Distinct changes in pulmonary surfactant homeostasis in common beta-chain- and GM-CSF-deficient mice

Pulmonary alveolar proteinosis (PAP) is caused by inactivation of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM receptor common beta-chain (beta(c)) genes in mice [GM(-/-), beta(c)(-/-)], demonstrating a critical role of GM-CSF signaling in surfactant homeostasis. To distinguish possible phenotypic differences in GM(-/-) and beta(c)(-/-) mice, surfactant metabolism was compared in beta(c)(-/-), GM(-/-), and wild-type mice. Although lung histology in beta(c)(-/-) and GM(-/-) mice was indistinguishable, distinct differences were observed in surfactant phospholipid and surfactant protein concentrations and clearance from lungs of beta(c)(-/-) and GM(-/-) mice. At 1-2 days of age, lung saturated phosphatidylcholine (Sat PC) pool sizes were higher in wild-type, beta(c)(-/-), and GM(-/-) mice compared with wild-type adult mice. In wild-type mice, Sat PC pool sizes decreased to adult levels by 7 days of age; however, Sat PC increased with advancing age in beta(c)(-/-) and GM(-/-) mice. Postnatal changes in Sat PC pool sizes were different in GM(-/-) compared with beta(c)(-/-) mice. After 7 days of age, the increased lung Sat PC pool sizes remained constant in beta(c)(-/-) mice but continued to increase in GM(-/-) mice, so that by 56 days of age, lung Sat PC pools were increased three- and sixfold, respectively, compared with wild-type controls. After intratracheal injection, the percent recovery of [(3)H]dipalmitoylphosphatidylcholine and (125)I-recombinant surfactant protein (SP) C was higher in beta(c)(-/-) compared with wild-type mice, reflecting decreased clearance in the receptor-deficient mice. The defect in clearance was significantly more severe in GM(-/-) than in beta(c)(-/-) mice. The ratio of SP Sat PC to SP-A, -B, and -C was similar in bronchoalveolar lavage fluid (BALF) from adult mice of all genotypes, but the ratio of SP-D to Sat PC was markedly increased in beta(c)(-/-) and GM(-/-) mice (10- and 5-fold, respectively) compared with wild-type mice. GM-CSF concentrations were increased in BALF but not in serum of beta(c)(-/-) mice, consistent with a pulmonary response to the lack of GM-CSF signaling. The observed differences in surfactant metabolism suggest the presence of alternative clearance mechanisms regulating surfactant homeostasis in beta(c)(-/-) and GM(-/-) mice and may provide a molecular basis for the range in severity of PAP symptoms. surfactant metabolism; alveolar macrophage; granulocyte-macrophage colony-stimulating factor     

Surfactant metabolism in SP-D gene-targeted mice

Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(-/-)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice. (125)I-SP-D was cleared with a half-life of 7 h from SP-D(-/-) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [(3)H]palmitic acid and [(14)C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(-/-) mice. Clearance rates of intratracheal [(3)H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(-/-) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.     

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.     

Surfactant phospholipid catabolic rate is pool size dependent in mice

We increased surfactant pool size by surfactant treatment in mice to test if the catabolism of the major component of surfactant, saturated phosphatidylcholine (Sat PC), was rate limited. By intratracheal instillation, we gave mice trace doses, doses of 45 or 110 micromol/kg, or three doses of 110 micromol/kg of Sat PC in surfactant that contained radiolabeled dipalmitoylphosphatidylcholine (DPPC) and a radiolabeled phospholipase A-resistant ether analog of DPPC. Two strains of mice with 2-fold differences in alveolar and total Sat PC pool sizes were used; the mice with the higher pool sizes had a 2.3-fold higher steady-state catabolic rate. Acute increases in alveolar surfactant given by intratracheal instillation increased catabolic rates approximately 2-fold over the steady-state rates in both strains. There was minimal loss of the ether analog of DPPC from the lungs, and the alveolar macrophages did not accumulate more than 10% of the ether analog. In these two strains of mice, the catabolism of Sat PC was not rate limited because catabolic rate increased when alveolar pool sizes were increased.     

