HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 34, 24496-24505, August 25, 2006

This Article Abstract Full Text (PDF) An addition or correction has been published All Versions of this Article: 281/34/24496    most recent M600651200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kingma, P. S. Articles by Whitsett, J. A. Search for Related Content PubMed PubMed Citation Articles by Kingma, P. S. Articles by Whitsett, J. A. Social Bookmarking                      What's this?

Correction of Pulmonary Abnormalities in Sftpd^-/- Mice Requires the Collagenous Domain of Surfactant Protein D^*

Paul S. Kingma, Liqian Zhang, Machiko Ikegami, Kevan Hartshorn, Francis X. McCormack^¶, and Jeffrey A. Whitsett¹

From the Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, Departments of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts 02118-3393, and ^¶Pulmonary/Critical Care Division, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0564

Received for publication, January 23, 2006 , and in revised form, June 19, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Surfactant protein D (SP-D) is a member of the collectin family of innate defense proteins. Members of this family share four distinct structural domains: an N-terminal cross-linking domain, a collagenous domain, a neck region, and a carbohydrate recognition domain. In this study, the function of the collagenous domain was evaluated by expressing a SP-D collagen deletion mutant protein (rSftpdCDM) in wild type and SP-D null mice (Sftpd^-/-). rSftpdCDM formed disulfide-linked trimers that further oligomerized into higher order structures. The mutant protein effectively bound carbohydrate and aggregated bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmonary morphology or surfactant phospholipid levels in wild type mice, the mutant protein failed to rescue the emphysema or enlarged foamy macrophages that are characteristic of Sftpd^-/- mice. Moreover, rSftpdCDM partitioned with small aggregate surfactant in a manner similar to SP-D, but rSftpdCDM did not correct the abnormal surfactant ultrastructure or phospholipid levels observed in Sftpd^-/- mice. In contrast, rSftpdCDM completely corrected viral clearance and the abnormal inflammatory response that occurs following pulmonary influenza A challenge in Sftpd^-/- mice. Our findings indicate that the collagen domain of SP-D is not required for assembly of disulfide-stabilized oligomers or the innate immune response to viral pathogens. The collagen domain of SP-D is required for the regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Surfactant protein D (SP-D)² is a member of the collectin family of C-type lectins. Members of this family include surfactant protein A (SP-A), SP-D, mannose-binding protein, conglutinin, and CL-43. SP-A and SP-D contribute to the innate immune system of the lung by binding and enhancing the clearance of a variety of viral, bacterial, and fungal pathogens (1-3). The collectins are defined by four structural domains shared by all family members: a short amino-terminal cross-linking domain, a triple helical collagenous domain, a neck domain, and a carbohydrate recognition domain (CRD) (4-8). Three neck domains associate to form a triple coiled-coil structure that facilitates the assembly of the remaining domains into a trimer (9). Final assembly of the trimer, thought to be the minimal functional unit of collectins, occurs through disulfide bonds between the cysteine-rich amino-terminal domains (10, 11). Trimers further combine into larger multimeric complexes through disulfide-stabilized, noncovalent interactions. Although larger structures are commonly observed, SP-D exists predominantly as a tetramer of trimeric subunits (dodecamer) assembled into a cruciform structure (10, 12).

Animal models of SP-D deficiency have revealed a complex role for SP-D in pulmonary immunity and alveolar homeostasis. Mice with a targeted deletion of the Sftpd gene (Sftpd^-/-) survived normally but developed gradually worsening pulmonary inflammation, emphysema, and surfactant phospholipid accumulations (13, 14). Sftpd^-/- mice accumulate apoptotic alveolar macrophages as well as enlarged, lipid-laden, macrophages that release metalloproteinases and reactive oxygen species (15-19). Uptake and clearance of viral pathogens, including influenza A and respiratory syncitial virus, were deficient in Sftpd^-/- mice (20, 21). In contrast, clearance of group B Streptococcus and Hemophilus influenza was unaltered in the absence of SP-D (19). However, oxygen radical release and production of the proinflammatory mediators, tumor necrosis factor-, IL-1, and IL-6, were increased in Sftpd^-/- mice when exposed to either viral or bacterial pathogens (19-21).

The roles of the various domains of SP-D in its complex functions have been studied by expressing SP-D point mutants, deletion mutants, or chimeric proteins of SP-D and other collectins. For example, whereas expression of the fulllength rat Sftpd gene (rSftpd) fully rescues the Sftpd^-/- mouse phenotype, expression of a fusion protein that included the N-terminal and collagen domains of SP-A fused to the neck and CRD of SP-D (rSftpa/d) was not sufficient to correct the emphysema or lipid accumulations characteristic of Sftpd^-/- mice, indicating that the collagenous and N-terminal domains of SP-D are essential for these functions (22). Substitution of serine for cysteine at positions 15 and 20 of the N-terminal domain (rSftpd^Ser15,20) results in a protein that corresponds to a single trimeric arm of the SP-D dodecamer but fails to correct the abnormal macrophages, emphysema, or lipid accumulations in Sftpd^-/- mice, suggesting that oligomers induced by the SP-D collagenous and N-terminal domains are required for complete function (23). However, intranasal administration of a truncated SP-D trimer (consisting of only the CRD and neck domains) into Sftpd^-/- mice partially corrects the apoptotic macrophages, lipid accumulations, and elevated inflammatory mediators, suggesting that the collagen domain is not essential for these activities (15, 24). Since most collectins form sulfhydryl-stabilized oligomers, the relatively large collagen domain of SP-D suggests that this domain serves a purpose that is beyond facilitating oligomerization of the SP-D CRD. Comparisons of SP-D, mannose-binding protein, and conglutinin fusion proteins suggest the large collagen domain promotes proper spacing of trimeric subunits in order to facilitate cross-linking of separate microorganisms and microbial aggregation (25). Therefore, to further investigate the function of the SP-D structural domains, we genetically introduced into wild type and Sftpd^-/- mice lungs an SP-D collagen deletion mutant protein (rSftpdCDM) that formed multimers via an intact N terminus. Whereas expression of rSftpdCDM elicited a protective response to influenza virus, the protein failed to correct the abnormal macrophage activation, emphysema, or lipid abnormalities in Sftpd^-/- mice, indicating that the SP-D collagenous domain is critical for normal regulation of lipid homeostasis, macrophage activity, and the structural integrity of peripheral airspaces.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Schematic representation of the rSftpdCDM transgene. The rSftpdCDM cDNA was generated using recombinant PCR to delete the 177-amino acid sequence of the collagen domain from rat SP-D. The rSftpdCDM cDNA was inserted into the EcoRI site of 3.7hSPC/SV40 expression vector and sequenced. The transgene was identified by transgene-specific PCR primers used to generate the transgenic mice.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of Transgenic Mice—rSftpdCDM cDNA was generated using recombinant PCR to delete a 177-amino acid sequence (Gly²⁶-Pro²⁰²) corresponding to the complete collagen domain from rat SP-D (Fig. 1). The rSftpdCDM cDNA was inserted into the EcoRI site of 3.7hSPC/SV40 expression vector and sequenced (26). The transgene was microinjected into FVB/N oocytes fertilized with Sftpd^-/- sperm by the Children's Hospital Transgenic Core facility, and founders were identified by transgene-specific PCR using upstream primer 5'-GGAGACAAAATCTTCAGGGCG-3' and downstream primer 5'-TTCGGATGGTGGCAGCATAG-3'. Transgenic animals were crossed with either Sftpd^-/- mice to generate rSftpdCDM^Tg+/Sftptd^-/- mice or wild type mice to generate rSftpdCDM^Tg+/Sftpd^+/+ mice (16).

