1 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6061; and 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School and Veterans Affairs Medical Center, Ann Arbor, Michigan 48109-0360
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ABSTRACT |
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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 × 105 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.
Pneumocystis carinii; surface tension; surfactant proteins; phospholipid
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INTRODUCTION |
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PNEUMOCYSTIS CARINII PNEUMONIA (PCP) remains a major cause of morbidity and mortality in patients suffering from acquired immunodeficiency syndrome, undergoing chemotherapy for cancer, or receiving immunosuppressive drugs for organ transplantation (39, 56, 61). This opportunistic infection is associated with impaired gas exchange and intrapulmonary shunting, resulting in clinically significant hypoxemia. Although the pathophysiological mechanisms of hypoxemia remain unclear, studies both in humans and in animal models of infection suggest that abnormalities in component expression and in the biophysical activity of pulmonary surfactant play an important role in the altered respiratory physiology associated with this infection (9, 28, 50, 57, 62).
Pulmonary surfactant is a surface-active mixture of phospholipid and protein secreted by the alveolar type II cell that reduces surface tension at the air-liquid interface and allows for maintenance of alveolar stability at low lung volumes (49). Surfactant is composed of ~80% phospholipid, 10% other lipids, and 10% proteins. Among the protein components, biochemical analysis of lung lavage material has identified four unique surfactant-associated proteins (SPs) designated SP-A, SP-B, SP-C, and SP-D. On the basis of structure, function, and solubility in organic solvents, SPs can be divided into two groups. The hydrophobic proteins SP-B and SP-C are extremely lipophilic and are thought to be involved in lipid packaging, organization, and augmentation of absorption at the air-liquid interface. The physiological contribution of SP-B and SP-C to lung function is underscored by several observations. First, intratracheal instillation of SP-B antibodies in rabbits creates respiratory distress syndrome (RDS) in vivo and inactivates replacement surfactants that contain SP-B (48). Second, congenital deficiency of SP-B (and commensurate absence of SP-C) uniformly results in chronic respiratory failure in term human infants (3, 41). Third, in preterm animal models of RDS, replacement therapy with synthetic surfactants containing either SP-B or SP-C as the sole protein component produces biophysical properties similar to natural surfactants (25, 27, 47).
In contrast, the hydrophilic proteins SP-A and SP-D do not appear to have a primary role in the reduction of surface tension but are important components of innate immunity (40, 64). SP-A and SP-D are members of the collectin family of C-type lectins that include a number of molecules with known host defense functions (reviewed in Ref. 26). Both proteins have been shown to enhance uptake of bacteria and viruses by alveolar macrophages and neutrophils and may influence macrophage function and signaling via specific receptors present on target cells. In vitro, SP-A and SP-D each selectively bind to P. carinii organisms (35, 66). SP-A-mediated augmentation of P. carinii clearance by macrophages has also been demonstrated (63).
Several abnormalities in surfactant composition have been described in PCP (9, 19, 28, 50, 57). Elevated phospholipase activities, decreases in total lipid contents, and alterations in relative proportions of individual lipid components (phosphatidylglycerol, dipalmitoylphosphatidylcholine, and monopalmitoylphosphatidylcholine) have been shown in surfactant from both humans and experimental animals with PCP (19, 28). Alterations in the minimum surface tension of surfactant recovered from a corticosteroid-treated rat model of PCP have been described (57). However, the reported changes in lipid composition also reported in that study are insufficient to explain the physiological alterations seen during P. carinii infection. Recently, Beers et al. (9) described selective downregulation of SP-B protein expression in both scid/scid mouse and CD4-depleted mouse models of PCP, but biophysical activity was not measured. Increases in the alveolar levels of SP-A and SP-D induced by PCP in corticosteroid-treated rats have each been reported separately (37, 43), but the role of any changes in these proteins in pathogenesis of surfactant dysfunction is unclear. In fact, knockout mice containing targeted disruption of either the SP-A or SP-D gene do not demonstrate alterations in surface tension (30, 31).
