INTRODUCTION

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Pulmonary surfactant is a highly surface-active material that lines the alveolar surface of the lung. It is a lipoprotein product of the type II pneumocyte, which reduces surface tension at the air-liquid interface in the alveolus, thereby preventing collapse of lung parenchyma at the end of expiration (1, 2). Lung immaturity with insufficient surfactant can lead to respiratory distress syndrome (RDS), a major cause of illness in premature infants (1, 3). Surfactant deficiencies or abnormalities are also believed to play a contributory role in the adult respiratory distress syndrome (ARDS) (3, 4). There is increasing evidence that surfactant also has a role in host defense mechanisms and immunologic function of the lung (5). Inherited mutations in surfactant apoprotein B (SP-B deficiency) have been identified recently as a cause of respiratory failure in term infants (6). The histopathological changes in the lungs of SP-B-deficient infants resemble the adult form of alveolar proteinosis or a mix of proteinosis and desquamative interstitial pneumonitis (6, 7). The lung parenchyma lacks SP-B in alveolar type II cells and on ultrastructural examination, the number and location of lamellar bodies appear abnormal (8). Several different mutations of the SP-B gene have been identified, which may account for the heterogeneity of clinical phenotypes (9, 10).

We report a cast of a full-term female infant with unexplained, severe respiratory failure, without SP-B deficiency, in whom ultrastructural findings suggest a defect in the assembly of lamellar bodies in alveolar type II cells.

	CASE REPORT

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This female infant was delivered at 42 wk to consanguineous parents after an uneventful pregnancy. The birth was a spontaneous vaginal delivery with Apgar scores of 9 at 1 min and 9 at 5 min. Minimal resuscitation was required and birth weight was 3,290 g. At 2 h of age she was noted to have signs of respiratory distress and was placed in oxygen. At 4 h of age a septic workup was carried out and ampicillin and gentamicin were commenced. A chest radiograph at that time revealed bilateral granular appearance throughout the lung fields with air bronchograms. Several hours later, she required intubation and mechanical ventilation because of increasing oxygen requirements. Surfactant (bovine lipid extract surfactant [BLES] produced by BLES Biochemicals Inc., London, ON, Canada) was given with some response (oxygen requirements decreased from 80% to 40%). A two-dimensional (2D) echocardiogram revealed normal anatomy, a small patent ductus, some tricuspid regurgitation, and pulmonary artery pressure just less than systemic pressure. On the third day of life, she was transferred to The Hospital for Sick Children because of increasing respiratory failure.

Repeat chest radiograph showed changes consistent with ARDS (Figure 1). She remained to severe respiratory failure (oxygenation index 48), and had only a transient response to repeat doses of surfactant. Bronchoalveolar lavage was negative for bacterial, fungal, or viral organisms. At 17 d of age, an open biopsy was performed complicated by pneumothoraces.

View larger version (157K): [in this window] [in a new window]   Figure 1.   Chest radiograph on third day of life showing extensive opacities and air bronchograms, consistent with ARDS.

Initial examination of the lung biopsy revealed a diffuse increase in the number of type II cells lining the alveoli with alveolar spaces filled by numerous macrophages, a picture suggestive of desquamative interstitial pneumonitis (DIP). Treatment with high-dose methylprednisolone and later chloroquine was commenced with no significant response. At 23 d of age, there was no significant improvement in her condition, and ventilatory support was withdrawn after discussion with the parents. Permission for postmortem examination was refused.

	METHODS

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Part of the lung biopsy was processed for histology after fixation in 10% neutral buffered formalin, snap-frozen for histochemical studies, or processed for transmission electron microscopy (TEM) using standard methods.

For histopathologic assessment, paraffin sections were stained with hematoxylin-eosin, periodic acid-Schiff (PAS) with and without diastase treatment, Masson trichrome and Van Gieson stains for connective tissue elements. Frozen sections were stained for neutral lipids using oil-red-O stain. Frozen sections were also used to demonstrate cellular acid or alkaline phosphatase content using standard enzyme histochemistry.