Western Blot Analysis—Animals were weighed, anesthetized by intraperitoneal injection of pentobarbital, and exsanguinated. Bronchoalveolar lavage was performed five times with 1 ml of normal saline. Typically, >90% of the instilled volume was recovered. For each lane, 40 µl of the bronchoalveolar lavage fluid (BALF) was dried, reconstituted in 15 µl of Laemmli buffer with or without sulfhydryl reduction with -mercaptoethanol, and resolved on 10-20% SDS/Tris/glycine/polyacrylamide gel (Novex, San Diego, CA). After separation and transfer to a nitrocellulose membrane, protein was detected with rabbit anti-mouse SP-D or guinea pig anti-rat SP-A antiserum (Seven Hills Bioreagents, Cincinnati, OH) diluted 1:5000 in Tris-buffered saline as previously described (27).

SP-D Purification—Previous studies demonstrated that inactivation of the granulocyte-macrophage colony-stimulating factor gene (Gmcsf^-/-) in mice impaired SP-D clearance and increased SP-D levels in BALF severalfold (28). Therefore, to increase the amount of starting material for protein purification, rSftpdCDM was purified from rSftpdCDM^Tg+/Sftptd^-/-/Gmcsf^-/- mice. Wild type mouse SP-D was purified from Sftpd^+/+/Gmcsf^-/- mice that also carried a deletion of the two expressed Sftpa genes in order to minimize the potential of SP-A contamination. A similar Sftpa deletion was not possible in the rSftpdCDM^Tg+/Sftptd^-/-/Gmcsf^-/- mice; however, contaminating SP-A was not detectable by silver stain gels in rSftpdCDM preparations.

SP-D- or rSftpdCDM-containing BALF was applied to a maltosyl-Sepharose (Sigma) column and selectively eluted with manganese as previously described (29). The pooled fractions were diluted 10-fold in 20 mM Tris-HCl, pH 7.4, and 30 mM CaCl₂ and applied to a 1-ml bed volume maltosyl-Sepharose column. The column was stripped of lipopolysaccharide with 20 mM Tris-HCl, pH 7.4, 20 mM n-octyl--D-glucopyranoside, 200 mM NaCl, 2 mM CaCl₂, 100 µg/ml polymyxin and washed with 20 mM Tris-HCl, pH 7.4, 0.5 mM CaCl₂, 200 mM NaCl. The protein was eluted with 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA. Under the conditions employed, lipopolysacharide concentration was typically 0.1 endotoxin units/µg of protein.

SP-D Sizing and Detection—The size of rSftpdCDM multimers was determined by gel filtration chromatography using Sepharose CL-6B equilibrated in 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 5 mM EDTA, and 0.02% sodium azide. Purified rSftpdCDM was diluted in the same buffer and applied to the column (1.5 x 90 cm). rSftpdCDM protein concentration was determined in each fraction using an enzyme-linked immunosorbent assay (ELISA). Plates were washed five times between incubations, and all washes and dilutions were carried out with Tris-buffered saline, 0.1% Tween 20. A mouse anti-SP-D monoclonal antibody was developed by exposing Sftpd^-/- mice to purified, full-length, human SP-D and selecting cell lines that demonstrated a high affinity for SP-D and minimal cross-reactivity with SP-A (Seven Hills Bioreagents, Cincinnati, OH). ELISA plates were coated with this monoclonal antibody (1 µg/ml, 100 µl/well) in 0.1 M carbonate buffer, pH 9.6, overnight at 4 °C and blocked with 1% bovine serum albumin for 1 h at room temperature. Plates were washed, and appropriate dilutions of standards and protein samples were added and incubated for 1 h at room temperature. Plates were washed and incubated with rabbit anti-mouse SP-D antiserum (100 µl/well diluted 1:750) for 1 h. This was followed by washing and incubation with donkey horseradish peroxidase-conjugated anti-rabbit IgG (100 µl/well diluted 1:10,000) (Jackson Immunoresearch, West Grove, PA) for 1 h. After washing plates again, TMB substrate (100 µl/well) (BioFx Laboratories, Owings Mills, MD) was added. The color reaction was stopped after 10 min with 2 M H₂SO₄, and plates were read at 450 nm. Typically, this assay results in absorbance changes that are equal, parallel, and linear for mouse SP-D, rSftpdCDM, and human SP-D concentrations between 10 and 150 ng/ml.