The current study extends previous work by our group by utilizing a well-characterized, non-steroid-treated murine model of PCP to provide a comprehensive assessment of the quantitative and functional changes in surfactant induced by P. carinii lung infection while simultaneously avoiding the confounding influence of corticosteroids on surfactant component expression. The steady-state contents of total phospholipid and all four major SPs were measured, and surface tension analysis was performed. For the first time in a non-steroid-treated model, our results demonstrate that PCP induces significant increases in minimum surface tension while simultaneously causing the selective inhibition of SP-B and SP-C expression. In addition, we demonstrate that alveolar levels of the lung collectin SP-D are markedly elevated.
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METHODS |
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Materials
Radioisotopes were purchased from NEN (Boston, MA). All other reagents were of electrophoretic grade and were purchased from Bio-Rad Laboratories (Hercules, CA) and/or Sigma (St. Louis, MO).SP antisera. Monospecific, polyclonal SP antisera against SP-A, SP-B, and SP-D were each produced in rabbits. Anti-SP-A (PA3) was produced with purified rat SP-A and recognizes murine, bovine, and human forms (60). The SP-B antibody (PT3) was produced with an organic extract of bovine surfactant (TA Surfactant, Ross Laboratories, Columbus, OH) as the immunizing antigen and recognizes bovine, murine, and human SP-B (10). A polyclonal anti-SP-D antibody produced with recombinant mouse SP-D was a generous gift from Dr. Jo Rae Wright (Duke University, Durham, NC).
cDNA probes. A cDNA probe for rat SP-C was generated by purification of a full-length insert from EcoR I digestion of the pGEM4Z prokaryotic expression vector containing a 0.9-kb full-length cDNA as previously published (21). A full-length cDNA probe for murine SP-D was a generous gift from Dr. Samuel Hawgood (University of California, San Francisco) and has been previously described (14).
Mouse Model of P. carinii Infection
Mice. For all studies in this report, we used the immunocompromised C.B-17 scid/scid mouse model of P. carinii lung infection previously described (7, 9). Male C.B-17 scid/scid mice (BALB/c background) purchased at 8 wk of age from Taconic (Germantown, NY) served as recipients of P. carinii inoculations. The colony of scid mice utilized in this study was pathogen free and without evidence of latent P. carinii infection as indicated by the absence of organisms in lung tissue sections stained with Gomori methenamine silver stain and in touch preparations of lung tissue stained with crystal violet and modified Giemsa stain. Histological sections of uninfected control animals were uniformly normal and showed no evidence of lipoproteinosis or other alveolar pathology.All mice were housed in an isolation room in the animal care facility of the Department of Veterans Affairs Medical Center (Ann Arbor, MI) in filter-top cages under laminar flow conditions. Mice received sterile rodent chow and sterile drinking water. Normal sentinel mice were routinely examined for the presence of unintended pathogens by culture and serology. All procedures were approved by the University Committee on the Use and Care of Animals (University of Michigan, Ann Arbor) and by the Institutional Animal Care and Use Committee (University of Pennsylvania, Philadelphia).
Organisms. Mouse P. carinii organisms were obtained from the lungs of athymic mice (nu/nu on a BALB/c background; Taconic Laboratories) in which P. carinii organisms are propagated by serial passage as previously described (6-9, 52). P. carinii organisms were harvested by homogenization of lungs with a Stomacher apparatus. After centrifugation, organisms in the resulting pellet were stained with modified Giemsa stain, counted, and diluted to a concentration of 2 × 106 organisms/ml. This preparation was used to intratracheally inoculate experimental mice as detailed below. Bacterial contamination was excluded by the routine use of gram staining of each preparation (7).
Mice were intratracheally inoculated with purified P. carinii organisms during pentobarbital sodium (75 mg/kg intraperitoneally) anesthesia. By direct visualization, 0.1 ml of the inoculum (2 × 105 P. carinii cysts) was injected at the level of the carina, immediately followed by an injection of 0.6 ml of air to ensure adequate dispersion of the inoculum into the distal lung.
