lamellar bodies usually appear as empty vacuoles due to the extraction of lipids. Previously, we showed that aldehyde fixation, freeze substitution with methanol containing uranyl acetate and low temperature embedding in Lowicryl HM 20 yielded good ultrastructural preservation of type II pneumocytes and lipid-rich surfactant forms (11). Nevertheless, lamellar bodies and tubular myelin figures are suboptimally fixed for architectural preparation as a compromise to preserve adequate antigenicity. Therefore, caution has to be given to potential artifacts that could lead to errors in interpretation. However, because the structural preservation of pulmonary surfactant forms in our preparations is similar to cryofixed tissue (19, 28, 29), it is unlikely that the organization of phospholipid lamellae reflects an artifact related to chemical fixation and resin embedding.

Surprisingly, mature SP-B was not uniformly distributed over the lamellar bodies in type II pneumocytes but restricted to the projection core in human lungs. At high magnification, this projection core consists of randomly arranged stacks of densely packed membrane segments. To exclude the possibility of the projection core in lamellar bodies in human lungs being fixation artifacts, we prepared rat lungs for immuno-EM according to the same protocol. In rat lungs, we never detected a projection core in lamellar bodies and mature SP-B was distributed over the lamellae. A similar distribution has been reported for the mouse lung in a previous immuno-EM study (30). Qualitative interspecies differences in the substructure of the lamellar bodies have been described by several authors. In many species, granular material, a matrix, or an amorphous core in lamellar bodies has been observed (2, 20, 31–33). However, except for a few monkey species and the lungs of Salamandra salamandra (20, 34), this material is finely granular or amorphic in contrast to randomly arranged stacks of densely packed membrane segments in lamellar bodies in human type II pneumocytes.

Following secretion into the alveolar space, lamellar bodies are converted into tubular myelin (2, 35). Within tubular myelin figures in human lungs, mature SP-B was also localized over dense core particles or target-like myelin structures. We never found those structures within tubular myelin figures in rat lungs, where mature SP-B was distributed over the lamellae of the lattice structure. Differences in the ultrastructural organization of intracellular as well as intra-alveolar surfactant forms in the human and rat lung are therefore reflected by a differential distribution of mature SP-B. Nevertheless, the importance of mature SP-B in the genesis of lamellar bodies and tubular myelin in human and animal lungs has been demonstrated in hereditary SP-B deficiency in humans, isolated human type II pneumocytes, and SP-B-/- mice (3, 36, 37).

Besides its lytic effects on membranes, activities of mature SP-B include: membrane binding, membrane fusion, promotion of lipid adsorption to the air–liquid interface, stabilization of monomolecular surface films, and respreading of films from a collapse phase (reviewed in Ref. 8). In vitro, the permeabilization of membranes by SP-B is associated with a morphologic rearrangement of the vesicular structure (38, 39). The dimeric nature of SP-B may allow it to bring two lipid bilayers into close proximity (40). At relatively high SP-B contents and under isothermal conditions (41, 42), small bilayer discs rapidly fuse to generate large membranous sheets that assemble into multilayered stacks with minimal interbilayer space (38) similar to the projection core in human lamellar bodies as well as the dense core particles or target-like myelin structures in tubular myelin figures. In vitro, fluorescence microscopy revealed highly condensed structures in planar films of phospholipids and bovine SP-B (43). Furthermore, it has been shown that dog and human SP-B binds to phospholipid membranes and appears to irreversibly cluster at the membrane surface (44). Because no ultrastructural investigation of "condensed structures" or the "clusters" has been performed, whether these structures correspond to the aggregates described by Williams and coworkers (38) or to dense core particles/target-like myelin structures of tubular myelin figures remains speculative.

In most in vitro surfactant models, surfactant from canine (45), pig (46), or bovine (43) lung was used. Previously, contradictory experimental data were reported for pig and canine SP-B. Despite ~ 80% sequence identity, pig SP-B remained associated with the surface film, whereas canine SP-B dissociated from the surface film during surface area cycling (45, 46). Furthermore, it is important to bear in mind that the in vitro properties of SP-B are influenced by the method of isolation, the incorporation of SP-B into liposomes, and the size as well as the phospholipid composition of liposomes (47–49). Given the interspecies differences in the function of SP-B (45), the sequence differences between human and rat as well as distinct distribution of SP-B, the function of rat or human SP-B in the formation of lb and tubular myelin might indeed be different.

The importance of SP-A and SP-B for the formation of tubular myelin figures in vivo has been demonstrated in SP-A– and SP-B–deficient mice, as well as in newborns with hereditary SP-B deficiency (36, 50, 51). To reorganize lipids into tubular myelin in vitro, both SP-B and SP-A are necessary (38, 39). To evaluate the relevance of SP-A in the formation of core particles of lb and tubular myelin figures, we performed a double labeling for SP-A and mature SP-B in human lungs. No colocalization of mature SP-B and SP-A was found over the projection core of lb or dense core particles/target-like myelin structures of tubular myelin figures. However, a few lb-like intra-alveolar surfactant forms were labeled for SP-A at their periphery, and SP-A was found in close association with the lamellae of the lattice structure of tubular myelin figures and the surface film as previously described (11). Based on biochemical data and ultrastructural observations, the tubular myelin formation might be an intermediate step in the formation of the surface film (11, 28, 52, 53). Despite clear evidence from in vitro studies that mature SP-B enhances the rate of adsorption of phospholipids from the aqueous subphase to the air–water interface (21), no mature SP-B was found in association with the surface film in human lungs. In line with biochemical data and the structural alterations of the pulmonary surfactant in hereditary SP-B deficiency, in human lungs mature SP-B might be necessary for the structural organization of lb and tubular myelin figures, forming core particles, whereas SP-A is involved in the unwinding of lamellae of secreted lb and the formation of the lattice structure of tubular myelin figures as well as the surface film.

In conclusion, our data are in line with previous results that the processing of proSP-B to mature SP-B occurs between the Golgi complex and mvb (9, 10). For the first time, we showed a colocalization of N-terminal propeptide fragments and mature SP-B in multivesicular, composite, and lamellar bodies. We identified a novel ~ 5-kD fragment of the N-terminal propeptide of proSP-B and provide evidence that this propeptide fragment and mature SP-B are transported together to the lb in type II pneumocytes. The ~ 5-kD N-terminal propeptide fragment might harbor the signal peptide necessary for targeting mature SP-B and SP-C to lb (6, 7, 27). In contrast to rat lungs, we localized mature SP-B over electron dense particles in lb and tubular myelin figures in adult human lungs. Differences in the ultrastructural organization of intracellular as well as intra-alveolar surfactant forms in the human and rat lungs are reflected by a differential distribution of mature SP-B. Mice and rats are generally accepted as animals for models of human pulmonary diseases, and SP-B–deficient mice provided many valuable data for the understanding of hereditary SP-B deficiency in newborns (27, 50). However, further biochemical and biophysical studies on human and animal SP-B are necessary to explain the differences in the structural organization of the lb as well as the tubular myelin.


   Acknowledgments
 
The excellent technical assistance of H. Hühn, S. Freese, A. Gerken (Göttingen), as well as M. Kochem, U. Thomek, and S. Geiger (Bochum) is highly appreciated. The authors thank Dr. Jeffrey Whitsett and collaborators (Cincinnati, OH) for their kind donation of polyclonal SP-B antiserum.

Received in original form July 11, 2003

Received in final form September 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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