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Am J Physiol Lung Cell Mol Physiol 291: L181-L190, 2006. First published February 24, 2006; doi:10.1152/ajplung.00015.2006
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Neither SP-A nor NH2-terminal domains of SP-A can substitute for SP-D in regulation of alveolar homeostasis

Liqian Zhang,1 Machiko Ikegami,1 Thomas R. Korfhagen,1 Francis X. McCormack,2 Mitsuhiro Yoshida,3 Robert M. Senior,4 J. Michael Shipley,4 Steven D. Shapiro,5 and Jeffrey A. Whitsett1

1Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, and 2Pulmonary/Critical Care Division, University of Cincinnati Medical Center, Cincinnati, Ohio; 3Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; 4Department of Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, Barnes-Jewish Hospital, St. Louis, Missouri; and 5Pulmonary and Critical Care, Brigham and Women’s Hospital, Boston, Massachusetts

Submitted 10 January 2006 ; accepted in final form 20 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surfactant proteins (SP)-A and -D are members of the collectin family of host defense proteins that share four distinct structural domains: NH2-terminal oligomerization, collagenous, neck, and carbohydrate recognition (CRD). To determine the specificity of the functions of these domains, the SFTPC promoter was used to express 1) full-length rat (r) Sftpa; 2) NH2-rSftpa/d, consisting of NH2-terminal and collagenous domains of SP-A with neck domain and CRD of SP-D; and 3) rSftpd/a, consisting of NH2-terminal and collagenous domains of SP-D with neck domain and CRD of SP-A, in Sftpd–/– mice. Increased expression of SP-A in Sftpd–/– mice did not correct the increased pulmonary saturated phosphatidylcholine levels, emphysema, or foamy alveolar macrophage and lymphocyte infiltrations characteristic of Sftpd–/– mice, indicating that the decreased SP-A level noted in Sftpd–/– mice does not account for the observed pulmonary abnormalities. The chimeric protein NH2-rSftpa/d was expressed and detected in the airways of transgenic mice, migrating as an SP-A-like oligomer that associated with large aggregate surfactant in a manner similar to that of SP-A rather than SP-D. NH2-rSftpa/d did not correct emphysema, foamy macrophage and lymphocyte infiltration, or the increased lipid accumulations characteristic of Sftpd–/– mice. Thus oligomerization and surfactant lipid association of SP-D requires its NH2-terminal and collagenous domains, which are needed for SP-D-dependent regulation of surfactant homeostasis in vivo. Attempts to express rSftpd/a fusion protein in vivo were unsuccessful. Mmp9–/–/Sftpd–/– and Mmp12–/–/Sftpd–/– mice developed air space enlargement similar to Sftpd–/– mice, supporting the concept that the increased expression of each metalloproteinase seen in Sftpd–/– lungs is not the major cause of emphysema.

Sftpd–/– mice; emphysema; lipid homeostasis; inflammation

SURFACTANT PROTEINS (SP)-A and -D are members of the collectin family expressed in the epithelial cells of the lung. The collectins share four distinct structural domains: 1) a short NH2-terminal domain mediating cysteine-dependent oligomerization, 2) a collagenous domain, 3) an {alpha}-helical neck region involved in trimer formation, and 4) a COOH-terminal carbohydrate recognition domain (CRD) (13, 30). The collectins are assembled as higher-ordered oligomers of trimeric subunits. SP-A is predominantly assembled as a bouquet of octadecamers consisting of six trimeric subunits. SP-D is predominantly assembled as dodecamers consisting of four trimeric subunits (8). In the lung, SP-A and SP-D are both synthesized and secreted by type II cells and nonciliated bronchiolar epithelial cells (6, 7, 35).

Previous studies of gene-targeted mice demonstrated that SP-A and SP-D have distinct roles in lung homeostasis. Lung morphology, SP-B, -C, and -D, lung tissue and alveolar phospholipid pool sizes, and lung compliance were not changed in Sftpa-null (Sftpa–/–) mice (18). Sftpd-null (Sftpd–/–) mice develop pulmonary abnormalities including lipid accumulations, emphysema, as well as foamy macrophage and lymphocyte infiltration (2, 19). The alveolar macrophages in Sftpd–/– mice have increased oxidant production and matrix metalloproteinase (MMP2, MMP9, and MMP12) activities that may contribute to the pulmonary abnormalities seen in this animal model (40). Decreased concentration of SP-A was observed in the lung and bronchoalveolar lavage fluid (BALF) of Sftpd–/– mice (16).

Previous studies in vivo demonstrated that SP-D functions in the regulation of surfactant lipids and prevention of emphysema is dependent on its NH2-terminal cysteine cross-linking domain (42). Substitution of the neck domain and CRD of SP-D with those of bovine conglutinin completely rescued viral clearance and cytokine production and partially rescued phospholipid abnormalities in Sftpd–/– mice (41). However, substitution of the NH2-terminal Cys-Cys cross-linking domain of SP-D in SP-A was not sufficient to correct the structural and lipid abnormalities in Sftpd–/– mouse lungs (26), indicating that the CRD or the collagen domain of SP-D was likely required for these activities. Because SP-A content is decreased in Sftpd–/– mice, it also remains unclear whether some or all of the abnormalities seen in Sftpd–/– mice are caused by changes in SP-A.

To investigate whether the decrease in SP-A levels in Sftpd–/– mice contributes to the structural and lipid abnormalities in the lung, we introduced full-length rat (r) rSftpa into the lungs of Sftpd–/– mice. To investigate whether the neck domain and CRD of SP-A and SP-D are functionally interchangeable, we expressed the fusion protein NH2-rSftpa/d consisting of NH2-terminal and collagenous domains of SP-A with neck domain and CRD of SP-D in Sftpd–/– mice. Neither the increased expression of SP-A nor the fusion protein NH2-rSftpa/d corrected the morphological and phospholipid abnormalities typical of Sftpd–/– mice. Our results indicated that SP-A and SP-D have distinct functions in lung homeostasis and that the function of the neck domain and CRD of SP-D is dependent on its own NH2-terminal and collagenous domains that cannot be complemented by those of SP-A. The NH2-terminal and collagenous domains determine the interaction of SP-D with lipids that is associated with the regulation of surfactant lipid pool sizes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse husbandry. Mice used in experimental procedures were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital. All mice had been maintained in the vivarium under barrier containment facilities. At the time of study, all mice appeared healthy with no evidence of infection. Sentinel mice in the colony were serologically negative for common murine pathogens.