Carbohydrate Binding—Direct binding of SP-D to carbohydrate was detected by mixing BALF from 6-8-week-old mice with maltosyl-Sepharose-linked beads at 4 °C for 2 h in 4 mM Tris-HCl, pH 7.4, and 5 mM CaCl₂ (23). Binding specificity and calcium dependence were confirmed by the addition of 100 mM maltose and 10 mM EDTA to the binding reaction, respectively. The samples were centrifuged at 10,000 x g for 1 min, and the supernatants were removed. The amount of unbound SP-D in the supernatant was determined by Western blot analysis.

Selective carbohydrate binding was performed as described previously (30). Briefly, microtiter plates were coated with 10 µg/ml mannan at 4 °C overnight, washed, and blocked with 1% bovine serum albumin. BALF was incubated with increasing concentrations of maltose, glucose, galactose, or GlcNAc. Samples were subsequently added to the coated plate and incubated with rabbit anti-SP-D antibody followed by peroxidase-conjugated goat anti-rabbit IgG antibody. After washing, o-phenylenediamine was added to each well, and the A_{490 nm} was measured. The concentration of sugar that inhibited 50% of SP-D binding to the mannan-coated plate was defined as the IC₅₀.

Bacterial Aggregation—Bacterial aggregation was assessed by measuring light transmission through a bacterial suspension after the addition of SP-D. Purified rSftpdCDM, mouse SP-D (150 nm based on the monomer molecular weight), or a control reaction that contained protein buffer without SP-D was mixed with 600 µlof Escherichia coli Y1088 (OD_{700 nm} 1) grown the previous night and resuspended in Tris-buffered saline plus 5 mM CaCl₂ (31). The OD_{700 nm} was measured every 2.5 min, and all values were reported as relative to the absorbance at t = 0. The extent of aggregation was determined by the decrease in the optical density of the bacterial suspension. Calcium dependence was confirmed by the inhibition of aggregation in the presence of 10 mM EDTA.

Lung Morphology—Lungs from 12-week old mice were fixed at 25 cm of water pressure with 4% paraformaldehyde in phosphate-buffered saline (PBS) and processed into paraffin blocks. Sections (5 µm) from each lobe were stained with hematoxylin and eosin. Immunohistochemistry for SP-D was performed at dilutions of 1:200 by using a rabbit polyclonal antibody generated to murine SP-D. Immune complexes were detected using an avidin-biotin-peroxidase technique (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA).

Metalloproteinase Activity—Alveolar macrophages (5 x 10⁵) were isolated by centrifuging BALF and cultured for 24 h in AIM-V medium (Invitrogen). Proteinases in the conditioned media were assayed by zymography as described previously (32).

Phospholipid Analysis—Saturated phosphatidylcholine (Sat PC) was measured in homogenized lung tissue and BALF from 6-8-week-old mice (n = 6-8 mice for each genotype) as previously described (33).

Isolation of Large and Small Aggregate Sufactant—Large and small aggregate surfactant lipids were isolated from rSftpdCDM^Tg+/Sftpd^+/+ mice (n = 6) as previously described (14). Briefly, bronchoalveolar lavage was performed five times with 1 ml of normal saline. BALF was centrifuged at 40,000 x g over a 0.8 M sucrose cushion for 15 min. Small aggregate surfactant was collected from the supernatant. Large aggregate surfactant was collected from the interface, diluted with normal saline, and centrifuged again at 40,000 x g for 15 min. The large aggregate pellet was dissolved in normal saline. An equal volume of each material was dried, diluted in Laemmli buffer, and analyzed by Western blot.

Ultrastructure of lipid aggregates was determined in pooled BALF samples (n = 5/pool) by electron microscopy from 6-8-week-old mice as previously described (14). Briefly, large and small aggregate surfactant was isolated from BALF; fixed with glutaraldehyde, paraformaldehyde, and CaCl₂ in 0.1 M sodium cacodylate buffer at 4 °C; and stained with osmium tetroxide, potassium ferrocyanide, and uranyl acetate. After dehydration, it was embedded in Embed812 resin (Electron Microscopy Sciences, Fort Washington, PA), and ultrathin sections (90 nm) were obtained. Random electron micrographs were taken, and ultrastructure was evaluated.

Influenza A Virus—Experiments utilized influenza A virus strain H₃N₂ A/Phillipines/82 (IAV) and were performed as previously described (34). Briefly, IAV was grown in chorioallantoic fluid of 10-day-old embryonated hen eggs. Virus was purified and stored frozen in PBS until use. Six-week-old (n = 6-8) mice were anesthetized with inhaled isoflurane and inoculated intratracheally with 5 x 10⁵ fluorescent foci in 80 µl of PBS. Quantitative IAV cultures were performed 3 days after inoculation. Lungs were removed and homogenized in 2 ml of PBS, and aliquots were frozen. IL-6, tumor necrosis factor-, and interferon- concentrations were determined by ELISA kits (R&D Systems, Minneapolis, MN). IAV titers were determined by incubating lung homogenates with Madin-Darby canine kidney monolayers for 7 h at 37°C. Monolayers were washed, fixed, and incubated with monoclonal antibody against IAV nucleoprotein followed by a rhodamine-labeled goat antimouse IgG. Fluorescent foci were counted, and the resulting viral titer was expressed as fluorescent foci/g lung weight.

SP-D binding to IAV was monitored by hemagglutination inhibition assays (n = 3). Purified mouse SP-D and rSftpdCDM were compared. Sample protein was serially diluted in 96-well round bottom plates with IAV in PBS with 0.5 mM CaCl₂ and 0.5 mM MgCl₂. After incubating the protein/IAV mixture at room temperature for 10 min, fertilized chicken egg erythrocytes were added, and the samples were incubated for an additional 2 h. The minimal amount of SP-D or rSftpdCDM needed to fully inhibit agglutination was observed, and the number of hemagglutination units inhibited per pmol of SP-D or rSftpdCDM was reported.