At intervals of up to 6 wk after inoculation, infected and uninfected mice were euthanized with a lethal dose of pentobarbital sodium (400 mg/kg) and were exsanguinated by aortic transection. The trachea was exposed through a midline incision and was cannulated with a polyethylene catheter. All lungs were lavaged with 0.5-ml aliquots of sterile, warmed, calcium- and magnesium-free 0.9% saline containing 0.6 mM EDTA to a total of 11 ml.
After lavage, total RNA was prepared from the tissue as detailed in mRNA analysis and analyzed for surfactant gene expression. For all experimental animals, portions of the lung were reserved for histological analysis to grade the intensity of P. carinii infection. Thus surfactant characterization and histological analysis were performed on lung samples from identical mice.
Scoring of P. carinii infection. Intensity of the P. carinii infection in scid mice was scored as previously described (8, 9, 51). The lungs were inflated with air through the trachea and immersion fixed in Formalin. Paraffin-embedded specimens were sectioned to 5 µm and stained with Gomori methenamine silver stain and hematoxylin and eosin stain for histological examination. Sections were graded for the extent of infection with P. carinii organisms in a blinded fashion with a scoring system previously described and validated (51). The scores range from 0 (absence of P. carinii cysts or foamy alveolar exudate in any section) to 4+ (abundant P. carinii cysts with large foamy extracellular alveolar exudates throughout the alveoli). The grading of tissue sections correlates highly with organism counts performed on homogenized lungs (8).
Immunohistochemistry
Paraffin sections prepared from the lungs of uninfected or P. carinii-infected scid mice killed 6 wk after inoculation were immunostained with the VECTASTAIN ABC Kit with polyclonal primary anti-SP-D (1:400) or nonimmune serum (1:400). Staining was visualized with 3,3'-diaminobenzidine as previously published (54).Preparation and Analysis of Surfactant Fractions
In this study, bronchoalveolar lavage (BAL) fluid was subfractionated into two surfactant fractions: the biophysically active large-aggregate (LA) form and the biophysically inactive small-aggregate (SA) form. Lavage returns averaging 10-11 ml/mouse were centrifuged at 1,000 g for 10 min at 4°C to remove cells. The cell-free supernatant was recentrifuged at 20,000 g for 40 min at 4°C for separation of LA surfactant in the pellet and SA surfactant in the supernatant fraction. The resulting LA pellets were resuspended in saline for biophysical and biochemical characterization. The total protein content of the samples from LA and SA fractions was determined with the method of Bradford (15), with bovine IgG as a standard.Phospholipid assay. LA and SA surfactant fractions were analyzed for total phospholipid content by extraction of total phospholipid (13) and determination of inorganic phosphorus content with the method of Bartlett (5). Briefly, total lipids were extracted from LA and SA surfactant fractions with chloroform-methanol, and total phospholipids in the lipid extracts in each fraction were determined by inorganic phosphorus analysis.
Measurement of surface tension. Surface tension of surfactant samples was determined with a pulsating bubble surfactometer with the method of Enhorning (20). Samples of LA surfactant from uninfected and infected mice were standardized to a fixed concentration of phospholipid (2.5 µg/µl) by dilution with 0.9% NaCl. Forty-microliter suspensions of LA surfactant fraction were placed in the chamber of a pulsating bubble surfactometer (Electronetic, Buffalo, NY) and heated to 37°C. The bubble was induced to fluctuate in radius between 0.40 and 0.55 mm at rate of 20 cycles/min. Bubble size was checked regularly and adjusted when necessary. The surface tension of each sample was monitored during cycling, and equilibrium minimum surface pressure was determined after 5 min. The bubble was then reformed, the sample was run twice more, and an average of minimum surface tension of each sample (expressed in mN/m) was calculated from the pressure and the radius according to the law of Laplace (20).