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.     

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice

Targeted disruption of the surfactant protein (SP) D (SP-D) gene caused a marked pulmonary lipoidosis characterized by increased alveolar lung phospholipids, demonstrating a previously unexpected role for SP-D in surfactant homeostasis. In the present study, we tested whether the local production of SP-D in the lung influenced surfactant content in SP-D-deficient [SP-D(-/-)] and SP-D wild-type [SP-D(+/+)] mice. Rat SP-D (rSP-D) was expressed under control of the human SP-C promoter, producing rSP-D, SP-D(+/+) transgenic mice. SP-D content in bronchoalveolar lavage fluid was increased 30- to 50-fold in the rSP-D, SP-D(+/+) mice compared with the SP-D(+/+) parental strain. Lung morphology, phospholipid content, and surfactant protein mRNAs were unaltered by the increased concentration of SP-D. Likewise, the production of endogenous mouse SP-D mRNA was not perturbed by the SP-D transgene. rSP-D, SP-D(+/+) mice were bred to SP-D(-/-) mice to assess whether lung-selective expression of SP-D might correct lipid homeostasis abnormalities in the SP-D(-/-) mice. Selective expression of SP-D in the respiratory epithelium had no adverse effects on lung function, correcting surfactant phospholipid content and decreasing phosphatidylcholine incorporation significantly. SP-D regulates surfactant lipid homeostasis, functioning locally to inhibit surfactant phospholipid incorporation in the lung parenchyma and maintaining alveolar phospholipid content in the alveolus. Marked increases in biologically active tissue and alveolar SP-D do not alter lung morphology, macrophage abundance or structure, or surfactant accumulation.     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits

Intrapulmonary surfactant catabolism was investigated by use of a phospholipase A1- and A2-resistant analogue of dipalmitoylphosphatidylcholine (DPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC ether). [14C]DPC ether, made into liposomes with [3H]DPC and associated with 32P-labeled rabbit surfactant, was given intratracheally to 1-kg rabbits, which were killed at preset times to 48 h. Recoveries of radiolabel as saturated phosphatidylcholine (Sat PC) isolated from alveolar wash (AW), postlavage lung homogenate (LH), and alveolar macrophages were measured. All groups had similar AW and LH Sat PC pool sizes, indicating no perturbation of endogenous Sat PC pools. Despite a nearly fivefold accumulation of [14C]DPC ether in the lung by 48 h (P less than 0.01), the three probes had similar alveolar clearance curves. Furthermore, the Sat PC reutilization efficiency (41.6%) and turnover time (5.9 h) calculated for DPC ether were not different from values for the DPC and rabbit surfactant. Of the DPC ether (0.7%) and DPC (9%) labels recovered as PC in organs outside the lung, greater than 85% was unsaturated, indicating de novo synthesis using precursors from degraded PC. DPC ether was a useful probe of intrapulmonary DPC catabolism, and after alveolar uptake there was no direct reentry of intact DPC from the catabolic compartment(s) into the secretory pathway.     

Correction of pulmonary abnormalities in Sftpd-/- mice requires the collagenous domain of surfactant protein D

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.     

Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.     

Activity of pulmonary surfactant protein-D (SP-D) in vivo is dependent on oligomeric structure

Pulmonary surfactant protein-D (SP-D) is a member of the collectin family of C-type lectins that is synthesized in many tissues including respiratory epithelial cells in the lung. SP-D is assembled predominantly as dodecamers consisting of four homotrimeric subunits each. Association of these subunits is stabilized by interchain disulfide bonds involving two conserved amino-terminal cysteine residues (Cys-15 and Cys-20). Mutant recombinant rat SP-D lacking these residues (RrSP-Dser15/20) is secreted in cell culture as trimeric subunits rather than as dodecamers. In this study, transgenic mice that express this mutant were generated to elucidate the functional importance of SP-D oligomerization in vivo. Expression of RrSP-Dser15/20 failed to correct the pulmonary phospholipid accumulation and emphysema characteristic of SP-D null (mSP-D-/-) mice. Expression of high concentrations of the mutant protein in wild-type mice reduced the abundance of disulfide cross-linked oligomers of endogenous SP-D in the bronchoalveolar lavage fluid and demonstrated a phenotype that partially overlapped with that of the SP-D-/- mice; the animals developed emphysema and foamy macrophages without the associated abnormalities in alveolar phospholipids typical of SP-D-/- mice. Development of foamy macrophages in SP-D-deficient mice is not secondary to the increased abundance of surfactant phospholipids. Disulfide cross-linked SP-D oligomers are required for the regulation of surfactant phospholipid homeostasis and the prevention of emphysema and foamy macrophages in vivo.     

Variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry

Antenatal exposure to intra-amniotic (i.a.) endotoxin initiates a complex series of events, including an inflammatory cascade, increased surfactant production, and alterations to lung structure. Using the low frequency forced oscillation technique as a sensitive tool for measurement of respiratory impedance, we aimed to determine which factors contributed most to measured changes in lung mechanics. Respiratory impedance data obtained from sedated preterm lambs exposed to either i.a. injection with saline or 20 mg of endotoxin 1, 2, 4, and 15 days before delivery at 125 days gestation were studied, and association with indexes of standard lung morphometry, inflammatory response, and alveolar surfactant-saturated phosphatidylcholine (Sat PC) pool size was demonstrated. Reduction in tissue impedance with increasing interval between exposure and delivery was evident as early as 4 days after i.a. endotoxin injection, coinciding with resolution of inflammatory reaction, increased alveolar surfactant pools, and contribution of alveolar ducts to the parenchymal fraction, and a later decrease in the tissue component of the parenchymal fraction. Decreases in tissue damping (resistance) were more marked than decreases in tissue elastance. Log alveolar Sat PC accounted for most variability in tissue damping (88.9%) and tissue elastance (73.4%), whereas tissue fraction contributed 2 and 6.4%, respectively. The alveolar Sat PC pool size was the sole factor contributing to change in tissue hysteresivity. No changes were observed in airway resistance. Despite the complex cascade of events initiated by antenatal endotoxin exposure, variability in lung tissue mechanics is associated primarily with changes in alveolar Sat PC pool and lung morphology.     

GM-CSF regulates protein and lipid catabolism by alveolar macrophages

Metabolism of surfactant protein (SP) A and dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar macrophages isolated from granulocyte-macrophage colony-stimulated factor (GM-CSF) gene-targeted [GM(-/-)] mice, wild-type mice, and GM(-/-) mice expressing GM-CSF under control of the SP-C promoter element (SP-C-GM). Although binding and uptake of (125)I-SP-A were significantly increased in alveolar macrophages from GM(-/-) compared with wild type or SP-C-GM mice, catabolism of (125)I-SP-A was markedly decreased in GM(-/-) mice. Association of [(3)H]DPPC with alveolar macrophages from GM(-/-), wild-type, and SP-C-GM mice was similar; however, catabolism of DPPC was markedly reduced in cells from GM(-/-) mice. Fluorescence-activated cell sorter analysis demonstrated decreased catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine by alveolar macrophages from GM(-/-) mice. GM-CSF deficiency was associated with increased SP-A uptake by alveolar macrophages but with impaired surfactant lipid and SP-A degradation. These findings demonstrate the important role of GM-CSF in the regulation of alveolar macrophage lipid and SP-A catabolism.     

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.     

The role of pulmonary collectin N-terminal domains in surfactant structure,function, and homeostasis in vivo