Data Analysis—Where appropriate, either a representative experiment from one mouse line was shown, or results from each line were averaged, and data were analyzed by unpaired Student's t tests.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transgenic Mouse Lines—rSftpdCDM^Tg+/Sftpd^-/- transgenic mice were produced by nuclear injection of the rSftpdCDM gene into FVB/N mice and backcrossed to Sftpd^-/- mice. The rat Sftpd gene is 92% identical (based on nucleotide and mature protein amino acid sequence) to the mouse Sftpd gene, and multiple studies indicate that mouse and rat SP-D have nearly identical properties. Four founder mice were identified using transgene-specific PCR on tail clip DNA. Germ line transmission was demonstrated in all four lines, and the transgene was inherited as an autosomal gene following Mendelian inheritance. Survival and breeding were not influenced by the rSftpdCDM^Tg+ transgene. Expression and secretion of transgenic protein rSftpdCDM was confirmed in all four mouse lines by Western blot analysis of mouse BALF using a rabbit anti-mouse SP-D antibody (data not shown). Two mouse lines with similar levels of rSftpdCDM protein expression were selected for further breeding and analysis. Experiments were done in parallel with both lines, and results were similar.

Expression of rSftpdCDM Protein—As determined by ELISA assays, levels of SP-D in BALF from wild type mice were 1.2 µg/ml compared with rSftpdCDM levels of 11 µg/ml in rSftpdCDM^Tg+/Sftpd^-/- mice. Rabbit anti-mouse SP-D antibody was used to detect SP-D and rSftpdCDM in BALF (Fig. 2). The monomeric form of the mutant protein migrated under reducing conditions at the predicted molecular mass of 22 kDa. rSftpdCDM migrated slightly slower than the 60-kDa standard under nonreducing conditions, indicating that the disulfide linkages normally observed between monomeric chains in the wild type N-terminal domain also formed in the mutant protein. To determine the ability of the rSftpdCDM to form high order complexes, the protein was analyzed by Sepharose column chromotography. Under these conditions, the majority of rSftpdCDM migrated between apoferritin and -amylase (molecular masses of 443 and 200 kDa, respectively) at a position equaling 270 kDa, which is the molecular mass expected if adding the N-terminal domain to the CRD trimer promoted noncovalent interactions between four trimeric subunits (dodecamer). Smaller peaks were observed at the expected positions of molecules composed of 1, 2, 30, and more trimers, suggesting that the purified mutant protein consists of a heterogeneous population of multimers. Whereas the mutant protein effectively self-assembled to higher order structures, it did not form intermediate molecular weight heteropolymers of mutant and wild type protein when expressed in a wild type SP-D background (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Analysis of bronchoalveolar lavage fluid and purified protein from mice expressing rSftpdCDM. A, BALF from wild type (lanes 1 and 3) and rSftpdCDMTg+/Sftpd-/- (lanes 2 and 4) mice were resolved on SDS-PAGE under reducing (Reduced) conditions. Disulfide-linked trimers were resolved under nonreducing (Non Reduced) conditions. Protein was detected with rabbit anti-mouse SP-D antibody. B, purified rSftpdCDM was resolved by column chromatography in Sepharose CL-6B. Protein levels were determined by ELISA and expressed as relative to the peak fraction. The arrows indicate the peak position of eluted standards: blue dextran (a), thyroglobulin (b), apoferritin (c), -amylase (d), alcohol dehydrogenase (e), albumin (f), and carbonic anhydrase (g).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. SP-D and rSftpdCDM proteins bind maltosyl-Sepharose. BALF from rSftpdCDMTg+/Sftpd+/+ mice was incubated with maltosyl-Sepharose beads followed by centrifugation to pellet the beads. The resulting supernatant was evaluated by SDS-PAGE and Western analysis. Binding of endogenous SP-D (SP-D) or mutant rSftpdCDM (CDM) was indicated by the absence of protein in the supernatant. BALF was incubated with (+) or without (-) beads or with beads and EDTA (EDTA) or excess free maltose (Maltose).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. rSftpdCDM aggregates E. coli. Bacterial suspensions (n = 3) were incubated with 150 nM purified rSftpdCDM (CDM, open triangles) or wild type SP-D (SP-D, closed circles). Absorbance values were recorded at the indicated times before (t = 0) and after the addition of the indicated protein and reported as relative to the absorbance value at t = 0. Aggregation was indicated by the clustering of bacterial particles and a subsequent drop in absorbance. Aggregation with rSftpdCDM was completely reversed by the addition of EDTA to the reaction mixture (EDTA, open circles). Control (closed circles) was protein buffer alone. S.E. values were <10% for all time points.

Lung Morphology—Deletion of the mouse Sftpd gene caused several distinct alterations in lung morphology, including emphysema and accumulations of enlarged foamy macrophages (13, 16-18). To determine if the mutant protein influenced these findings, pulmonary structure was assessed in wild type and Sftpd^-/- mice expressing rSftpdCDM^Tg+ (Fig. 5). Immunostaining revealed marked expression of rSftpdCDM^Tg+ in alveolar type II cells and bronchiolar epithelial cells. However, rSftpdCDM did not alter the lung morphology in 12-week-old wild type mice. Moreover, the mutant protein did not correct the abnormal morphology typically observed in Sftpd^-/- mice. Enlarged, foamy macrophage accumulations and emphysema were detected readily in both transgenic mouse lines expressing rSftpdCDM^Tg+ in a Sftpd^-/- background. Therefore, whereas targeted expression of the full-length rat Sftpd gene in Sftpd^-/- mice fully rescues the Sftpd^-/- phenotype (36), expression of rSftpdCDM^Tg+ does not correct the abnormal lung morphology in Sftpd^-/- mice.

View larger version (141K): [in this window] [in a new window]   FIGURE 5. rSftpdCDM does not correct lung morphology in Sftpd-/- mice. Lungs were fixed and stained with hematoxylin and eosin. A, lungs from wild type mice; B, rSftpdCDMTg+ in a wild type background; C, immunostaining of SP-D in wild type mice; D, Sfpd-/- mice; E, rSftpdCDMTg+ in Sftpd-/- background; F, immunostaining of rSfpdCDM in a Sftpd-/- background. The arrowheads point to enlarged, foamy macrophages. The arrows point to SP-D immunostaining in type II cells and bronchiolar epithelial cells. Bars, 100 µm. The insets show the same tissue under higher magnification.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. rSftpdCDM does not correct increased metalloproteinase activity in Sftpd-/- mice. Proteinase activity in conditioned media from alveolar macrophages isolated from wild type (WT), rSftpdCDMTg+/Sftpd-/-, and Sftpd-/- mice was evaluated on zymogram gels. BALF was diluted as shown, and MMP-2 and MMP-9 activity was indicated by a clear band at 72 and 92 kDa, respectively.