Generation of transgenic mice. NH2-rSftpa/d cDNA was cloned, using recombinant PCR to link the NH2-terminal and collagenous domains of rSP-A with the neck domain and CRD of rSP-D. NH2-rSftpa/d cDNA was inserted into the EcoRI site of 3.7SFTPC/SV40 expression vector (39) and sequenced. The transgene was microinjected into FVB/N oocytes fertilized with Swiss Black Sftpd–/– sperm by the Cincinnati Children’s Hospital Transgenic Core facility, and founders were identified by transgene specific PCR using upstream primer 5'-CCCTCTTCTTGACTGTTGTCGC-3' and downstream primer 5'-TTCGGATGGTGGCAGCATAG-3'. Founders were crossed with Swiss Black Sftpd–/– mice (19) to generate NH2-rSftpa/dtg+/Sftpd–/– mice. rSftpatg+ (FVB/N) (10), Mmp9–/– (Swiss Black) (21), and Mmp12–/– (Swiss Black) mice (32) were mated to Swiss Black Sftpd–/– mice to generate rSftpatg+/Sftpd–/–, Mmp9–/–/Sftpd–/–, and Mmp12–/–/Sftpd–/– mice. Mice were of mixed strains derived from FVB/N and Swiss Black. A summary of these transgenic mice is shown in Table 1. Littermates were used for comparisons in each experiment to minimize differences related to strain.


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  		Table 1. Transgenic mice

 
Western blot analysis. Animals were weighed, anesthetized by intraperitoneal injection of pentobarbital, and exsanguinated by severing the distal aorta. Bronchoalveolar lavage (BAL) was performed five times with saline for each lung, and the collected volume was measured (18). BALF (25 µl) from each mouse was vacuum dried and reconstituted in 15 µl of nonreduced or reduced (with beta-mercaptoethanol) Laemmli sample buffer (Bio-Rad, Hercules, CA). After resolution with a 10–20% SDS-Tris-glycine-polyacrylamide gel (Novex, San Diego, CA) and transfer to nitrocellulose membrane, blots were blocked with 5% nonfat milk and then incubated at room temperature (RT) overnight with either rabbit anti-mouse SP-D antibody (1:5,000) or guinea pig anti-rat SP-A antibody (1:10,000) in Tris-buffered saline (TBS) with 0.1% Tween 20. Blots were washed with TBS-Tween and incubated at RT for 4 h with 1:5,000 dilution of peroxidase-conjugated goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody (Calbiochem, La Jolla, CA). After washing, blots were developed with a chemiluminescence detection system (Amersham Pharmacia Biotech, Chalfont St. Giles, UK). Generally, four to eight mice from each genotype were analyzed for each mouse line, and representative results are shown.

Lung morphology. Mouse lungs (12 wk old) were fixed at 25-cmH2O pressure with 4% paraformaldehyde in phosphate-buffered saline (PBS) and processed into paraffin blocks. Three to five mice from each genotype were analyzed for each experiment. Five-micrometer sections from each lobe were stained with hematoxylin and eosin. Immunostaining of endogenous SP-D and transgenic NH2-rSftpa/d was performed in lung tissue with an avidin-biotin-peroxidase technique (Vectastain Elite ABC kit, Vector Laboratories). The rabbit anti-mouse SP-D antibody was generated against purified mouse SP-D and affinity absorbed against lung homogenates from Sftpd–/– mice (33). Immunostaining of endogenous SP-A and transgenic rSP-A used rabbit anti-rat SP-A antibody as reported previously (10).

Phospholipid analysis. BAL was performed on 8-wk old-mice by repeated installation of five aliquots of 1 ml of saline on each lung. After lavage, lung tissue was homogenized in saline. The amount of saturated phosphatidylcholine (Sat PC) in BALF and in the lung homogenate was measured as previously described (19). Mice (7–10) from each genotype were analyzed, and the differences between genotypes were evaluated by ANOVA Fisher analysis. Differences of P < 0.05 were considered significant.

Ultrastructure of surfactant lipid aggregate. Large (LA)- and small (SA)-aggregate surfactant were isolated from pooled BALF samples (n = 5) by sucrose gradient centrifugation as described previously (15). Samples were processed for electron microscopy. Electron micrographs were randomly taken from ultrathin sections (90 nm).

Zymography assay for MMP2 and MMP9. BAL was performed in adult mice by instillation of 10 aliquots of 1 ml of PBS. Alveolar macrophages (5 x 105) from each genotype were cultured in AIM-V medium (GIBCO, Carlsbad, CA) for 24 h in a 24-well plate at 37°C. Cultured media were concentrated with gelatin Sepharose 4B beads (Amersham Pharmacia Biotech) and suspended in 2x Laemmli buffer without beta-mercaptoethanol. After incubation at 37°C for 1 h, gelatin beads were loaded onto 10% zymogram gels (Invitrogen, Carlsbad, CA). The gels were renatured in 2.5% Triton X-100 at room temperature, incubated in developing buffer (Invitrogen) at 37°C overnight, and stained with 0.1% Coomassie blue at room temperature. After destaining with 7.5% acetic acid-5% methanol, MMP9 and MMP2 generated a clear band at 92 and 72 kDa, respectively.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Increased SP-A does not correct pulmonary abnormalities in Sftpd–/– mice. Because SP-A levels are decreased in the lungs of Sftpd–/– mice (16), we determined whether increased expression of SP-A corrected pulmonary abnormalities in Sftpd–/– mice. Sftpd–/– mice were mated to mice that express increased levels of rSP-A in the airway (10). SP-A levels in BALF were increased in rSftpatg+/Sftpd–/– compared with Sftpd–/– mice (Fig. 1).