Analytic Methods
PAGE and immunoblotting. One-dimensional SDS-PAGE was performed in 16.5% polyacrylamide gels with a Tris-Tricine buffer system as previously described (11) or in 12% gels with the method of Laemmli (33). Gels containing electrophoresed samples were transferred to 0.2-µm nitrocellulose at 60 mA/cm2 for 12-18 h for subsequent immunoblotting or autoradiography. Immunoblotting was performed by incubation at room temperature in primary antiserum at indicated titers for 2 h followed by incubation with goat anti-rabbit antiserum conjugated to horseradish peroxidase (Bio-Rad). The bands were visualized by enhanced chemiluminescence with the ECL Kit (Amersham, Arlington Heights, IL).[14C]iodoacetamide assay for mature SP-C. Monospecific antisera recognizing mature SP-C are not available. Therefore, the level of SP-C in surfactant was determined with a modification of the method of Qanbar and Possmayer (46). Because SP-C is the only palmitoylated protein found in surfactant, this method specifically detects mature SP-C by stoichiometric derivatization of two thioester-linked palmitate residues. Samples of LA surfactant from control and P. carinii-infected mice (10 µg of total protein each) were mixed with triethylamine (3 M) and 150 nCi of [14C]iodoacetamide (Dupont NEN) in chloroform-methanol (1:1), and the mixture (final volume = 170 µl) was incubated for 3 h at 37°C. After separation of proteins on 16.5% Tricine gel electrophoresis and transfer to nitrocellulose, the relative amount of mature SP-C was determined by quantitation of [14C]SP-C bands with a phosphorimager (Bio-Rad). An internal standard consisting of 10 µg of rat surfactant reacted as described above was included on each gel to normalize for variations in efficiency of radiolabel coupling. A negative labeling control consisting of 10 µg of rat surfactant reacted in the absence of triethylamine was performed to demonstrate specificity of the coupling reaction.
mRNA analysis. Total RNA was isolated from lung fluid after lavage based on the method of Chomczynski and Sacchi (16) by homogenization of lavaged lungs with TRIzol Reagent following the manufacturer's instructions (GIBCO BRL, Life Technologies, Gaithersburg, MD). Quantitation of recovered RNA was done by determining absorbance at 260 nm. Ten micrograms of isolated total RNA was size-fractionated by electrophoresis through a 1% formaldehyde agarose gel, transferred to nitrocellulose by capillary action, and baked for 2 h at 80°C in a vacuum oven.
Specific mRNA content was determined by cDNA hybridization with
Northern blot as previously described (9).
[32P]cDNAs for SP-C and SP-D were
prepared from purified plasmid inserts by labeling with
[32P]dCTP with a random-primer labeling
technique (Ready-to-Go Kit, Pharmacia, Piscataway, NJ) to specific
activities of 6-8 × 106
counts · min1 · µg
DNA
1. Nitrocellulose blots were hybridized under
high stringency with QuikHyb (Stratagene, La Jolla, CA). Normalization
of the specific SP-C and SP-D signals for variability in RNA loading
was performed by prehybridization of each blot with an 32P
end-labeled ([
-32P]ATP) 28S rRNA
oligonucleotide probe based on the method of Barbu and Dautry (4).
Blots were exposed to film for 1-7 days at
70°C. Counts
in specific mRNA bands were quantitated either by utilizing direct
beta counting with an AMBIS 4000 radioanalyzer (Scanolytics, San Diego, CA) or by densitometric scanning of
exposed film and quantitation with Quantity 1 (PDI, Huntington Station, NY).
