SP-D-dependent regulation of NO metabolism in lipopolysaccharide-stimulated peritoneal macrophages.

This work was designed to study the role of surfactant protein D in the regulation of NO synthesis by "non-alveolar" microphages. We evaluated whether the effects of surfactant protein D depend on the phenotype of macrophages. In the absence of surfactant protein D, the LPS-induced iNOS response was shown to decrease in macrophages of native and proinflammatory phenotypes by 30%, and in macrophages of the antiinflammatory phenotype (by 63%). Under the influence of lipopolysaccharide in high doses (500 ng/ml), NO(2)*- production by mouse macrophages without surfactant protein D was reduced in native cells (by 25%), but increased in proinflammatory (by 40%) and antiinflammatory phenotypes (by 12% compared to mouse macrophages with surfactant protein D). Our results suggest that surfactant protein D is involved in the immune response in the whole organism, but not only in the lungs. The effect of surfactant protein D depends on the phenotype of macrophages.

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D.

BACKGROUND: Ozone (O3), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.

Surfactant proteins in pulmonary alveolar proteinosis in adults.

Pulmonary alveolar proteinosis (PAP) is a rare disorder characterised histologically by an intra-alveolar accumulation of fine granular eosinophilic and periodic acid-Schiff positive material. In a retrospective study, the composition of the intra-alveolarly accumulated material of adult patients with PAP was analysed by means of immunohistochemistry and Western blotting. In patients with PAP, the current authors found an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, SP-B, variable amounts of mono-, di-, and oligomeric SP-C forms, as well as SP-D. Only in one patient was a precursor of SP-C detected. By means of immuno-electron microscopy, the current authors identified not only transport vesicles labelled for precursors of SP-B and SP-C, but also transport vesicles containing either precursors of SP-B or SP-C in type-II pneumocytes in normal human lungs. It is concluded that pulmonary alveolar proteinosis in adults is characterised by an intra-alveolar accumulation of surfactant protein A, precursors of surfactant protein B, and surfactant proteins B, C and D. The current data provide evidence that not only an impairment of surfactant clearance by alveolar macrophages, but also an abnormal secretion of transport vesicles containing precursors of surfactant protein B (but not surfactant protein C) and an insufficient palmitoylation of surfactant protein C, which may lead to the formation of di- and oligomeric surfactant protein C forms, play a role in the pathogenesis of pulmonary alveolar proteinosis.

Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene.

Mutations in the surfactant protein C gene (SFTPC) were recently reported in patients with interstitial lung disease. In a 13-month-old infant with severe respiratory insufficiency, a lung biopsy elicited combined histological patterns of nonspecific interstitial pneumonia and pulmonary alveolar proteinosis. Immunohistochemical and biochemical analyses showed an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, mature SP-B, aberrantly processed proSP-C, as well as mono- and dimeric SP-C. Sequencing of genomic DNA detected a de novo heterozygous missense mutation of the SFTPC gene (g.1286T>C) resulting in a substitution of threonine for isoleucine (173T) in the C-terminal propeptide. At the ultrastructural level, abnormal transport vesicles were detected in type-II pneumocytes. Fusion proteins, consisting of enhanced green fluorescent protein and wild-type or mutant proSP-C, were used to evaluate protein trafficking in vitro. In contrast to wild-type proSP-C, mutant proSP-C was routed to early endosomes when transfected into A549 epithelial cells. In contrast to previously reported mutations, the 173T represents a new class of surfactant protein C gene mutations, which is marked by a distinct trafficking, processing, palmitoylation, and secretion of the mutant and wild-type surfactant protein C. This report heralds the emerging diversity of phenotypes associated with the expression of mutant surfactant C proteins.

Synthesis and post-translational processing of surfactant protein C.

Traditional thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. Accumulated data now suggests that the four major surfactant proteins (SPs) are a biochemically and functionally diverse group of mammalian peptides that have function beyond modification of alveolar surface tension. Alveolar SP-C (SP-C3.7, Mr 21,000) is 35 amino acid peptide isolated from lung surfactant that is synthesized and processed from a 191-197 amino acid precursor (proSP-C21). Although its solubility in organic solvents and avidity for lipid membranes impart properties important for its biophysical activity, SP-C represents a structurally and functionally challenging protein for the alveolar type II cell that must synthesize and traffic the peptide through the regulated secretory pathway. Despite technical and analytical difficulties imposed by its unique structure, our current understanding of SP-C biosynthesis has evolved over the past 10 years. Recent data now require us to consider proSP-C21 as a hybrid molecule incorporating structural and functional features both of bitopic integral membrane proteins as well us more classically recognized propeptide hormones. Our article highlights major developments related to characterization of molecular and cellular mechanisms underlying expression, post-translational processing, and targeting of proSP-C21 that result in production of secreted SP-C3.7.

Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.

Aspergillus fumigatus-induced allergic airway inflammation alters surfactant homeostasis and lung function in BALB/c mice.

The differential regulation of pulmonary surfactant proteins (SPs) is demonstrated in a murine model of Aspergillus fumigatus (Af )-induced allergic airway inflammation and hyperresponsiveness. BALB/c mice were sensitized intraperitoneally and challenged intranasally with Af extract. Enzyme-linked immunosorbent assay analysis of serum immunoglobulin (Ig) levels in these mice showed markedly increased total IgE and Af-specific IgE and IgG1. This was associated with peribronchial/perivascular tissue inflammation, airway eosinophilia, and secretion of interleukin (IL)-4 and IL-5 into the bronchoalveolar lavage fluid (BALF). Functional analysis revealed that in comparison with nonsensitized mice, allergic sensitization and challenge resulted in significant increases in acetylcholine responsiveness. To analyze levels of SPs, the cell-free supernate of the BALF was further fractionated by high-speed (20,000 x g) centrifugation. After sensitization and challenges, the pellet (large-aggregate fraction) showed a selective downregulation of hydrophobic SPs SP-B and SP-C by 50%. This reduction was reflected by commensurate decreases in SP-B and SP-C messenger RNA (mRNA) expression of the lung tissue of these animals. In contrast, there was a 9-fold increase in SP-D protein levels in the 20,000 x g supernate without changes in SP-D mRNA. The increased levels of SP-D showed a significant positive correlation with serum IgE (r = 0.85, P < 0.001). Tissue mRNA and protein levels of SP-A in either the large- or the small-aggregate fractions were unaffected. Our data indicate that allergic airway inflammation induces selective inhibition of hydrophobic SP synthesis accompanied by marked increases in the lung collectin SP-D protein content of BALF. These changes may contribute significantly to the pathophysiology of Af-induced allergic airway hyperresponsiveness.

Pneumocystis carinii pneumonia alters expression and distribution of lung collectins SP-A and SP-D.

Surfactant proteins SP-A and SP-D, members of the collectin family, have been shown to play a significant role in lung host defense. Both proteins selectively bind Pneumocystis carinii (PC) organisms and modulate the interaction of this pathogen with alveolar macrophages. We hypothesized that the expression and distribution of lung collectins SP-A and SP-D is altered by PC lung infection. PC organisms (2 x 10(5)) were inoculated intratracheally into C.B-17 scid/scid mice that do not require steroids for immunosuppression. Four weeks after inoculation, bronchoalveolar lavage (BAL) fluid was fractionated into three fractions-cell pellet, large aggregate (LA), and small aggregate (SA) surfactant-and each fraction was analyzed for the expression of surfactant components. In uninfected mice, the majority of SP-A (62% +/- 10%) was found in association with lipids in the LA fraction, while 55% +/- 14% of SP-D was distributed in the SA fraction. In contrast, both hydrophobic proteins SP-B and SP-C were associated exclusively with LA. PC infection resulted in major changes in the expression of all surfactant components. Total protein content of LA was unchanged by PC infection (115% +/- 18% of control), whereas SA protein content markedly increased (240% +/- 18% of control level, P <.001). In contrast, the phospholipid content of LA was significantly decreased (53% +/- 5% of control level, P <.001), whereas the SA phospholipid content of infected mice was increased (172% +/- 16% of control level, P <.001). By Western blotting, PC pneumonia (PCP) induced a 3-fold increase in the total alveolar SP-D protein that was reflected mainly in increases in SA SP-D (454% +/- 135% of control, P <.05). The total alveolar SP-A protein content was also increased in PCP because of a large increase in SP-A in SA (720% +/- 115% of control, P <.05); SP-A levels in LA were unchanged. The increases in lung collectin expression were selective, because PCP resulted in the down-regulation of both SP-B and SP-C in LA (5% +/- 2% and 13% +/- 2% of control, respectively, P <.001). We conclude that PCP induces marked elevations in alveolar collectin levels because of increased expression and accumulation of SP-A and SP-D protein in SA surfactant.

Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.

Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.

Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues.

Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1-194)), C-terminally deleted proSP-C (EGFP/SP-C(1-185); EGFP/SP-C(1-191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1-194) and EGFP/SP-C(1-191 )were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1-185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (&ggr;)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.

Very low surfactant protein C contents in newborn Belgian White and Blue calves with respiratory distress syndrome.

We have studied a respiratory distress syndrome (RDS) occurring in newborn calves of the Belgian White and Blue (BWB) breed that represents the large majority of beef cattle in Belgium. Pulmonary surfactant isolated from 14 BWB newborn calves that died from RDS and from 7 healthy controls was analysed for composition and surface activity. An extremely low content or, in some instances, an absence of surfactant protein C (SP-C) was detected in the RDS samples by Western blotting and differential amino acid analysis [0.03+/-0.01% (w/w) relative to total phospholipids, compared with 0.39+/-0.06% for healthy controls (means+/-S.E.M., P < 0.001)]. The contents of surfactant protein B (SP-B) were similar in RDS and control samples. The crude surfactant samples isolated from RDS calves had higher ratios of total protein to total phospholipid, altered phospholipid profiles and lower SP-A contents. Both crude and organic extracts of RDS surfactant samples showed increased dynamic surface tension compared with healthy controls when evaluated with a pulsating-bubble surfactometer. The addition of purified SP-C to organic extracts of RDS surfactant samples lowered surface tension. Strongly decreased levels of mature SP-C associated with fatal RDS and altered surface activity in vitro have, to the best of our knowledge, not been previously reported. The mechanisms underlying RDS and the decrease in SP-C in BWB calves remain to be established.

Pulmonary surfactant metabolism in infants lacking surfactant protein B.

Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.

P. carinii induces selective alterations in component expression and biophysical activity of lung surfactant.

Studies of Pneumocystis carinii pneumonia (PCP) suggest an important role for the surfactant system in the pathogenesis of the hypoxemic respiratory insufficiency associated with this infection. We hypothesized that PCP induces selective alterations in alveolar surfactant component expression and resultant biophysical properties. PCP was induced by intratracheal inoculation of 2 x 10(5) P. carinii organisms into C.B-17 scid/scid mice. Six weeks after inoculation, large (LA)- and small (SA)-aggregate surfactant fractions were prepared from bronchoalveolar lavage fluids and analyzed for expression of surfactant components and for biophysical activity. Total phospholipid content was significantly reduced in LA surfactant fractions from mice infected with PCP (53 +/- 15% of uninfected mice; P < 0.05). Quantitation of hydrophobic surfactant protein (SP) content demonstrated significant reductions of alveolar SP-B and SP-C protein levels in mice with PCP compared with those in uninfected mice (46 +/- 7 and 19 +/- 6%, respectively; P < 0.05 for both). The reductions in phospholipid, SP-B, and SP-C in LA fractions measured during PCP were associated with an increase in the minimum surface tension of LAs as measured by pulsating bubble surfactometer (13.1 +/- 1.1 vs. 5.4 +/- 1.8 mN/m; P < 0.05). In contrast to decreases in the hydrophobic SPs, SP-D content in the SA fraction was markedly increased (343 +/- 30% of control value; P < 0. 05) and SP-A levels in LA surfactant were maintained (93 +/- 26% of control value) during P. carinii infection. In all cases, the changes in SP content were reflected by commensurate changes in the levels of mRNA. We conclude that PCP induces selective alterations in surfactant component expression, including profound decreases in hydrophobic protein contents and resultant increases in surface tension. These changes, demonstrated in an immunologically relevant animal model, suggest that alterations in surfactant could contribute to the hypoxemic respiratory insufficiency observed in PCP.

Structural requirements for intracellular targeting of SP-C proprotein.