For immunohistochemical studies, indirect immunoperoxidase method was employed on formalin-fixed paraffin sections. The following primary monoclonal antibodies (mab) were used: mab against low-molecular-weight keratin (Mol. no. 8, 18, 19; Becton-Dickinson, Mountain View, CA), mabs against actin, the leukocyte marker CD45RB, the macrophage marker CD68 (all from Dako Corporation, Carpinteria, CA), and proliferating cell nuclear antigen (PCNA) (marker of cell proliferation; Novocastra, Newcastle upon Tyne, UK).

Immunohistochemical analysis of surfactant protein expression was performed in the Molecular Morphology Core, Divisions of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio. Paraffin sections were immunostained for surfactant protein A (SP-A), B (SP-B), pro-SP-B, and pro-SP-C using rabbit polyclonal antibodies (provided by Drs. Jeffrey Whitsett and Timothy Weaver) and a Vectastain ABC Peroxidase Elite Rabbit IgG Kit (Vector Laboratories Inc., Burlingame, CA). The enzymatic reaction product was enhanced with nickel cobalt to give a black precipitate, and the sections were counterstained with nuclear fast red (11).

For TEM studies the sample of lung was fixed in "universal fixative" (1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer), postfixed in 2% osmium tetroxide, and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate followed by lead citrate and examined using a Phillips 400 or 201 electron microscope.

Genomic DNA from the patient's blood sample was examined for the presence of the 121ins2 and R236C mutations by polymerase chain reaction (PCR) amplification and restriction analysis using SfuI and BstU1 as previously described (12, 13). DNA was analyzed for possible novel SP-B gene mutations by direct sequencing of the translated exons of the SP-B gene. Briefly, PCR products corresponding to SP-B gene exons 1 to 10 were generated with the PCR. The amplimers were sequenced by end labeling a specific primer with radioactive phosphorus-deoxyadenosine triphosphate (³³P-dATP) using T₄ polynucleotide kinase (New England BioLabs, Beverly, MA), and cycle sequencing was performed using a Circumvent kit (New England BioLabs). Sequencing reaction products were separated on a 6% polyacrylamide gel, the gel dried, and exposed to Kodak X-ray film overnight. Patient SP-B genomic sequences were compared with published SP-B DNA sequence (14).

	RESULTS

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Light Microscopy

Routine histologic examination of the lung biopsy showed diffuse consolidation of the lung parenchyma caused by septal thickening and an increase in cuboidal type II cells lining the air spaces (Figure 2a). No normal lung parenchyma was present. The alveolar lumen contained clusters of cells with characteristics of macrophages. Occasional acute inflammatory cells and minimal hyaline membrane material were also present. The alveolar septae showed early fibroplasia with minimal fibrosis and scanty inflammatory cells. The histopathologic pattern was reminiscent of DIP with no features of alveolar proteinosis.

View larger version (179K): [in this window] [in a new window]   Figure 2.   (a) Peripheral lung tissue in a lung biopsy shows DIP-like picture with prominent cuboidal cells lining the alveolar spaces. The interstitium contains spindle-shaped fibroblasts and scattered inflammatory type cells. (Hematoxylin-eosin stain; original magnification: ×250.) (b) Immunostaining for low-molecular-weight cytokeratin shows strong positivity in alveolar lining cells. The cells filling the alveolar lumen are negative as expected for macrophages. (Immunperoxidase method for cytokeratin; magnification: ×250.)Figure 3. (a) Histochemical stain for acid phosphatase on a frozen section of lung biopsy shows strong reactivity in the cytoplasm of macrophages within alveolar spaces (AS). The apical cytoplasm of alveolar lining cells shows mild to moderate reactivity. (Acid phosphatase histochemistry; original magnification: ×250.) (b) Histochemistry for alkaline phosphatase, marker for type II cells, shows strong positive reactivity in alveolar lining cells. (Alkaline phosphatase histochemistry; original magnification: ×250.)Figure 4. (a) Immunostaining for SP-A on paraffin sections shows positive reaction in alveolar lining cells and is also associated with alveolar space (AS) contents. (Immunoperoxidase method for SP-A; magnification: ×250.) (b) Immunostaining for SP-B on paraffin sections shows positive reactivity in a similar distribution to that shown in (a) with localization of SP-B in alveolar lining cells and within alveolar space contents. (Immunoperoxidase method for SP-B; magnification: ×250.)