The N-terminal domains of the lung collectins, surfactant proteins A (SP-A) and D (SP-D), are critical for surfactant phospholipid interactions and surfactant homeostasis, respectively. To further assess the importance of lung collectin N-terminal domains in surfactant structure and function, a chimeric SP-D/SP-A (D/A) gene was constructed by substituting nucleotides encoding amino acids Asn(1)-Ala(7) of rat SP-A with the corresponding N-terminal sequences from rat SP-D, Ala(1)-Asn(25). Recombinant D/A migrated as a 35-kDa band on reducing SDS-PAGE and as a ladder of disulfide-linked multimers under nonreducing conditions. The recombinant D/A bound and aggregated phosphatidylcholine containing vesicles as effectively as rat SP-A. Mice in which endogenous pulmonary collectins were replaced with D/A were developed by human SP-C promoter-driven overexpression of the D/A gene in SP-A(-/-) and SP-D(-/-) animals. Analysis of lavage fluid from SP-A(-/-,D/A) mice revealed that glycosylated, oligomeric D/A was secreted into the air spaces at levels that were comparable with the authentic collectins and that the N-terminal interchange converted SP-A from a "bouquet" to a cruciform configuration. Transmission electron microscopy of surfactant from the SP-A(-/-,D/A) mice revealed atypical tubular myelin containing central "target-like" electron density. Surfactant isolated from SP-A(-/-,D/A) mice exhibited elevated surface tension both in the presence and absence of plasma inhibitors, but whole lung compliance of the SP-A(-/-,D/A) animals was not different from the SP-A(-/-) littermates. Lung-specific overexpression of D/A in the SPD(-/-) mouse resulted in hetero-oligomer formation with mouse SP-A and did not correct the air space dilation or phospholipidosis that occurs in the absence of SP-D. These studies indicate that the N terminus of SP-D 1) can functionally replace the N terminus of SP-A for lipid aggregation and tubular myelin formation, but not for surface tension lowering properties of SP-A, and 2) is not sufficient to reverse the structural and metabolic pulmonary defects in the SP-D(-/-) mouse.     

Inhibitors of inflammation and endogenous surfactant pool size as modulators of lung injury with initiation of ventilation in preterm sheep

Background

Increased pro-inflammatory cytokines in tracheal aspirates correlate with the development of BPD in preterm infants. Ventilation of preterm lambs increases pro-inflammatory cytokines and causes lung inflammation.

Objective

We tested the hypothesis that selective inhibitors of pro-inflammatory signaling would decrease lung inflammation induced by ventilation in preterm newborn lambs. We also examined if the variability in injury response was explained by variations in the endogenous surfactant pool size.

Methods

Date-mated preterm lambs (n = 28) were operatively delivered and mechanically ventilated to cause lung injury (tidal volume escalation to 15 mL/kg by 15 min at age). The lambs then were ventilated with 8 mL/kg tidal volume for 1 h 45 min. Groups of animals randomly received specific inhibitors for IL-8, IL-1, or NF-κB. Unventilated lambs (n = 7) were the controls. Bronchoalveolar lavage fluid (BALF) and lung samples were used to quantify inflammation. Saturated phosphatidylcholine (Sat PC) was measured in BALF fluid and the data were stratified based on a level of 5 μmol/kg (~8 mg/kg surfactant).

Results

The inhibitors did not decrease the cytokine levels or inflammatory response. The inflammation increased as Sat PC pool size in BALF decreased. Ventilated lambs with a Sat PC level > 5 μmol/kg had significantly decreased markers of injury and lung inflammation compared with those lambs with < 5 μmol/kg.

Conclusion

Lung injury caused by high tidal volumes at birth were decreased when endogenous surfactant pool sizes were larger. Attempts to decrease inflammation by blocking IL-8, IL-1 or NF-κB were unsuccessful.     

Neither SP-A nor NH2-terminal domains of SP-A can substitute for SP-D in regulation of alveolar homeostasis

Surfactant proteins (SP)-A and -D are members of the collectin family of host defense proteins that share four distinct structural domains: NH(2)-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) NH(2)-rSftpa/d, consisting of NH(2)-terminal and collagenous domains of SP-A with neck domain and CRD of SP-D; and 3) rSftpd/a, consisting of NH(2)-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 NH(2)-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. NH(2)-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 NH(2)-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.     

Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung

Mice lacking surfactant protein (SP)-A (SP-A-/-) or SP-D (SP-D-/-) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A-/- mice but not in SP-D-/- mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A-/- mice generated significantly less, whereas those from SP-D-/- mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D-/- mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D-/- mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.