Lung Phospholipids and Surfactant Structure—SP-D selectively interacts with small aggregate surfactant in adult mice and regulates the uptake and catabolism of surfactant lipids by alveolar type II cells (13, 14). Consequently, mice lacking SP-D have 2-5-fold higher surfactant pool sizes (13, 16). Moreover, surfactant isolated from Sftpd^-/- mice has abnormally large aggregate lipid structures and small aggregates consisting of atypical multilamellated forms (14). To evaluate if rSftpdCDM corrected surfactant phospholipid pool sizes, Sat PC in BALF and lung homogenates were assessed in wild type and Sftpd^-/- mice with and without rSftpdCDM^Tg+ (Fig. 7). Expression of the mutant protein did not alter alveolar, tissue, or total Sat PC levels in wild type mice, and it did not correct the elevated Sat PC levels in Sftpd^-/- mice. In addition, to determine if rSftpdCDM corrected the abnormal surfactant ultrastructure characteristic of Sftpd^-/- mice, large and small aggregate surfactant was examined by electron microscopy (Fig. 8). The large aggregate fraction from Sftpd^-/- and rSftpdCDM^Tg+/Sftpd^-/- mice contained abnormal, large lamellated lipid structures. The small aggregate fraction in wild type mice consisted of single layer sheets or vesicles, whereas atypical multilayered structures predominated in the small aggregate fraction from Sftpd^-/- and rSftpdCDM^Tg+/Sftpd^-/- mice. Despite the failure of rSftpdCDM to correct surfactant lipid structure, analysis of large and small aggregate surfactant from rSftpdCDM^Tg+/Sftpd^+/+ mice revealed that rSftpdCDM partitioned with small aggregate surfactant in a manner that was similar to SP-D (Fig. 9). This is in contrast to SP-A, which segregated primarily with large aggregate surfactant. Therefore, these results demonstrate that the collagen domain of SP-D is not required for the selective partitioning of SP-D with small aggregate surfactant, but it is required for normal surfactant ultrastructure that in turn influences uptake by alveolar type II cells (14).

Correction of Influenza A Infection by rSftpdCDM—Previous in vitro studies demonstrated that SP-D binds IAV and enhances IAV binding and uptake by neutrophils (31, 37, 38). Decreased viral clearance and enhanced inflammation was observed in Sftpd^-/- mice exposed to intratracheal IAV (21). As determined by hemagglutination inhibition assays, rSftpdCDM effectively bound IAV and inhibited IAV-mediated hemagglutination (Fig. 10). Moreover, hemagglutination inhibition activity of rSftpdCDM was 2-fold greater than that observed with an equal molar (based on the molecular weight of a SP-D or rSftpdCDM monomer) amount of wild type SP-D. To further evaluate the anti-IAV activity of rSftpdCDM, IAV was administered to mouse lungs intratracheally, and the viral titers and cytokine response (Fig. 11) were measured 3 days later. In contrast to Sftpd^-/- mice, no detectable IAV was recovered from the wild type or rSftpdCDM^Tg+/Sftpd^-/- lung homogenates. In addition, the increased IL-6, tumor necrosis factor-, and interferon- levels observed in IAV-challenged Sftpd^-/- mice were restored to wild type levels in rSftpdCDM^Tg+/Sftpd^-/- mice. Taken together, these results demonstrate that rSftpdCDM completely corrects viral binding, clearance, and inflammatory responses observed in Sftpd^-/- mice.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Increased lung saturated phosphatidylcholine in rSftpdCDMTg+/Sftpd-/- mice. Alveolar, tissue, and total Sat PC levels were determined in wild type (Sftpd+/+) and SP-D null (Sftpd-/-) mice with (open bars)or without (closed bars) the rSftpdCDM transgene (n = 6-8 mice for each genotype). Values were normalized for body weight. Sat PC levels in Sftpd-/- mice were significantly (p 0.05) higher than wild type controls. The addition of rSftpdCDM did not significantly (p = 0.48, 0.47, and 0.40 for alveolar, tissue, and total, respectively) correct this increase. The error bars indicate S.E.

View larger version (120K): [in this window] [in a new window]   FIGURE 8. rSftpdCDM does not correct surfactant ultrastructure in Sftpd-/- mice. Ultrastructure of large aggregate (LA) and small aggregate (SA) surfactant isolated from wild type (Sftpd+/+CDM-), Sftpd-/- (Sftpd-/-CDM-), and rSftpdCDMTg+/Sftpd-/- (Sftpd-/- CDM+) mice. The closed arrowheads point to normal large aggregate lamellar bodies and small aggregate single layer sheets and vesicles. The open arrowheads point to abnormal large lipid structures in large aggregate surfactant. The arrows point to atypical multilayered structures in small aggregate surfactant. Photomicrographs are representative of samples pooled from three mice of each genotype.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Deletion of the collagen domain resulted in the secretion of an 22-kDa protein that migrated as a trimer under nonreducing conditions. These findings are consistent with in vitro studies describing an SP-D collagen deletion mutant that was expressed in Chinese hamster ovary cells by Ogasawara and Voelker (35, 39). In contrast to these earlier studies, the majority of the present rSftpdCDM formed higher order complexes of four trimeric subunits (dodecamer) when expressed in the mouse respiratory system. The reason for this disparity is unclear, but given the close proximity of the regions deleted, it probably reflects differences in the protein expression systems utilized. In addition, the finding that adding the N terminus to the trimeric CRD facilitates dodecamer formation supports previous results that indicate that the structural predilection for dodecamers is contained within the N-terminal domain (40).