Immunostaining for low-molecular-weight cytokeratin showed strong positive staining of alveolar lining cells and airway epithelium, highlighting the interstitial thickening, whereas cells filling the alveoli were negative (Figure 2b). Immunostaining for actin showed focal staining of smooth muscle in peribronchial areas, vessel walls, and focally within alveolar septae. As expected, the cells filling the alveoli were immunoreactive for CD68 (marker of alveolar macrophages), whereas CD45RD (marker of leukocytes) showed focal staining of septal cells (data not shown).

The cell proliferation marker, PCNA, showed positive reactivity mostly in nuclei of interstitial cells and focally in alveolar lining cells, indicating that the most active replication was taking place in the interstitium (data not shown).

Histochemical studies on frozen sections showed focal positive staining for neutral lipids in alveolar macrophages without staining of epithelial cells. Strong reactivity for acid phosphatase was present in alveolar macrophages and a weak positive reactivity was observed in the apical cytoplasm of alveolar lining cells (Figure 3a). Alkaline phosphatase, a marker of type II cells, was strongly positive in alveolar lining cells but negative in alveolar macrophages (Figure 3b).

Immunostaining for surfactant-associated proteins showed positive reactivity for SP-A, SP-B, pro-SP-B, and pro-SP-C in the lung biopsy sample. Immunoreactivity for SP-A (Figure 4a) and mature SP-B (Figure 4b) was detected in cuboidal cells lining the alveolar spaces and in association with the lumenal contents of alveoli. Pro-SP-B was also detected in alveolar cells and in the lumenal contents (data not shown). Pro-SP-C was restricted to alveolar type II cells; immunoreactive pro-SP-C was not detected in the alveolar spaces (data not shown). Extracellular staining for pro-SP-C is typically observed in infants with hereditary SP-B deficiency, corresponding to partially processed pro-SP-C peptides (6, 11). Analysis of DNA from a blood sample from this patient was negative for known mutations in the SP-B gene, and sequence analysis of the coding exons of the SP-B genes did not reveal any deviations from the published SP-B gene sequence (14).

Electron Microscopy

At the ultrastructural level, the alveolar lining cells consisted mostly of cuboidal cells with surface microvilli characteristic of alveolar type II cells (Figure 5a). The most striking finding was an apparent absence of cytoplasmic lamellar bodies which are typically composed of electron-dense, concentric lamellae with ultrastructural features of phospholipid membranes. Instead, the apical and basolateral cytoplasm contained round to oval electron-dense bodies with ultrastructural features of lysosomes or phagolysosomes and occasional clear or electron-dense multivesicular bodies (Figure 5b). Other cytoplasmic organelles including the mitochondria, endoplasmic reticulum, and Golgi apparatus showed no apparent ultrastructural abnormalities. There was no evidence of glycogen deposits or lipid droplets. The nuclei exhibited finely dispersed chromatin and some contained prominent nucleoli. The alveolar lumen lacked tubular myelin and focally contained finely granular material. The alveolar macrophages contained numerous electron-dense, pleomorphic bodies, some forming concentric lamellae whereas other contained mutlivesicular structures (Figure 5a). The ultrastructural appearance of this material was consistent with engulfed exogenous surfactant, previously administered to this infant. There were no unusual ultrastructural abnormalities noted in any other pulmonary cell type represented in the biopsy material.