Figure 1
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  		Fig. 1. Increased expression of surfactant protein (SP)-A is detected in bronchoalveolar lavage fluid (BALF) of rSftpatg+/Sftpd–/– mice. BALF from individual wild-type (WT; lanes 1 and 2), rSftpatg+/Sftpd–/– (lanes 3–5), and Sftpd–/– (lanes 6–8) mice were separated by SDS-PAGE (reduced gel) and analyzed with guinea pig anti-rat SP-A antibody to detect both mouse and rat SP-A protein.

 
In normal adult mice, SP-A staining was detected in type II alveolar epithelial cells, in macrophages, and in the bronchiolar epithelium (Fig. 2A). In Sftpd–/– mice (Fig. 2E), SP-A staining of type II alveolar and bronchiolar epithelial cells was decreased. In rSftpatg+/Sftpd–/– mice, SP-A staining was increased in type II alveolar and bronchiolar epithelial cells, consistent with the activity of the SFTPC promoter that was used to express the rSftpa transgene (Fig. 2C). Enlarged alveolar spaces and foamy macrophage and lymphocyte infiltrates typical of Sftpd–/– mice were observed in rSftpatg+/Sftpd–/– mice (Fig. 2, B, D, and F). Alveolar and tissue Sat PC levels were similarly increased in rSftpatg+/Sftpd–/– and Sftpd–/– mice compared with wild-type (WT) mice (Fig. 3), indicating that SP-A did not correct the increased Sat PC levels associated with SP-D deficiency. The increase in Sat PC levels in rSftpatg+/Sftpd–/– mice compared with Sftpd–/– mice is likely related to differences in the genetic backgrounds of these mice. Sat PC levels in both Sftpd–/– and WT mice are higher in FVB/N background than in Swiss Black background (data not shown). These results demonstrate that increased levels of SP-A do not correct the structural and surfactant abnormalities typical of Sftpd–/– mice.


Figure 2
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  		Fig. 2. Increased expression of SP-A does not correct lung pathology in rSftpatg+/Sftpd–/– mice. Lung sections (5 µm) from WT (A and B), rSftpatg+/Sftpd–/– (C and D), and Sftpd–/– (E and F) mice were stained with rabbit anti-rat SP-A antibody that detects both mouse and rat SP-A (left) or hematoxylin and eosin (H&E; right). Type II cells (thin arrows), alveolar macrophages (thick arrows), and bronchiolar cells (black arrowheads) were stained with anti-rat SPA antibody. Thick arrows, enlarged, foamy macrophages in the alveoli; open arrowheads, peribronchiolar lymphocyte infiltrates; stars, enlarged alveolar spaces. Bar, 100 µm.

 

Figure 3
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  		Fig. 3. Rat SP-A does not correct surfactant lipid abnormalities in Sftpd–/– mice. Alveolar, tissue, and total lung saturated phosphatidylcholine (Sat PC) were determined in WT, rSftpatg+/Sftpd–/–, and Sftpd–/– mice and normalized for body weight. Values are means ± SD; n = 8–10 for each genotype. *Significant statistical differences (P < 0.05) between WT and rSftpatg+/Sftpd–/– and between WT and Sftpd–/– mice by ANOVA Fisher analysis.

 
Expression of NH2-rSftpa/d fusion protein in vivo. NH2-rSftpa/d transgenic mice were produced by nuclear injection of fertilized oocytes and backcrossed to Sftpd–/– mice. The transgene was inherited as an autosomal gene following Mendelian inheritance. Survival and breeding were not influenced by the SFTPC-NH2-rSftpa/d transgene. To determine whether the NH2-rSftpa/d fusion protein was synthesized and secreted into the alveolar space, BALF from NH2-rSftpa/dtg+/Sftpd–/– and Sftpd–/– mice was analyzed by Western blot using rabbit anti-mouse SP-D antibody that detects both the native SP-D protein and the NH2-rSftpa/d fusion protein. After reduction in beta-mercaptoethanol, SP-D from normal control mice migrated as a protein of 43 kDa, consistent with the size of the mouse SP-D monomer; lesser amounts of a 32-kDa degradation product were also observed (Fig. 4A). Immunoreactive proteins were detected with anti SP-D antibody in BALF from NH2-rSftpa/dtg+/Sftpd–/– mice. Two bands of 34 and 29 kDa, the lower band consistent with the calculated molecular mass of the NH2-rSftpa/d fusion protein, were detected (Fig. 4A, lanes 1 and 3). After N-glycanase treatment, the proportion of the 34-kDa band was decreased and that of the 29-kDa band was increased (Fig. 4A, lanes 5–7), indicating that the 34-kDa band was an N-glycosylated form of the NH2-rSftpa/d fusion protein. As expected, SP-D was not detected in the BALF of Sftpd–/– mice (Fig. 4A, lanes 2 and 4).


Figure 4
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  		Fig. 4. NH2-rSftpa/d fusion protein is detected in mouse BALF. A: reduced Western blot of BALF from NH2-rSftpa/dtg+/Sftpd–/– and Sftpd–/– mice. Rabbit anti-mouse SP-D antibody was used to detect 29-kDa NH2-rSftpa/d fusion protein and 43-kDa native SP-D protein. BALF from NH2-rSftpa/dtg+/Sftpd–/– mice was treated with N-glycanase in lanes 5–7. B: unreduced Western blot of BALF from NH2-rSftpa/dtg+/Sftpd–/–, Sftpd–/–, and WT mice. Rabbit anti-mouse SP-D antibody was used to detect the polymerization pattern of NH2-rSftpa/d fusion protein (*) and native SP-D protein (-) (lanes 1–5). Guinea pig anti-rat SP-A antibody was used to detect the polymerization pattern of SP-A protein ({circ}) and rSftpa/d (lanes 6–8). C: reduced Western blot of surfactant large aggregate (LA) and small aggregate (SA) surfactant from NH2-rSftpa/dtg+/Sftpd–/– mouse. Rabbit anti-mouse SP-D antibody was used to detect NH2-rSftpa/d fusion protein. Guinea pig anti-rat SP-A antibody was used to detect SP-A protein.