Statistics
Mean data from multiple groups were compared with ANOVA and either Fisher's protected least significant difference or Student-Newman-Keuls follow-up testing. Infection scores are expressed as median values, and experimental groups were compared with the Kruskal-Wallis test. All testing was performed with SigmaStat software (Jandel Scientific). Significance was accepted at P < 0.05. ![]() |
RESULTS |
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PCP Decreases Total Phospholipid Content and Increases Surface Tension of Surfactant
Surfactant fractions from uninfected and infected mice were analyzed for total phospholipid content as described in METHODS. Infection with P. carinii (median infection score 3 for infected mice vs. 0 for uninfected mice; P < 0.001; n = 23 mice/group) resulted in significantly decreased phospholipid content in the LA fraction of infected mice, reaching 53% of baseline levels at 6 wk after inoculation (Fig. 1A).
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Importantly, PCP significantly altered the biophysical activity of
surfactant isolated from infected mice. Samples of LA surfactant fractions representing pooled material (2-3 mice/point) from
uninfected and P. carinii-infected mice were analyzed
separately for surface tension with the pulsating bubble surfactometer.
All samples were normalized to a fixed phospholipid content (2.5 mg/ml). LA surfactant from scid mice with PCP (median infection
score 3 for infected mice vs. 0 for uninfected mice; P < 0.001; n = 18 mice/group) demonstrated a significantly higher
minimum surface tension than LA surfactant from control mice (Fig.
1B). PCP-induced alterations in surface tension were associated
with conversion of surfactant from LA to SA forms. Phospholipid
analysis performed on LA and SA surfactant fractions of uninfected and
infected mice demonstrated an increase in the relative content of total
phospholipid in SA forms in infected mice (Table
1). Collectively, these data demonstrate that PCP induces a physiologically significant aberration in surface tension of surfactant, with a shift of total phospholipid to the biophysically inactive SA pool.
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PCP Decreases Hydrophobic SPs SP-B and SP-C in Surfactant
Because the hydrophobic SPs have been shown to be responsible for augmenting the biophysical function of surfactant phospholipids, we then analyzed the LA surfactant fraction for hydrophobic surfactant SP-C and SP-B content. Because of its hydrophobicity, monospecific antisera against mature SP-C are not available. Therefore, we utilized the selective substitution of thioester-linked palmitic acid residues with [14C]iodoacetamide. After derivatization of mature SP-C with triethylamine and [14C]iodoacetamide, autoradiography detected expression of a 3.7-kDa band (mature SP-C) in the LA surfactant fraction of uninfected mice that was markedly decreased 6 wk after inoculation with P. carinii organisms (Fig. 2A). Quantitation by densitometric scanning of multiple samples demonstrated significant decreases in SP-C content compared with those in uninfected mice (Fig. 2B).
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To confirm that the downregulation of SP-C expression observed during
PCP also occurred for SP-B, nitrocellulose blots from the
[14C]iodoacetamide assay were then probed with
antibody against SP-B. Western blots showed expression of the 8-kDa
mature SP-B protein in the LA fraction of uninfected mice, and this
expression was markedly decreased 6 wk after inoculation (Fig.
3A). Densitometric quantitation of
multiple samples demonstrated significant decreases in SP-B protein
content during PCP (Fig. 3B).
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The observed changes in hydrophobic protein content in LA surfactant
were not attributable to a dilutive effect from changes in protein
content of this fraction (Table 2).
Although the total protein content of BAL fluid (LA plus SA pools)
increases twofold during PCP, protein levels in LAs from uninfected
mice were unchanged during PCP. The increase in BAL fluid protein
levels during PCP was thus due to changes in the SA fraction protein
content that were markedly increased 6 wk after P. carinii
inoculation. Taken together, these data indicate that PCP decreases
SP-B and SP-C protein content independent of alterations in LA total
protein content.