View larger version (204K): [in this window] [in a new window]   View larger version (192K): [in this window] [in a new window]   Figure 5.   (a) Low-magnification electron micrograph of part of an alveolar space lined by cuboidal type II cells lacking lamellar body inclusions. These cells have prominent surface membrane microvilli (Mv), large vesicular nuclei (Nu) some with prominent nucleoli (Nc). The basal aspect of type II cells is in contact with the basement membrane (arrow head ) and in close proximity to an alveolar capillary (Cap). An alveolar macrophage in the lumen shows pleomorphic electron-dense cytoplasmic inclusions (arrow) representing phagolysosomes. (TEM; original magnification: ×10,000.) (b) Higher magnification of apical cytoplasm of a type II cell. The plasma membrane shows surface microvilli (Mv). The cytoplasm contains scattered electron-dense inclusions, some with features of primary lysosomes (Ly). Few electron-lucent (arrow) as well as an electron-dense multivesicular bodies (arrow head ) are present. Mitochondria (Mi), Golgi complex (Go), and a nucleus (Nu) show no abnormalities. (TEM; original magnification: ×30,000.)

Because of this unusual ultrastructural appearance of the alveolar type II cells, the findings in this case were compared with other cases of neonatal lung disease and normal controls available in our files (approximately 1,000 cases of pediatric lung biopsies, 80% with TEM studies). Alveolar type II cells from a full-term neonate with no evidence of lung disease (control) showed well-developed concentric lamellar bodies. Similarly, several cases of DIP were reviewed. These cases included DIP caused by rubella infection and an idiopathic case (both responded to treatment with steroids). In both instances well-developed lamellar bodies were identified. Present, but somewhat reduced number of lamellar bodies was noted in type II cell hyperplasia in cases of usual interstitial pneumonitis or in the regenerative phase of diffuse alveolar damage. In our cases of confirmed SP-B deficiency, type II cells showed distinctive alterations characterized by numerous, abnormally large, multivesicular bodies (unpublished observations). Therefore, the ultrastructural features of alveolar type II cells in our index case appear to be unusual and perhaps represent a unique form of surfactant deficiency.

	DISCUSSION

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The most striking finding in our case of a full-term neonate with lethal respiratory disease was an apparent absence of mature lamellar bodies in alveolar type II cells, the principal source of pulmonary surfactant. Transient improvement after administration of exogenous surfactant in our patient is consistent with a notion of primary surfactant deficiency as a basis for this fatal respiratory disease. However, the basic cellular and molecular defect in this case remains unknown. Based on current understanding of alveolar type II cell development and the pathophysiology of the pulmonary surfactant system, several possibilities exist that might explain this potential defect.

The intrauterine development and differentiation of alveolar epithelium, including type II cells, has been reviewed recently (15). In human fetal lung, alveolar type II cells with recognizable lamellar bodies appear between 20 to 24 wk gestation (16). These early type II cells (precursor cells) are characterized by large pools of cytoplasmic glycogen (17). With advancing gestation the synthesis of surfactant increases, while there is a proportional decrease in cytoplasmic glycogen (17). At the time of birth, fully mature type II cells contain numerous well-developed lamellar bodies and lack cytoplasmic glycogen. Therefore, the possibility that our case may simply represent "immature" lung alveolar epithelial cells is unlikely as no glycogen was identified in alveolar cells. In addition, the alveolar cells in our case exhibited classic features of type II cells, i.e., cuboidal shape, apical microvilli, alkaline phosphatase activity (18), and expression of surfactant proteins A, B, and pro-C. It should be noted that during lung development expression of messenger RNA (mRNA) for surfactant-associated proteins in type II cell precursors precedes the appearance of lamellar bodies (19). Likewise, surfactant-associated protein can be detected immunohistochemically before the formation of lamellar bodies (20). These results are consistent with our finding of apparently normal expression of SP-B and SP-A protein in our patient with absent lamellar bodies. The possibility that absent lamellar bodies are a result of type II cell hyperplasia also appears unlikely as our cases of infants with documented regenerative phase of diffuse alveolar damage showed normal lamellar bodies in hyperplastic type II cells.