 
To determine whether the NH2-rSftpa/d fusion protein formed SP-A- or SP-D-like oligomers, BALF from NH2-rSftpa/dtg+/Sftpd–/–, Sftpd–/–, and WT mice were analyzed after separation in the absence of reducing agents. Rabbit anti-mouse SP-D antibody was used to detect the oligomerization of SP-D and NH2-rSftpa/d. Guinea pig anti-rat SP-A antibody was used to identify oligomers of SP-A. In the absence of reducing agent, SP-D and SP-A have distinct oligomerization patterns in WT mice (Fig. 4B, lanes 1 and 6). SP-D forms oligomers of 2-, 3-, and 4-mers, whereas SP-A forms oligomers of 2-, 3-, 4-, 5-, and 6-mers. The NH2-rSftpa/d fusion protein oligomerized in a pattern similar to that of SP-A, with oligomers of 2-, 3-, 4-, 5-, and 6-mers (Fig. 4B, lanes 2–5). Quantitation of the density volumes of bands on Western blot with ImageQuant5.2 software showed no statistical differences between the levels of expression of NH2-rSftpa/d protein in BALF and that of SP-D in WT mice (P = 0.40; n = 3 from each genotype).

NH2-rSftpa/d associates with LA surfactant. To determine the association of the NH2-rSftpa/d protein with surfactant lipids, LA and SA lipid fractions were isolated from BALF from NH2-rSftpa/dtg+/Sftpd–/– mice. The NH2-rSftpa/d fusion protein partitioned in LA surfactant (Fig. 4C), consistent with the distribution of SP-A rather than SP-D, suggesting that the oligomeric structure of the chimeric protein was determined by the collagenous and/or NH2-terminal domains that influence its association with LA surfactant lipids.

NH2-rSftpa/d does not correct lung morphology and phospholipid abnormalities in Sftpd–/– mice. Immunohistochemical staining of SP-D was assessed with a rabbit anti-mouse SP-D antibody in rSftpa/dtg+/Sftpd–/– and WT mice. SP-D staining was detected in type II epithelial cells and alveolar macrophages of WT mice (Fig. 5A). Intense NH2-rSftpa/d staining was detected in type II epithelial cells and alveolar macrophages in lungs of NH2-rSftpa/dtg+/Sftpd–/– mice (Fig. 5C). There was no detectable NH2-rSftpa/d staining in the bronchiolar epithelium. As expected, SP-D was not detected in the lungs of Sftpd–/– mice (Fig. 5E).


Figure 5
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  		Fig. 5. Expression of NH2-rSftpa/d fusion protein in type II cells does not correct pulmonary abnormalities in Sftpd–/– mice. Lung sections (5 µm) from WT (A and B), NH2-rSftpa/dtg+/Sftpd–/– (C and D), and Sftpd–/– (E and F) mice were stained with rabbit anti-mouse SP-D antibody that detects both the SP-D and the NH2-rSftpa/d protein (left) or hematoxylin and eosin (right). Type II cells (thin arrows) and alveolar macrophages (thick arrows) were stained with anti-SP-D antibody in WT and NH2-rSftpa/dtg+/Sftpd–/– mice but not in Sftpd–/– mice. Thick arrows, enlarged, foamy macrophages in the alveoli; open arrowheads, peribronchiolar lymphocyte infiltrates; stars, enlarged alveolar spaces. Bar, 100 µm.

 
Abnormalities in lung morphology typical of Sftpd–/– mice were not corrected by expression of NH2-rSftpa/d (Fig. 5D). Air space enlargement, foamy alveolar macrophages, and peribronchiolar lymphocytic infiltrations were observed in lungs of both Sftpd–/– and NH2-rSftpa/dtg+/Sftpd–/– mice. Alveolar, tissue, and total Sat PC levels were similarly increased in the NH2-rSftpa/dtg+/Sftpd–/– and Sftpd–/– littermates compared with WT control mice of similar genetic background (Fig. 6). Thus the oligomeric structure, determined by the NH2-terminal and/or collagenous domain of SP-D is required for the regulation of surfactant lipids and suppression of lung inflammation.


Figure 6
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  		Fig. 6. NH2-rSftpa/d does not correct lipid abnormalities in Sftpd–/– mice. Alveolar, tissue, and total lung Sat PC were determined in WT, NH2-rSftpa/dtg+/Sftpd–/–, and Sftpd–/– mice and were normalized for body weight. Values are means ± SD; n = 7–9 for each genotype. *Statistical differences (P < 0.05) between WT and NH2-rSftpa/dtg+/Sftpd–/– and between WT and Sftpd–/– mice by ANOVA Fisher analysis.

 
Ultrastructure of surfactant lipids. Ultrastructures of LA and SA surfactant lipids were determined by electron microscopy (Fig. 7). Abnormally large and dense lamellar bodies were observed in LA fraction from Sftpd–/– mice (Fig. 7G) compared with WT mice (Fig. 7A). In LA fractions from both rSftpatg+/Sftpd–/– (Fig. 7C) and NH2-rSftpa/dtg+/Sftpd–/– mice (Fig. 7E), abnormally large and dense lamellated forms were observed. The ultrastructure of LA surfactant from both rSftpatg+/Sftpd–/– and NH2-rSftpa/dtg+/Sftpd–/– mice was similar to that from Sftpd–/– mice, indicating that neither SP-A nor NH2-rSftpa/d fusion protein could substitute for SP-D in regulating surfactant ultrastructure.


Figure 7
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  		Fig. 7. Neither rSP-A nor NH2-rSftpa/d fusion protein corrects the abnormal ultrastructure of surfactant lipids in Sftpd–/– mice. LA (left) and SA (right) surfactant lipids from WT (A and B), rSftpatg+/Sftpd–/– (C and D), NH2-rSftpa/dtg+/Sftpd–/– (E and F), and Sftpd–/– (G and H) mice were analyzed by electron microscopy. Arrowheads indicate lamellar bodies.