View larger version (81K): [in this window] [in a new window]   FIGURE 9. rSftpdCDM partitions with small aggregate surfactant. BALF was isolated from rSfptdCDMTg+/Sftpd+/+ mice, and surfactant lipids were separated into small (SA) and large (LA) aggregate fractions by centrifugation. A, SP-D (SP-D) and rSftpdCDM (CDM) were detected in the BALF prior to lipid isolation (BAL) and in small aggregate lipids by Western blot with anti-SP-D antibody. B, identical samples were analyzed with anti-SP-A antibody. SP-A (SP-A) was detected in BALF and in large aggregate surfactant.

View larger version (10K): [in this window] [in a new window]   FIGURE 10. rSftpdCDM binds influenza A virus. Binding to influenza A virus was determined by hemagglutination inhibition assays (n = 3). The number of hemagglutination units inhibited (HAI) per pmol of rSftpdCDM (CDM) or wild type SP-D (SP-D) is shown. Error bars, S.E.

Despite the considerable binding affinity of rSftpdCDM, expression of the mutant protein failed to correct the aberrant alveolar macrophage activity characteristic of Sftpd^-/- mice (13, 16-18). Foamy macrophages, increased secretion of metalloproteinases, and emphysema persisted despite expression of high levels of rSftpdCDM. In contrast, influenza virus binding, clearance, and the associated cytokine response mediated by rSftpdCDM were similar to or better than observed in wild type mice. Similar findings of abnormal base-line macrophage activation in the setting of a normal response to influenza virus were also described in studies utilizing an SP-D conglutinin (SftpdCong) fusion protein consisting of the N terminus and collagen domains of rat SP-D and the neck and CRD of conglutinin (27). Several interesting considerations regarding SP-D are raised from these observations. First, although the rSftpdCDM^Tg+/Sftpd^-/- alveolar macrophages have an abnormal histological appearance and base-line level of activity, they still are able to mount an effective and relatively normal inflammatory response to the infectious pathogen, influenza A. Second, although limited by the fact that only one infectious pathogen was tested, the normal cytokine response observed following influenza A viral infection suggests that the elevated base-line macrophage activity in Sftpd^-/- mice is not due to the inability to clear a persistent low level infection. Finally, the divergence of macrophage base-line activity and the inflammatory response to viral infection in rSftpdCDM^Tg+/Sftpd^-/- mice suggests that SP-D regulates these processes through two independent pathways.

View larger version (12K): [in this window] [in a new window]   FIGURE 11. rSftpdCDM corrects response to Influenza A virus in Sftpd-/- mice. IL-6, tumor necrosis factor- (TNF), and interferon- (IFN) levels as well as influenza A viral titers were determined in lung homogenates 3 days after intratracheal installation of virus into wild type (WT), rSftpdCDMTg+/Sftpd-/- (CDM), or Sftpd-/- (Sftpd-/-) mice (n = 6-8 mice for each genotype). Viral titers are expressed as relative values with Sftpd-/- mice normalized to 100%. Inflammatory cytokine levels and viral titers were significantly higher (p < 0.05) in Sftpd-/- mice when compared with wild type or rSftpdCDMTg+/Sftpd-/- mice. Error bars, S.E.

The failure of rSftpdCDM to correct the enlarged foamy macrophages, emphysema, and pulmonary phospholipid accumulations observed in Sftpd^-/- mice is in contrast to earlier work with a recombinant fragment of SP-D consisting of a trimeric CRD and neck domain, which partially corrected the abnormal macrophages and surfactant pool sizes in Sftpd^-/- mice (15). The reason why a single trimeric CRD would partially resolve the abnormalities that an oligomerized trimeric CRD fails to correct is uncertain, but it may reflect unanticipated changes that sometimes occur in mutant proteins or differences in the concentration of the mutant SP-Ds utilized in each study. Alternatively, this inconsistency may suggest a complex interplay between the N-terminal domain and the CRD in macrophage regulation and control of phospholipid pool sizes.

Although alveolar macrophages and type II epithelial cells equally contribute to surfactant phospholipid catabolism, previous studies demonstrated that SP-D regulates surfactant pool size by enhancing surfactant uptake by alveolar type II cells (14). Specifically, SP-D maintains normal surfactant ultrastructure by selectively interacting with small aggregate surfactant, thereby facilitating surfactant uptake by type II cells. The lipid binding specificity of collectins is mediated by the CRD in vitro (42-45) and intranasal administration of trimeric CRDs decreased intra-alveolar lipid accumulations in Sftpd^-/- mice (15, 24). In contrast, the SP-D CRD and neck domain in the rSftpa/d fusion protein failed to correct surfactant lipid ultrastructure or lower the phospholipid levels when expressed in Sftpd^-/- mice (22). In the present study, the addition of the SP-D N terminus to the CRD failed to correct surfactant ultrastructure or increased phospholipid levels. Therefore, normal surfactant ultrastructure and the uptake of surfactant by type II cells depend on the collagen domain.

Although rSftpa/d included the neck and CRD of SP-D, analysis of large and small aggregate surfactant lipids demonstrated that rSftpa/d partitioned with large aggregate surfactant in a manner similar to SP-A (22). In contrast, rSftpdCDM, which included the neck, CRD, and N-terminal domains of SP-D, partitioned with small aggregate surfactant like the full-length SP-D protein. Taken together, these studies suggest that whereas phospholipid binding in vitro may be mediated by the CRD, the structural features of SP-D (and SP-A) that influence partitioning between small versus large aggregate surfactant are contained within the N-terminal domain.