The cellular and molecular events involved in the synthesis, release, and recycling of surfactant by alveolar type II cells have been recently reviewed (21). It is now well established that alveolar type II cells synthesize, store, and secrete a complex phospholipid-protein material, pulmonary surfactant, which is a mixture of neutral and anionic phospholipids (90%) and proteins (10%) (22). Before exocytosis, this material is stored in lamellar bodies, which contain, in addition to surfactant, lysosomal enzymes (23). The final secretory product is segregated as concentric lamellar bodies which are destined to be secreted into the alveolar space to become part of the surfactant lining layer (24). Once released into the alveolar space, the lamellar body is transformed into a highly ordered, calcium-dependent, lattice structure called tubular myelin, which is believed to be the precursor of the surface-active phospholipid monolayer at the air-liquid interface within the alveolus (25). Williams has found that type II cells can take up substances from the alveoli by the process of adsorptive endocytosis and incorporate this material into lamellar bodies (26). Likewise, both phospholipid and surfactant-associated proteins are taken up by type II cells and recycled back into lamellar bodies (27).

Surfactant proteins are essential for normal surfactant function. Four surfactant-associated proteins have been identified and characterized to date: SP-A, SP-B, SP-C, and SP-D (28). SP-A and SP-B are known to enhance the rate of absorption of phospholipids to the surface monolayer (29, 30). In vitro studies of phospholipid mixtures and surfactant proteins have shown that both SP-A and SP-B are required for the formation of tubular myelin (31). The role of SP-B in lung function has been investigated in animal models in which SP-B activity was blocked with monoclonal antibodies (32) or using mice with targeted deletion of the SP-B gene (33). Mice with homozygous deletion of the SP-B gene show increased number and size of multivesicular and composite bodies (33). These findings indicate that SP-B is essential for the assembly of mature lamellar bodies. Targeted deletion of the SP-A gene, on the other hand, had no effect on the formation of lamellar bodies, although tubular myelin was absent (34).

The clinical course of patients with SP-B deficiency is that of persistent, severe respiratory distress with transient response to administration of exogenous surfactant (6, 9). The histopathology of the lung in SP-B deficiency is characterized by accumulation of proteinaceous material in the alveoli, i.e., alveolar proteinosis and/or features of DIP (6). The latter feature is most likely related to the length of survival and/or secondary to ventilatory treatment as no such changes are present in SP-B deficient mice which die within minutes after birth (33).

In our case the clinical course and histopathologic findings in the lung biopsy were initially suggestive of SP-B deficiency, but SP-B was detected by immunohistochemical analysis. A novel SP-B gene mutation resulting in immunoreactive but nonfunctional SP-B was excluded by direct sequencing of the SP-B gene. The outstanding feature of our case was the virtual absence of lamellar bodies within type II pneumocytes, although the cells did contain numerous electron-dense bodies. Although many details of surfactant processing are not fully understood, fusion of the surfactant-protein-containing vesicular body with lipid is thought to produce a membrane-bound organelle, the composite lamellar body (19, 28). Two populations of multivesicular bodies have been demonstrated in type II pneumocytes, larger ones with an electron-lucent matrix and small ones with an electron-dense matrix (21). The electron-dense bodies that we identified could represent lysosomes and/or multivesicular bodies. The absence of mature lamellar bodies indicates disrupted surfactant processing.

A similar case reported recently (35) suggests that this or a related disorder may be the underlying cause of other rare cases of respiratory failure in the term infants. Because our patient was a product of consanguineous marriage, this disorder is likely to be genetically based with autosomal recessive mode of inheritance. The elucidation of the molecular defect and of the exact mechanisms accounting for the above morphological abnormalities awaits further studies.