 
Failure to express rSftpd/a fusion protein in lung in vivo. To further validate the requirement for the NH2-terminal and collagenous domains of SP-D, the fusion protein rSftpd/a (consisting of the NH2-terminal and collagen domains of rSP-D and neck and CRD domains of rSP-A) was introduced into Sftpd–/– mice. Even though the mRNA was readily detected in lung tissue, the rSftpd/a fusion protein was not detected in adult mouse BALF (data not shown). Failure to produce the rSftpd/a fusion protein may be related to its misfolding, degradation, or cell injury.

Mmp9–/–/Sftpd–/– and Mmp12–/–/Sftpd–/– double knockout mice develop emphysema and foamy macrophage infiltrations. To determine whether NH2-rSftpa/d fusion protein or increased expression of rSP-A influences MMP9 and MMP2 activities, alveolar macrophages from WT, NH2-rSftpa/dtg+/Sftpd–/–, rSftpatg+/Sftpd–/–, and Sftpd–/– mice were cultured for 24 h and the media analyzed by zymography. Expression of NH2-rSftpa/d partially corrected the increased MMP9 and MMP2 activities characteristic of Sftpd–/– mice (Fig. 8A). Production of MMP9 and MMP2 were similarly increased in alveolar macrophages from rSftpatg+/Sftpd–/– and Sftpd–/– mice, compared with WT mice (Fig. 8B). Thus neither SP-A nor NH2-rSftpa/d fully corrected metalloproteinase expression and emphysema.


Figure 8
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  		Fig. 8. NH2-rSftpa/d partially corrects matrix metalloproteinase (MMP9 and MMP2) activities of alveolar macrophages. Conditioned media from cultured alveolar macrophages (5 x 105) isolated from WT, NH2-rSftpa/dTg+/Sftpd–/–, and Sftpd–/– mice (A) or from WT, rSftpatg+/Sftpd–/–, and Sftpd–/– mice (B) were serially diluted and analyzed by zymogram gels. MMP9 (92 kDa) and MMP2 (72 kDa) production was increased in Sftpd–/– mice compared with WT and were partially corrected by NH2-rSftpa/dtg transgene. Increased MMP9 (92 kDa) and MMP2 (72 kDa) activities were not corrected by expression of rSftpa. Images are representative of 3 or 4 separate experiments.

 
Increased matrix metalloproteinase (MMP9, MMP2, and MMP12) activities were previously observed in alveolar macrophages from Sftpd–/– mice (40). To assess whether increased metalloproteinase (MMP9 and MMP12) activities played an important role in the pathogenesis of pulmonary disease in Sftpd–/– mice, Mmp9–/– and Mmp12–/– mice were mated with Sftpd–/– mice. Lung morphology was normal in Mmp9–/– and Mmp12–/– mice at the light microscopic level (data not shown). Neither deletion of Mmp9 nor deletion of Mmp12 corrected lung abnormalities in Sftpd–/– mice. Enlarged alveolar spaces and foamy macrophage infiltrates, typical of Sftpd–/– mice, were readily observed in lungs from the double knockout Mmp9–/–/Sftpd–/– and Mmp12–/–/Sftpd–/– mice (Fig. 9). However, in Mmp9–/–/Sftpd–/– mice, the peribronchiolar lymphocyte infiltrates typically seen in Sftpd–/– mice were not detected. Thus deletion of Mmp9 or Mmp12 was not sufficient to correct emphysema or inflammation in the Sftpd–/– mice.


Figure 9
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  		Fig. 9. Deletion of Mmp9 and Mmp12 does not correct pulmonary abnormalities in Sftpd–/– mice. Lung sections from Mmp9–/–/Sftpd–/– (A and B), Mmp12–/–/Sftpd–/– (C and D), and Sftpd–/– (E and F) mice (12 wk old) were stained with hematoxylin and eosin. Arrows, enlarged foamy macrophage accumulation in the alveoli; arrowheads, peribronchiolar lymphocyte infiltrates; stars, enlarged alveolar space. Bars, 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although the domains shared by the pulmonary collectins SP-A and SP-D are structurally conserved, the functions of these proteins in the regulation of surfactant homeostasis are quite distinct. SP-A plays a critical role in the formation of tubulin myelin and host defense but does not regulate surfactant phospholipid pool sizes and is not required for surfactant function or maintenance of pulmonary homeostasis (18). In contrast, SP-D is required for the regulation of surfactant pool sizes, the proportion of LA/SA surfactant lipids, the maintenance of alveolar structure, and the suppression of inflammation in the absence of infection or injury (19, 38). Despite increased understanding of their structure and functions, the precise mechanisms and domains mediating the various activities of the pulmonary collectins remain poorly understood. The present findings demonstrate that increased SP-A cannot compensate for SP-D-dependent pulmonary abnormalities. Expression of a chimeric NH2-rSftpa/d protein failed to rescue air space remodeling and lipid abnormalities in the Sftpd–/– mice, demonstrating that the activity of SP-D requires its collagenous and NH2-terminal regions, which cannot be replaced by those from SP-A. The pulmonary collectins play distinct roles in alveolar homeostasis that are determined by the integrated functions of both the NH2- and COOH-terminal regions of each molecule.

Increased SP-A fails to rescue Sftpd–/– dependent abnormalities. SP-A concentrations were decreased in BALF and lung tissue in the Sftpd–/– mice (16). Increased expression of rSP-A in WT mice did not change the surfactant pool sizes, alveoli sizes, and surface activity of surfactant (10). Expression of rSP-A in Sftpd–/– mice did not affect the alveolar and tissue Sat PC pool sizes, surfactant ultrastructure, alveolar macrophage infiltration, and air space abnormalities or suppress MMP activity. Thus decreased SP-A does not account for the pulmonary abnormalities in association with Sftpd deficiency. The findings that neither SP-A nor the chimeric NH2-rSftpa/d influenced the increased lipid pools in Sftpd–/– mice were consistent with previous findings in mice where the deletion of Sftpa or increased expression of Sftpa did not alter lung phospholipid pool sizes (10, 18). Sat PC levels in both Sftpd–/– and WT mice are also influenced by genetic backgrounds, being higher in FVB/N background than in Swiss Black background (data not shown). In the present study, Sat PC levels in NH2-rSftpa/dtg+/Sftpd–/– mice were compared with those of littermate Sftpd–/– mice to control for differences in genetic background. Likewise, Sat PC in lungs of rSftpatg+/Sftpd–/– mice was compared with that of Sftpd–/– mice of similar strain mix.