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PCP Increases SP-D Expression
To determine whether PCP specifically downregulated SP-B and SP-C or inhibited all epithelial cell SP expression, surfactant fractions were analyzed for SP-A and SP-D (Fig. 4). In contrast to the hydrophobic SPs (SP-B and SP-C), SP-D levels increased significantly during the development of PCP. Western blots of SA surfactant showed expression of a 43-kDa SP-D band in the BAL fluid of uninfected mice, and this expression was markedly increased 6 wk after inoculation with P. carinii organisms (Fig. 4A). Although SP-D was detectable in LA fractions of infected and uninfected mice, the relative signal intensity in the LA fraction was minor compared with the total in the SA fraction (data not shown). After SP-D detection, blots from uninfected and infected mice were stripped and reprobed with an antibody against SP-A. Western blotting demonstrated that in LAs, SP-A protein was maintained in the setting of P. carinii infection (Fig. 4A). SP-A was not detectable in SA fractions in either uninfected or infected mice (data not shown). Densitometric quantitation of multiple samples (Fig. 4B) confirmed a marked increase in the levels of 43-kDa SP-D. Under these conditions, SP-A levels in LA surfactant were maintained, consistent with previously published data by Beers et al. (9) regarding tissue SP-A levels during P. carinii infection.
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Because of the marked upregulation of alveolar SP-D,
immunohistochemistry was performed to provide spatial localization of SP-D protein expression (Fig. 5). Staining
of paraffin-embedded sections with anti-SP-D demonstrated low basal
expression in alveolar type II cells, with marked local upregulation of
SP-D occurring during PCP in regions of atelectasis and consolidation.
Methenamine silver staining of histological sections from the same
mouse demonstrated numerous P. carinii cysts in similar areas
of consolidation (data not shown).
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PCP Induces Commensurate Changes in Surfactant mRNA Expression
The alterations in SP levels induced by P. carinii lung infection were reflected in commensurate changes in mRNA levels (Figs. 6 and 7). Total lung RNA was prepared from the lungs of mice infected with P. carinii for 6 wk and probed for SP-C mRNA content by Northern blot analysis. Densitometric evaluation of the 0.9-kb SP-C band obtained from multiple mice revealed a marked decrease in normalized SP-C mRNA content compared with that in uninoculated control mice (Fig. 6).
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In contrast to SP-C mRNA expression, Northern blot analysis showed that SP-D mRNA expression increased during P. carinii infection (Fig. 7A). Quantitation of multiple samples (Fig. 7B) demonstrated changes that were similar in magnitude to the observed changes in SP-D protein.
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DISCUSSION |
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Severe pulmonary infection of the immunocompromised host with P. carinii results in hypoxemic respiratory failure. One likely mechanism for this occurrence is that PCP induces alterations in biophysical activity and in component expression of pulmonary surfactant. In this report, we utilized a well-characterized mouse model of PCP to determine the selective effects of PCP on each major component of the surfactant system. Previously, Beers et al. (9) had reported that PCP induces downregulation of SP-B. The current study extends the previous work, demonstrating that PCP causes downregulation of SP-C protein in surfactant. Furthermore, the loss of SP-B and SP-C proteins that occurs during infection accompanies physiologically important changes in the biophysical properties of surfactant. Importantly, in contrast to the hydrophobic proteins, differential expression of the lung collectins SP-A and SP-D occurred, which included a marked upregulation of SP-D protein and RNA. Therefore, the observed changes induced by PCP were selective and unlikely due to nonspecific alveolar damage.
The PCP-induced decreases in phospholipid content and hydrophobic SP levels measured in surfactant fractions from infected mice were associated with a loss of biophysical surface activity. Because surface tension assessment was measured in vitro with equal phospholipid concentrations, the loss of biophysical activity cannot be explained by differences in total phospholipid levels. Our results also demonstrate that the functional increase in minimal surface tension that occurs during PCP is accompanied by decreases in levels of both SP-B and SP-C. The magnitude of the decrease in SP-B that we observed is similar to that observed for heterozygous SP-B knockout mice (17) and in sepsis patients shown to be at risk for acute RDS (ARDS) (22). In both cases, abnormalities in either biophysical activity or lung mechanics were also demonstrated. Furthermore, during PCP in scid mice, we also observed downregulation of SP-C protein in which relative levels decreased by nearly 80%. The absolute levels of SP-C required for maintenance of normal function are largely unknown; however, replacement therapy with surfactant containing recombinant SP-C alone has been shown to restore biophysical function in a surfactant-depleted rat model of lung injury (25). This is the first reported occurrence of commensurate loss of both hydrophobic proteins and changes in surfactant biophysics in a model of lung injury.