In summary, our study provides further support for the importance of the distinct molecular domains of SP-D in mediating the complex functions of this protein. The finding that the collagen deletion mutant fails to correct the abnormal lung morphology or surfactant lipid homeostasis of Sftpd^-/- mice supports a role of the collagen domain that is beyond proper spacing of trimeric subunits for bacterial aggregation. Assimilating the results of this study with those of prior reports on SP-D reveals that whereas our understanding of SP-D is improving, inconsistencies still exist. The CRD is critical for binding to lipopolysaccharide, viruses, bacteria, fungus, and lipids. Nonetheless, SP-D interactions with small aggregate surfactant and uptake of surfactant by alveolar type II cells in vivo do not depend on the CRD alone. The collagen domain is required for surfactant lipid structure and metabolism, but it is not needed to effectively aggregate bacteria or to suppress inflammatory responses to influenza A virus. The N-terminal domain is critical for partitioning with large versus small aggregate surfactant lipids in adult mice, and interactions between interchain N-terminal domains are essential for oligomerization, which, in turn, influences CRD binding affinity, phospholipid catabolism, and the inflammatory response to infectious pathogens. Finally, at the time of this writing, all SP-D mutant proteins that delete or substitute even a single domain of SP-D fail to fully correct the enlarged, foamy macrophages that are characteristic of Sftpd^-/- mice, suggesting that this function of SP-D is dependent on a full-length, multimeric protein.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health (NIH) Grants HL56387 (to J. A. W.), HL63329 (to M. I.), and HL68861 (to F. X. M.) and NHLBI, NIH, Grant HL60931 (to K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-4830; Fax: 513-636-7868; E-mail: jeff.whitsett{at}cchmc.org.

² The abbreviations used are: SP-D, surfactant protein D; SP-A, surfactant protein A; CRD, carbohydrate recognition domain; IL, interleukin; BALF, bronchoalveolar lavage fluid; PBS, phosphate-buffered saline; IAV, influenza A virus; ELISA, enzyme linked immunosorbent assay; MMP, matrix metalloproteinase; Sat PC, saturated phosphatidylcholine; SIRP, signal-inhibitory protein.