Glycosylation and secretion of NH2-rSftpa/d protein. In the present study, the NH2-rSftpa/d protein was produced in the type II alveolar cells and was secreted into the alveolar spaces, where it formed SP-A like oligomers. N-glycanase treatment demonstrated that NH2-rSftpa/d protein was N-glycosylated. The conserved N-glycosylation site in SP-D is in the collagen domain (31), and the N-glycosylation of SP-A is in the CRD (27). The fusion of SP-A and SP-D domains in NH2-rSftpa/d did not create a new N-glycosylation site. Because N-glycosylation sites are not present on the CRD of rat or human SP-D (34), the N-linked carbohydrate must be attached to the collagenous or NH2-terminal domain of the chimeric protein. A potential N-glycosylation site is present in the NH2-terminal region of rSP-A, at 30Asn-Val-Thr (23, 29). It is unclear whether this finding represents glycosylation that is normally present in the NH2-terminal region of rat SP-A or the domain is aberrantly glycosylated in the chimeric protein.

NH2-terminal domain of SP-D is required for correction of surfactant abnormalities. SP-A partitioned with LA surfactant whereas SP-D partitioned with SA surfactant during fractionation by ultracentrifugation (15). The lipid binding specificity of the collectins is thought to be mediated by the CRD (3, 9, 17, 24, 25, 28). However, those studies assessed SP-D binding to isolated lipids bound to a solid phase rather than to lipid vesicles or surfactant particles. SP-D influences surfactant ultrastructure and regulates surfactant uptake by type II cells (15). The lipid binding specificity of NH2-rSftpa/d is similar to that of SP-D in vitro (24). A chimeric protein with the NH2-terminal and collagenous domains of SP-D linked to the neck and CRD domains of conglutinin partitioned with SA surfactant like SP-D and partially corrected lipid abnormalities in Sftpd–/– mice (41), raising the possibility that the SP-D CRD, or its specific lipid binding activity, may not be required in this activity. SP-D interacts selectively with SA surfactant in a process that influences surfactant uptake by type II cells (15). The present study supports the concept that interaction with SA lipids is dependent on the NH2-terminal and collagenous domains of SP-D and cannot be substituted with those from SP-A. NH2-rSftpa/d protein did not correct the elevated Sat PC levels or the abnormal surfactant ultrastructure typical of Sftpd–/– mice. NH2-rSftpa/d also failed to correct foamy macrophage accumulation, peribronchiolar lymphocyte infiltrations, or development of air space abnormalities, indicating the requirement of the NH2-terminal and collagenous domains of SP-D for these activities. These results indicate that the function of rSP-D CRD requires the context of its own NH2-terminal and collagenous domains for full activity. Together, these data suggested a model in which oligomeric structures determined by the NH2-terminal and collagenous domains of SP-D play in important role in its interaction with surfactant lipid particles rather than a model dependent on lipid binding specificity conferred by the CRD.

NH2-rSftpa/d partially suppressed MMP expression but did not alter air space remodeling. Matrix metalloproteinases are a family of calcium- and zinc-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue-remodeling events. MMP9 is normally expressed by a number of cell types including neutrophils, macrophages, osteoclasts, and trophoblasts. MMP9 cleaves extracellular matrix proteins (36, 37). Increased expression of rSP-A or expression of a chimeric molecule consisting of the NH2-terminal and collagen domains of SP-D linked to the neck and CRD domains of conglutinin did not correct air space abnormalities or reduce macrophage MMP9 and MMP2 activities, indicating the need for the CRD of SP-D for these functions (41). Thus the neck domain and the CRD of SP-D require the proper context of its own NH2-terminal and collagenous domains for the normal regulation of MMP expression. Previous studies demonstrated that proper oligomerization of SP-D is required in the regulation of MMP activities and the prevention of air space remodeling in vivo (42).

Because NH2-rSftpa/d fusion protein partially corrected MMP2 and MMP9 expression but failed to correct air space abnormalities in Sftpd–/– mice, we assessed the potential roles of MMP9 or MMP12 proteinases that are known to influence air space remodeling in animal models (1). Mmp9–/–/Sftpd–/– and Mmp12–/–/Sftpd–/– mice developed air space abnormalities identical to those in Sftpd–/– mice, indicating that increased MMP9 or MMP12 was not required for the development of emphysema in Sftpd–/– mice. In contrast, an IL-13-dependent emphysema was ameliorated by deficiency of Mmp9 or Mmp12 (20). In human diseases such as asthma, chronic obstructive pulmonary disease, and emphysema, increased secretion of MMP9 and MMP12 are associated with the pathogenesis of the diseases (5). The mechanism of air space enlargement in Sftpd–/– mice is not obvious. Potential mechanisms might include epithelial damage from macrophage-derived oxidants or impaired alveolar VEGF expression with associated alveolar epithelial apoptosis.

MMP9 modifies lymphocytic infiltration in Sftpd–/– mice. Sftpd–/– mice develop peribronchial and perivascular lymphocytic infiltration. FACS analysis showed that the relative distribution of lymphocyte classes (CD4+,CD8+ T cells, NK cells, or B cells) was not different in Sftpd–/– and WT mice. In Sftpd–/– mice, the CD4+,CD8+ lymphocytes were activated and an increase in memory CD4+ lymphocytes was observed (11). In Mmp9–/–/Sftpd–/– double knockout mice lungs, lymphocytic infiltration was not observed, even though foamy macrophage infiltration and emphysema persisted, indicating that MMP9 is required for lymphocytic infiltration in Sftpd–/– mice. MMP9 has been shown to be critical for the release of kit-ligand to recruit stem and progenitor cells from the bone marrow niche (14). However, in IL-13-induced inflammation, neutrophil infiltration was increased and lymphocyte infiltration persisted (20). Thus the lymphocytic infiltration seen in Sftpd–/– mice is dependent on MMP9 and is distinct from that induced by IL-13.