In this study, BAL fluid was subfractionated into LA and SA pools. Veldhuizen et al. (59) have shown morphological and functional differences between surfactant LAs and SAs. Tubular myelin, large multilamellar vesicles, and lamellar bodies are observed in the LA fraction, which has also been shown to account for the biophysical properties of surfactant. In addition, SP-A, SP-B, and SP-C are found in this fraction. In contrast, SA fractions contain only small unilamellar vesicles and show poor surface activity both in vitro and in vivo. The majority of SP-D has been shown to isolate with this fraction. The analysis of these two pools for phospholipid content in the infected mice demonstrates that PCP induced an increase in the amount of total phospholipid in the SA surfactant fraction and a decrease in phospholipid content in the LA surfactant fraction. The "conversion" of surfactant subfractions (i.e., a decrease in the percentage of phospholipids in the LA fraction and an increase in the SA fraction) has been noted in other models of experimental lung injury and in human patients with ARDS (23, 24, 59).
Although the conversion of LA surfactant occurs in PCP, the downregulation of SP-B and SP-C during P. carinii infection is selective compared with that in ARDS. The differential expression of the lung collectins SP-A and SP-D in our model indicates that loss of hydrophobic protein expression is not due to global impairment of type II cell function. In this study, we show that the SP-A content of LA surfactant remains preserved under the conditions of PCP. These results are in agreement with the previous results by Beers et al. (9) in which they analyzed tissue levels of SP-A protein and mRNA and showed no change despite progressive PCP and loss of SP-B. In other animal models of PCP, SP-A levels in BAL fluid were reported to be increased (43). However, the animals in these studies were treated with corticosteroids to achieve immunosuppression. Similarly, BAL fluid from PCP in human immunodeficiency virus-infected patients also showed increases in SP-A (42, 55); however, the use of steroids was not documented, and clusters of P. carinii were noted in the BAL fluid after low-speed centrifugation. Because SP-A has been shown to bind to P. carinii, it is possible that P. carinii-associated SP-A contributed to the elevated levels in those studies. In the current study, we utilized centrifugation of BAL fluid at a slightly higher relative centrifugal force and for longer periods of time (500 g for 10 min) to remove all cellular debris.
Additionally, the administration of corticosteroids to rats has been shown to alter the expression of other surfactant components including phospholipid and SP-D (36, 58, 65). Young and Silbajoris (65) have demonstrated that administration of dexamethasone for 7 days increased the phospholipid pool in alveolar surfactant. Limper et al. (36) have shown that in the corticosteroid-treated rat model of PCP, SP-D content of surfactant increased both in the control (uninfected) animals and in the P. carinii-infected animals. In contrast, Dichter et al. (19) have recently reported that administration of adjuvant methylprednisolone to human immunodeficiency virus-infected patients with PCP did not alter the rate of recovery of surfactant lipid levels after 10 days of therapy. However, both in human fetal lung preparations cultured in vitro and in human fetuses of mothers in preterm labor in vivo, administration of steroids have been shown to have an effect on the regulation of both SPs and lipids (1, 12, 34). Therefore, although steroid administration can be a confounding variable in the interpretation of studies of surfactant component expression, we have demonstrated selective changes in individual component expression induced by PCP in a relevant non-steroid-treated animal model of infection.