Rat surfactant protein (SP) C is synthesized as a 194-amino acid proprotein that is proteolytically processed to a 35-amino acid mature form in subcellular compartments distal to the medial Golgi compartment. To identify domains of SP-C proprotein (proSP-C) necessary for endoplasmic reticulum translocation and for targeting to cytosolic processing compartments, we characterized expression patterns of heterologous SP-C fusion proteins in A549 lung epithelial cells and in the rat pheochromocytoma cell line PC-12. cDNA constructs were produced; these constructs encoded fusion proteins consisting of enhanced green fluorescent protein (EGFP) and wild-type proSP-C (EGFP/SP-C(1-194)), mature SP-C (EGFP/SP-C(24-59)), or progressive deletions of the NH(2)- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/SP-C(1-194) transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain Phe(24) to His(59), whereas a motif for targeting to cytosolic vesicular compartments was localized to the NH(2) flanking domain Met(10) to Gln(23). Truncations of the distal COOH terminus were retained in the endoplasmic reticulum/Golgi compartment; however, the COOH flanking region alone was insufficient for targeting. In PC-12 cells, EGFP/SP-C(1-194) was expressed in peripheral cytosolic vesicles, whereas EGFP/SP-C(24-194) and EGFP/SP-C(24-59) were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C (Phe(24) to Leu(58)) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas Met(10) to Gln(23), but not the COOH flanking peptide, is required for targeting to late vesicular compartments.

Inhibition of lung surfactant protein B expression during Pneumocystis carinii pneumonia in mice.

The pathogenesis of Pneumocystis carinii pneumonia (PCP) suggests an important role for dysfunction of the pulmonary surfactant system in the hypoxemic respiratory insufficiency associated with this infection. Surfactant protein B (SP-B) is a hydrophobic protein shown to be essential for normal surfactant function in vivo. Therefore, we hypothesized that the inhibition of SP-B expression occurs during PCP, and we tested this hypothesis in two immunodeficient animal models. PCP was induced in C.B-17 scid/scid mice by intratracheal inoculation of P. carinii organisms. Infected lung homogenates, obtained at time points up to 6 weeks after inoculation, were analyzed for SP-B and mRNA content. When a comparison was made with uninfected scid controls, the densitometric quantitation of Western blots of lung homogenates demonstrated significant reductions in 8 kd SP-B in mice infected with P. carinii 4 weeks after inoculation (16% of the control value). Northern blot analysis showed a concomitant decrease in SP-B mRNA to 24% of the control level. The decrease in SP-B and mRNA levels in lung homogenates of infected mice was reflected in lower SP-B levels in the surfactant. An enzyme-linked immunosorbent assay for the SP-B level in surfactant prepared from bronchoalveolar lavage samples of infected scid mice demonstrated a significant reduction in alveolar SP-B content (45% of the control value). In contrast to the results with SP-B, neither the SP-A protein content nor the mRNA level was significantly altered by PCP infection. To confirm these observations, SP-B expression was studied in an additional animal model of PCP. The SP-B content of lung homogenates from BALB/c mice depleted of CD4+ T cells and infected with P. carinii was also reduced (51% of the control value). We conclude that P. carinii induces selective inhibition of the expression of SP-B in two mouse models of PCP and that this down-regulation is mediated at the level of mRNA expression. Therefore, an acquired deficiency of SP-B is likely to be an important contributor to the pathogenesis of hypoxemic respiratory failure that is observed in patients with PCP.

TGF-beta1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.

Surfactant protein B processing in human fetal lung.

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8 (Phe201-Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145-Leu160, Gln186-Gln200, and Gly284-Ser304). Western blot analysis revealed a novel intermediate at approximately 9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of approximately 10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8 increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.

Synthetic processing of surfactant protein C by alevolar epithelial cells. The COOH terminus of proSP-C is required for post-translational targeting and proteolysis.

Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59). Using in vitro transcription/translation, each of the seven constructs produced a 35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events.

Pneumocystis carinii glycoprotein A inhibits surfactant phospholipid secretion by rat alveolar type II cells.

Pneumocystis carinii pneumonia (PCP) remains a major cause of morbidity in AIDS. The pathogenesis of PCP is poorly understood, but evidence of surfactant abnormalities is mounting. The role of the major surface glycoprotein of P. carinii, gpA, in producing surfactant abnormalities was investigated. Rat type II pneumocytes were incubated with [3H]choline, purified gpA, and modulators. Lipid was extracted, and [3H]dipalmitoyl phosphatidylcholine (DPPC) secretion was calculated. Contaminating endotoxin had no effect on DPPC secretion. gpA inhibited basal and ATP-stimulated DPPC secretion in a dose- and time-dependent manner. An anti-gpA monoclonal antibody attenuated inhibition of DPPC secretion. Unglycosylated recombinant gpA inhibited secretion, suggesting that functional activity resides in the protein moiety of gpA. These results suggest that gpA is a specific trigger for abnormalities of surfactant lipids in PCP. This is a unique role for a microbial product in disease pathogenesis and a potentially exploitable therapeutic target.