Summary. The NH2-terminal and collagenous domains of SP-A were not sufficient to substitute for those of SP-D in vivo. The NH2-terminal domains of SP-D determine its oligomerization state and its association with SA rather than LA surfactant lipids. Increased SP-A or an NH2-rSftpa/d fusion protein did not restore surfactant homeostasis or protect from air space remodeling in Sftpd–/– mice, indicating that the CRD of SP-D requires its NH2-terminal and collagenous domains for its full biological function. At present, analysis of various chimeric and mutated SP-D proteins (see Table 2) supports the concept that the NH2-terminal and collagenous domains of SP-D play important roles in surfactant structure and metabolism. The findings that the CRD of conglutinin partially restored surfactant homeostasis when linked to the NH2-terminal and collagenous domains of SP-D suggest that the specific interactions of the SP-D CRD with surfactant lipids are not required for its influence on surfactant homeostasis and that the NH2-terminal and collagenous domains of SP-D play an important role in the interaction of SP-D with surfactant lipid particles. The ability of SP-D to regulate surfactant homeostasis may not rely on its interaction with specific phospholipids, but rather on its interaction with small aggregate surfactant.


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  		Table 2. Effect of SP-A, SP-D, and mutant collectins in Sftpd–/– mice in vivo

 

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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56387 (J. A. Whitsett), HL-63329 (M. Ikegami), HL-58795 (T. R. Korfhagen), HL-68861 (F. X. McCormack), and HL-47328 (R. M. Senior).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Whitsett, Cincinnati Children’s Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (e-mail: jeff.whitsett{at}cchmc.org)

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. Section 1734 solely to indicate this fact.