The presence of abundant SP-D in the lung during PCP coupled with the demonstration that this protein modulates the interaction of P. carinii with macrophages (36, 58) strongly suggests that SP-D accumulation may represent a significant element of the host-organism relationship during infection. Unlike SP-A, SP-B, and SP-C, the majority of SP-D is localized to the SA fraction (high-speed supernatant) of BAL fluid and does not bind surfactant phospholipid (18). Strikingly, in our model, in contrast to the other three proteins, PCP induced a significant upregulation of SP-D protein. Immunohistochemistry demonstrated that in PCP, the upregulation of SP-D was localized to regions of atelectasis and cellular infiltration that routinely contain P. carinii organisms. SP-D as well as SP-A is a member of a novel, growing family of proteins that are believed to play a role in non-antibody-mediated innate immune response (26). This family consists of SP-A and SP-D in the lung as well as mannose binding protein (MBP) and collectin-43 in the blood. Termed collectins, for collagen-like lectin, the primary function of these proteins appears to be in modulation of host defense and inflammation. The prototypical MBP selectively binds to some bacteria, fungi, and viruses. Low levels of MBP are associated with recurrent infections in children and provide the most compelling evidence for its role in host defense.
In this study, the relative levels of expression of SP-C and SP-D mRNAs
during PCP were quantitated. When combined with the published data by
Beers et al. (9) for the expression of SP-A and SP-B, these data
demonstrate that the differential regulation of the four SPs is
reflected by commensurate changes at the mRNA level. Table
3 summarizes the expression of mRNA for all
four SPs 6 wk after P. carinii infection. Normalized data show
that SP-D mRNA was upregulated, SP-B and SP-C mRNAs were significantly decreased, and SP-A mRNA levels were unaffected during development of
P. carinii infection. Levels of SP-A, SP-B, SP-C, and SP-D expression are known to be altered by a variety of hormones and second messengers including dexamethasone, insulin, cAMP, and phorbol
ester (1, 32). In addition, several immunoregulatory mediators
known to be present in PCP have been shown to regulate SP expression in
a differential fashion. Interferon- has been shown to upregulate
SP-A production in vitro but has no effect on SP-B, SP-C, or SP-D (2).
In vivo studies in mice using intratracheal administration of
recombinant tumor necrosis factor-
have demonstrated decreases in
SP-B and SP-C RNA expression but failed to show a consistent effect on
SP-A message regulation (44, 45). Thus the differential regulation of
these components likely represents a complex interaction between
several mediators.
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In summary, we have shown that PCP induces selective downregulation of SP-B and SP-C, with corresponding changes in surface tension. Furthermore, the selective upregulation of SP-D demonstrated in this model during PCP raises new questions for the role of lung collectins in the host defense and inflammatory response to this opportunistic infection. The alterations in hydrophobic protein content raise the possibility that surfactant replacement could be useful as adjunctive therapy in PCP. Recently, Hughes et al. (29) have shown the efficacy of exogenous surfactant replacement in a rat model of PCP. In humans, this has been attempted on a limited scale, with several case reports and a small series showing some benefit (38, 53). Based on results from our study, the role of altered surfactant composition and biophysical function cannot be underestimated, and the recognition of these changes raises the possibility of surfactant as a new therapeutic intervention in the treatment of this infection.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jo Rae Wright (Duke University, Durham, NC) for providing mouse anti-surfactant protein D antiserum and Dr. Samuel Hawgood (University of California, San Francisco) for supplying the cDNA probe for mouse surfactant protein D. We also thank Dr. Avinash Chander and Graham Vigiotta (Thomas Jefferson University, Philadelphia, PA) for assistance with surface tension measurements in the pulsating bubble surfactometer. We acknowledge the technical support of Scott Russo and the helpful comments of Dr. Jeffrey Curtis.
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FOOTNOTES |
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* E. N. Atochina and M. F. Beers contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-59867 (to M. F. Beers), R01-HL-57011, and R01-HL-59823 (both to J. M. Beck).
Address for reprint requests and other correspondence: M. F. Beers, Pulmonary and Critical Care Division, Dept. of Medicine, Univ. of Pennsylvania School of Medicine, 807 BRB II/III Bldg., 421 Curie Blvd., Philadelphia, PA 19104-6061 (E-mail: mfbeers{at}mail.med.upenn.edu).
Received 7 July 1999; accepted in final form 19 October 1999.
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