	ACKNOWLEDGMENTS

 
We are grateful to Mitchell R. White for invaluable advice and technical assistance in influenza A viral assays and to Sarah K. Yoshimura for critical reading of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Hawgood, S., and Poulain, F. R. (2001) Annu. Rev. Physiol. 63, 495-519[CrossRef][Medline] [Order article via Infotrieve]
2. Crouch, E., and Wright, J. R. (2001) Annu. Rev. Physiol. 63, 521-554[CrossRef][Medline] [Order article via Infotrieve]
3. Whitsett, J. A. (2005) Biol. Neonate 88, 175-180[CrossRef][Medline] [Order article via Infotrieve]
4. Rust, K., Grosso, L., Zhang, V., Chang, D., Persson, A., Longmore, W., Cai, G. Z., and Crouch, E. (1991) Arch. Biochem. Biophys. 290, 116-126[CrossRef][Medline] [Order article via Infotrieve]
5. Shimizu, H., Fisher, J. H., Papst, P., Benson, B., Lau, K., Mason, R. J., and Voelker, D. R. (1992) J. Biol. Chem. 267, 1853-1857[Abstract/Free Full Text]
6. Crouch, E., Rust, K., Veile, R., Donis-Keller, H., and Grosso, L. (1993) J. Biol. Chem. 268, 2976-2983[Abstract/Free Full Text]
7. Lu, J., Wiedemann, H., Holmskov, U., Thiel, S., Timpl, R., and Reid, K. B. (1993) Eur. J. Biochem. 215, 793-799[Medline] [Order article via Infotrieve]
8. van de Wetering, J. K., van Golde, L. M., and Batenburg, J. J. (2004) Eur. J. Biochem. 271, 1229-1249[Medline] [Order article via Infotrieve]
9. Zhang, P., McAlinden, A., Li, S., Schumacher, T., Wang, H., Hu, S., Sandell, L., and Crouch, E. (2001) J. Biol. Chem. 276, 19862-19870[Abstract/Free Full Text]
10. Crouch, E., Persson, A., Chang, D., and Heuser, J. (1994) J. Biol. Chem. 269, 17311-17319[Abstract/Free Full Text]
11. Brown-Augsburger, P., Hartshorn, K., Chang, D., Rust, K., Fliszar, C., Welgus, H. G., and Crouch, E. C. (1996) J. Biol. Chem. 271, 13724-13730[Abstract/Free Full Text]
12. Crouch, E., Chang, D., Rust, K., Persson, A., and Heuser, J. (1994) J. Biol. Chem. 269, 15808-15813[Abstract/Free Full Text]
13. Ikegami, M., Whitsett, J. A., Jobe, A., Ross, G., Fisher, J., and Korfhagen, T. (2000) Am. J. Physiol. 279, L468-L476
14. Ikegami, M., Na, C. L., Korfhagen, T. R., and Whitsett, J. A. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 288, L552-L561[Abstract/Free Full Text]
15. Clark, H., Palaniyar, N., Strong, P., Edmondson, J., Hawgood, S., and Reid, K. B. (2002) J. Immunol. 169, 2892-2899[Abstract/Free Full Text]
16. Korfhagen, T. R., Sheftelyevich, V., Burhans, M. S., Bruno, M. D., Ross, G. F., Wert, S. E., Stahlman, M. T., Jobe, A. H., Ikegami, M., Whitsett, J. A., and Fisher, J. H. (1998) J. Biol. Chem. 273, 28438-28443[Abstract/Free Full Text]
17. Botas, C., Poulain, F., Akiyama, J., Brown, C., Allen, L., Goerke, J., Clements, J., Carlson, E., Gillespie, A. M., Epstein, C., and Hawgood, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11869-11874[Abstract/Free Full Text]
18. Wert, S., Jones, T., Korfhagen, T., Fisher, J., and Whitsett, J. (2000) Chest 117, Suppl. 1, 248[Free Full Text]
19. LeVine, A. M., Whitsett, J. A., Gwozdz, J. A., Richardson, T. R., Fisher, J. H., Burhans, M. S., and Korfhagen, T. R. (2000) J. Immunol. 165, 3934-3940[Abstract/Free Full Text]
20. LeVine, A. M., Elliott, J., Whitsett, J. A., Srikiatkhachorn, A., Crouch, E., DeSilva, N., and Korfhagen, T. (2004) Am. J. Respir. Cell Mol. Biol. 31, 193-199[Abstract/Free Full Text]
21. LeVine, A. M., Whitsett, J. A., Hartshorn, K. L., Crouch, E. C., and Korfhagen, T. R. (2001) J. Immunol. 167, 5868-5873[Abstract/Free Full Text]
22. Zhang, L., Ikegami, M., Korfhagen, T. R., McCormack, F. X., Yoshida, M., Senior, R. M., Shipley, J. M., Shapiro, S. D., and Whitsett, J. A. (2006) Am. J. Physiol. 291, L181-L190
23. Zhang, L., Ikegami, M., Crouch, E. C., Korfhagen, T. R., and Whitsett, J. A. (2001) J. Biol. Chem. 276, 19214-19219[Abstract/Free Full Text]
24. Clark, H., Palaniyar, N., Hawgood, S., and Reid, K. B. (2003) Ann. N. Y. Acad. Sci. 1010, 113-116[CrossRef][Medline] [Order article via Infotrieve]
25. Hartshorn, K. L., White, M. R., and Crouch, E. C. (2002) Infect Immun. 70, 6129-6139[Abstract/Free Full Text]
26. Wert, S. E., Glasser, S. W., Korfhagen, T. R., and Whitsett, J. A. (1993) Dev. Biol. 156, 426-443[CrossRef][Medline] [Order article via Infotrieve]
27. Zhang, L., Hartshorn, K. L., Crouch, E. C., Ikegami, M., and Whitsett, J. A. (2002) J. Biol. Chem. 277, 22453-22459[Abstract/Free Full Text]
28. Reed, J. A., Ikegami, M., Robb, L., Begley, C. G., Ross, G., and Whitsett, J. A. (2000) Am. J. Physiol. 278, L1164-L1171
29. Strong, P., Kishore, U., Morgan, C., Lopez Bernal, A., Singh, M., and Reid, K. B. (1998) J. Immunol. Methods 220, 139-149[CrossRef][Medline] [Order article via Infotrieve]
30. Lim, B. L., Wang, J. Y., Holmskov, U., Hoppe, H. J., and Reid, K. B. (1994) Biochem. Biophys. Res. Commun. 202, 1674-1680[CrossRef][Medline] [Order article via Infotrieve]
31. Hartshorn, K. L., Crouch, E., White, M. R., Colamussi, M. L., Kakkanatt, A., Tauber, B., Shepherd, V., and Sastry, K. N. (1998) Am. J. Physiol. 274, L958-L969
32. Wert, S. E., Yoshida, M., LeVine, A. M., Ikegami, M., Jones, T., Ross, G. F., Fisher, J. H., Korfhagen, T. R., and Whitsett, J. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5972-5977[Abstract/Free Full Text]
33. Korfhagen, T. R., Bruno, M. D., Ross, G. F., Huelsman, K. M., Ikegami, M., Jobe, A. H., Wert, S. E., Stripp, B. R., Morris, R. E., Glasser, S. W., Bachurski, C. J., Iwamoto, H. S., and Whitsett, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9594-9599[Abstract/Free Full Text]
34. Hartshorn, K. L., Sastry, K. N., Chang, D., White, M. R., and Crouch, E. C. (2000) Am. J. Physiol. 278, L90-L98
35. Ogasawara, Y., and Voelker, D. R. (1995) J. Biol. Chem. 270, 19052-19058[Abstract/Free Full Text]
36. Zhang, L., Ikegami, M., Dey, C. R., Korfhagen, T. R., and Whitsett, J. A. (2002) J. Biol. Chem. 277, 38709-38713[Abstract/Free Full Text]
37. Hartshorn, K. L., Crouch, E. C., White, M. R., Eggleton, P., Tauber, A. I., Chang, D., and Sastry, K. (1994) J. Clin. Invest. 94, 311-319[Medline] [Order article via Infotrieve]
38. Hartshorn, K., Chang, D., Rust, K., White, M., Heuser, J., and Crouch, E. (1996) Am. J. Physiol. 271, L753-L762
39. Ferguson, J. S., Voelker, D. R., Ufnar, J. A., Dawson, A. J., and Schlesinger, L. S. (2002) J. Immunol. 168, 1309-1314[Abstract/Free Full Text]
40. Palaniyar, N., Zhang, L., Kuzmenko, A., Ikegami, M., Wan, S., Wu, H., Korfhagen, T. R., Whitsett, J. A., and McCormack, F. X. (2002) J. Biol. Chem. 277, 26971-26979[Abstract/Free Full Text]
41. Gardai, S. J., Xiao, Y. Q., Dickinson, M., Nick, J. A., Voelker, D. R., Greene, K. E., and Henson, P. M. (2003) Cell 115, 13-23[CrossRef][Medline] [Order article via Infotrieve]
42. Ogasawara, Y., McCormack, F. X., Mason, R. J., and Voelker, D. R. (1994) J. Biol. Chem. 269, 29785-29792[Abstract/Free Full Text]
43. Kishore, U., Wang, J. Y., Hoppe, H. J., and Reid, K. B. (1996) Biochem. J. 318, 505-511
44. Sano, H., Kuroki, Y., Honma, T., Ogasawara, Y., Sohma, H., Voelker, D. R., and Akino, T. (1998) J. Biol. Chem. 273, 4783-4789[Abstract/Free Full Text]
45. DeSilva, N. S., Ofek, I., and Crouch, E. C. (2003) Am. J. Respir. Cell Mol. Biol. 29, 757-770[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Ikegami, S. Grant, T. Korfhagen, R. K. Scheule, and J. A. Whitsett Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes J Appl Physiol, May 1, 2009; 106(5): 1545 - 1552. [Abstract] [Full Text] [PDF]

				M. White, P. Kingma, T. Tecle, N. Kacak, B. Linders, J. Heuser, E. Crouch, and K. Hartshorn Multimerization of Surfactant Protein D, but Not Its Collagen Domain, Is Required for Antiviral and Opsonic Activities Related to Influenza Virus J. Immunol., December 1, 2008; 181(11): 7936 - 7943. [Abstract] [Full Text] [PDF]

				A. V. Andreeva, M. A. Kutuzov, and T. A. Voyno-Yasenetskaya Regulation of surfactant secretion in alveolar type II cells Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L259 - L271. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) An addition or correction has been published All Versions of this Article: 281/34/24496    most recent M600651200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kingma, P. S. Articles by Whitsett, J. A. Search for Related Content PubMed PubMed Citation Articles by Kingma, P. S. Articles by Whitsett, J. A. Social Bookmarking                      What's this?