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 ABSTRACT
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 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Atkinson JJ and Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 28: 12–24, 2003.[Abstract/Free Full Text]
  2. Botas C, Poulain F, Akiyama J, Brown C, Allen L, Goerke J, Clements J, Carlson E, Gillespie AM, Epstein C, and Hawgood S. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc Natl Acad Sci USA 95: 11869–11874, 1998.[Abstract/Free Full Text]
  3. Chiba H, Pattanajitvilai S, Evans AJ, Harbeck RJ, and Voelker DR. Human surfactant protein D (SP-D) binds Mycoplasma pneumoniae by high affinity interactions with lipids. J Biol Chem 277: 20379–20385, 2002.[Abstract/Free Full Text]
  4. Clark H, Palaniyar N, Strong P, Edmondson J, Hawgood S, and Reid KB. Surfactant protein D reduces alveolar macrophage apoptosis in vivo. J Immunol 169: 2892–2899, 2002.[Abstract/Free Full Text]
  5. Cataldo DD, Gueders M, Rocks N, Sounni NE, Evrard B, Bartsch P, Louis R, Noel A, and Foidart JM. Pathogenic role of matrix metalloproteases and their inhibitors in asthma and chronic obstructive pulmonary disease and therapeutic relevance of matrix metalloproteases inhibitors. Cell Mol Biol (Noisy-le-grand) 49: 875–884, 2003.
  6. Crouch E, Parghi D, Kuan SF, and Pesson A. Surfactant protein D: subcellular localization in nonciliated bronchiolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 263: L60–L66, 1992.[Abstract/Free Full Text]
  7. Crouch E, Rust K, Marienchek W, Parghi D, Chang D, and Persson A. Developmental expression of pulmonary surfactant protein D (SP-D). Am J Respir Cell Mol Biol 5: 13–18, 1991.
  8. Crouch EC. Collectins and pulmonary host defense. Am J Respir Cell Mol Biol 19: 177–201, 1998.[Abstract/Free Full Text]
  9. DeSilva NS, Ofek I, and Crouch EC. Interactions of surfactant protein D with fatty acids. Am J Respir Cell Mol Biol 29: 757–770, 2003.[Abstract/Free Full Text]
  10. Elhalwagi BM, Zhang M, Ikegami M, Iwamoto HS, Morris RE, Miller ML, Dienger K, and McCormack FX. Normal surfactant pool sizes and inhibition-resistant surfactant from mice that overexpress surfactant protein A. Am J Respir Cell Mol Biol 21: 380–387, 1999.[Abstract/Free Full Text]
  11. Fisher JH, Larson J, Cool C, and Dow SW. Lymphocyte activation in the lungs of SP-D null mice. Am J Respir Cell Mol Biol 27: 24–33, 2002.[Abstract/Free Full Text]
  12. Fisher JH, Sheftelyevich V, Ho YS, Fligiel S, McCormack FX, Korfhagen TR, Whitsett JA, and Ikegami M. Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 278: L365–L373, 2000.[Abstract/Free Full Text]
  13. Hakansson K and Reid KB. Collectin structure: a review. Protein Sci 9: 1607–1617, 2000.[Abstract]
  14. Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, and Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109: 625–637, 2002.[CrossRef][ISI][Medline]
  15. Ikegami M, Na CL, Korfhagen TR, and Whitsett JA. Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 288: L552–L561, 2005.[Abstract/Free Full Text]
  16. Ikegami M, Whitsett JA, Jobe A, Ross G, Fisher J, and Korfhagen T. Surfactant metabolism in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 279: L468–L476, 2000.[Abstract/Free Full Text]
  17. Kishore U, Wang JY, Hoppe HJ, and Reid KB. The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem J 318: 505–511, 1996.
  18. Korfhagen TR, Bruno MD, Ross GF, Huelsman KM, Ikegami M, Jobe AH, Wert SE, Stripp BR, Morris RE, Glasser SW, Bachurski CJ, Iwamoto HS, and Whitsett JA. Altered surfactant function and structure in SP-A gene targeted mice. Proc Natl Acad Sci USA 93: 9594–9599, 1996.[Abstract/Free Full Text]
  19. Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD, Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M, Whitsett JA, and Fisher JH. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 273: 28438–28443, 1998.[Abstract/Free Full Text]
  20. Lanone S, Zheng T, Zhu Z, Liu W, Lee CG, Ma B, Chen Q, Homer RJ, Wang J, Rabach LA, Rabach ME, Shipley JM, Shapiro SD, Senior RM, and Elias JA. Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13-induced inflammation and remodeling. J Clin Invest 110: 463–474, 2002.[CrossRef][ISI][Medline]
  21. Liu Z, Shipley JM, Vu TH, Zhou X, Diaz LA, Werb Z, and Senior RM. Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid. J Exp Med 188: 475–482, 1998.[Abstract/Free Full Text]
  22. Madan T, Kishore U, Singh M, Strong P, Clark H, Hussain EM, Reid KB, and Sarma PU. Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens. J Clin Invest 107: 467–475, 2001.[CrossRef][ISI][Medline]
  23. McCormack FX, Calvert HM, Watson PA, Smith DL, Mason RJ, and Voelker DR. The structure and function of surfactant protein A. Hydroxyproline- and carbohydrate-deficient mutant proteins. J Biol Chem 269: 5833–5841, 1994.[Abstract/Free Full Text]
  24. Ogasawara Y, McCormack FX, Mason RJ, and Voelker DR. Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction. J Biol Chem 269: 29785–29792, 1994.[Abstract/Free Full Text]
  25. Ogasawara Y and Voelder DR. Altered carbohydrate recognition specificity engineered into surfactant protein D reveals different binding mechanisms for phosphatidylinositol and glucosylceramide. J Biol Chem 270: 14725–14732, 1995.[Abstract/Free Full Text]
  26. Palaniyar N, Zhang L, Kuzmenko A, Ikegami M, Wan S, Wu H, Korfhagen TR, Whitsett JA, and McCormack FX. The role of pulmonary collectin N-terminal domains in surfactant structure, function, and homeostasis in vivo. J Biol Chem 277: 26971–26979, 2002.[Abstract/Free Full Text]
  27. Ross GF, Meuth J, Ohning B, Kim Y, and Whitsett JA. Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain. Biochim Biophys Acta 870: 267–278, 1986.[CrossRef][Medline]
  28. Sano H, Kuroki Y, Honma T, Ogasawara Y, Sohma H, Voelker DR, and Akino T. Analysis of chimeric protein identifies the regions in the carbohydrate recognition domains of rat lung collectins that are essential for interactions with phospholipids, glycolipids and alveolar type II cells. J Biol Chem 273: 4783–4789, 1998.[Abstract/Free Full Text]
  29. Sano K, Fisher J, Mason RJ, Kuroki Y, Schilling J, Benson B, and Voelker D. Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A). Biochem Biophys Res Commun 144: 367–374, 1987.[CrossRef][ISI][Medline]
  30. Sastry K and Ezekowitz RA. Collectins: pattern recognition molecules involved in first line host defense. Curr Opin Immunol 5: 59–66, 1993.[CrossRef][ISI][Medline]
  31. Shimizu H, Fisher JH, Papst P, Benson B, Lau K, Mason RJ, and Voelker DR. Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence. J Biol Chem 267: 1853–1857, 1992.[Abstract/Free Full Text]
  32. Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, and Shapiro SD. Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci USA 93: 3942–3946, 1996.[Abstract/Free Full Text]
  33. Stahlman MT, Gray ME, Hull WM, and Whitsett JA. Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 50: 651–660, 2002.[Abstract/Free Full Text]
  34. Van Eijk M, van de Lest C, Batenburg JJ, Vaandrager AB, Meschi J, Hartshorn KL, van Golde LM, and Haagsman HP. Porcine surfactant protein D is N-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers. Am J Respir Cell Mol Biol 26: 739–747, 2002.[Abstract/Free Full Text]
  35. Voorhout WF, Beenendaal T, Kuroki Y, Ogasawara Y, van Golde LM, and Geuze HJ. Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung. J Histochem Cytochem 40: 1589–1597, 1992.[Abstract]
  36. Vu TH and Werb Z. Gelatinase B: structure, regulation, and function. In: Matrix Metalloproteinases, edited by Parks WC and Mecham RP. San Diego, CA: Academic, 1998, p. 115–148.
  37. Welgus HG, Campbell EJ, Cury JD, Eisen AZ, Senior RM, Wilhelm SM, and Goldberg GI. Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development. J Clin Invest 86: 1496–1502, 1990.[ISI][Medline]
  38. Wert SE, Jones T, Korfhagen T, Fisher J, and Whitsett JA. Spontaneous emphysema in surfactant protein D gene-targeted mice (Abstract). Chest 117: 248S, 2000.[Free Full Text]
  39. Wert SE, Glasser SW, Korfhagen TR, and Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156: 426–443, 1993.[CrossRef][ISI][Medline]
  40. Wert SE, Yoshida M, LeVine AM, Ikegami M, Jones T, Ross GF, Fisher JH, Korfhagen TR, and Whitsett JA. Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. Proc Natl Acad Sci USA 97: 5972–5977, 2000.[Abstract/Free Full Text]
  41. Zhang L, Hartshorn KL, Crouch EC, Ikegami M, and Whitsett JA. Complementation of pulmonary abnormalities in SP-D(–/–) mice with an SP-D/conglutinin fusion protein. J Biol Chem 277: 22453–22459, 2002.[Abstract/Free Full Text]
  42. Zhang L, Ikegami M, Crouch EC, Korfhagen TR, and Whitsett JA. Activity of pulmonary surfactant protein-D (SP-D) in vivo is dependent on oligomeric structure. J Biol Chem 276: 19214–19219, 2001.[Abstract/Free Full Text]
  43. Zhang L, Ikegami M, Dey CR, Korfhagen TR, and Whitsett JA. Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo. J Biol Chem 277: 38709–38713, 2002.[Abstract/Free Full Text]




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