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

Samuel Hawgood, Jennifer Akiyama, Cynthia Brown, Lennell Allen, Gordon Li, and Francis R. Poulain

Department of Pediatrics, Cardiovascular Research Institute, University of California, San Francisco, California 94118-1245


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

lung surfactant; granulocyte-macrophage colony-stimulating factor; type II cells; inflammation; surfactant protein D


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SURFACTANT LIPIDS ACCUMULATE abnormally after birth in the tissue and airspaces of mice with targeted disruption of the surfactant protein D gene (SP-D; see Refs. 5 and 22). The alteration in surfactant metabolism was unexpected because in vitro studies have not shown any direct effects of SP-D on surfactant lipid secretion (23) or clearance (14). Instead, many studies suggest SP-D is a component of the innate immune response (reviewed in Ref. 7). SP-D is a large oligomeric protein with collagen-like arms connected to globular trimeric carbohydrate-recognition domains. SP-D belongs to the collectin subfamily of C-type lectins that includes mannose-binding protein, conglutinin, and surfactant protein A (SP-A). All collectins are thought to have immunomodulatory functions (reviewed in Refs. 9 and 42). In vitro, SP-D has antimicrobial properties, including the ability to agglutinate and opsonize bacteria, fungi, and viruses (7). SP-D also has anti-inflammatory properties, including the ability to inhibit stimulus-induced T-lymphocyte proliferation (4) and allergen-induced histamine release (38). These observations raise the possibility that SP-D alters surfactant metabolism by modulating lung inflammation rather than by directly influencing surfactant synthesis, secretion, or clearance.

Several recent studies show that surfactant homeostasis can be significantly perturbed during active inflammation of the lung (10, 19, 25, 30). In addition to an increase in tissue and airspace surfactant pools, SP-D-deficient mice accumulate numerous, large, multinucleated, foamy alveolar macrophages and develop hypertrophic type II cells containing giant lamellar bodies (5, 22). Changes in surfactant pool size, alveolar macrophage number and appearance, and type II cell and lamellar body size also occur in different models of lung inflammation (10, 15, 20, 30). The factors leading to the striking changes in type II cell and alveolar macrophage morphology in the SP-D-deficient mouse are unknown, but we reasoned that one or more proinflammatory cytokines might play a role.

Several cytokines, including interleukin (IL)-1, IL-4, transforming growth factor-beta , tumor necrosis factor-alpha (TNF-alpha ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), have been shown to influence surfactant metabolism in type II cells and/or alveolar macrophages (12, 16, 17, 19, 40). For this study, we focused on GM-CSF as a possible mediator of some of the cellular changes seen in the SP-D-deficient mouse. GM-CSF and both subunits of the GM-CSF receptor are expressed in the lung in type II epithelial cells and alveolar macrophages, the primary cells involved in surfactant metabolism (15). GM-CSF is chemotactic for macrophages and regulates both proliferation and differentiation (6, 24). Overexpression of GM-CSF in pulmonary epithelial type II cells causes type II cell hypertrophy and hyperplasia, increased phospholipid synthesis, and a marked increase in the number of activated, multinucleated alveolar macrophages with a concomitant increase in surfactant clearance (15, 16). Several of these changes are also seen in SP-D-deficient mice.

The importance of GM-CSF for the catabolism of surfactant by alveolar macrophages is highlighted by findings in the GM-CSF-deficient mouse. Mice deficient in either GM-CSF (8, 33) or the GM-CSF/IL-3/5 beta c-receptor (28, 32) develop alveolar tissue and airspace lipidosis and proteinosis. Metabolic studies in the GM-CSF-deficient mouse show normal rates of surfactant synthesis and secretion but markedly decreased rates of clearance from the alveolar space (17). The lung phenotype of the GM-CSF/IL-3/5 beta c-receptor-deficient mouse is almost entirely corrected by selectively restoring macrophage responses to GM-CSF by transplantation using normal bone marrow (29). These results suggest that a block in surfactant degradation by alveolar macrophages is the primary defect underlying the GM-CSF-deficient phenotype. Although the lipidosis that develops in the SP-D-deficient and GM-CSF-deficient mice is similar in amount and composition (5, 22), it is not known whether the foamy macrophages in SP-D-deficient mice also have a decreased ability to clear and degrade surfactant. To determine if GM-CSF plays a significant role in the phenotype of SP-D-deficient mice, we crossed SP-D- and GM-CSF-deficient mice to obtain littermate double null, single null, and wild-type mice. We measured alveolar surfactant phospholipid and apoprotein pools and examined the morphology of the alveolar type II cells and macrophages in each of the genotypes from 4 to 16 wk of age.


    METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Transgenic strains. SP-D-deficient mice were developed in an outbred C57BL6/CD-1 background as previously described (5). GM-CSF-deficient mice fully backcrossed to a C57BL6 background were obtained from Dr. Glenn Dranoff (Harvard University, Cambridge, MA) (8). Homozygous SP-D-deficient and GM-CSF-deficient were first bred to obtain mice heterozygous for both the SP-D and GM-CSF alleles. Double heterozygous mice were then intercrossed to produce study mice that were wild type for both genes (D+G+), null for SP-D alone (D-G+) or GM-CSF alone (D+G-), or null for both genes (D-G-). In addition, five other genotypes involving heterozygosity at one or both alleles were obtained. Littermates were screened by sequential PCRs using primers specific for the SP-D- and GM-CSF-targeted alleles. Mice were kept in isolator cages in a barrier facility for the duration of this study. The Committee on Animal Research at the University of California, San Francisco approved all experimental protocols.

Bronchoalveolar lavage fluid lipid and protein measurements. Four mice of each genotype at 4, 8, 12, and 16 wk postnatal age were lavaged with 4 × 1-ml aliquots of 10 mM Tris, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4. Bronchoalveolar lavage (BAL) fluid (BALF) was centrifuged at 250 average g for 5 min at 4°C. The total protein concentration in the cell-free BALF supernatant was determined using bicinchoninic acid as a substrate. The cell-free BALF was extracted into chloroform-methanol, and the total phospholipid content was derived from the phosphorus concentration (2).

SP-A and SP-D mRNA and BALF protein measurements. Total RNA isolated from the lungs of 8-wk-old mice of each genotype by the guanidine isothiocyanate method was hybridized with [32P]cDNA probes for mouse SP-D, SP-A, and 18S as described. Signals were quantified in a phosphorimager (Molecular Dynamics, Sunnyvale, CA) and normalized to 18S. Serial dilutions of cell-free BALF from mice of each genotype at 8 wk were analyzed for SP-A and SP-D content with a quantitative dot blot assay using monospecific polyclonal antibodies against recombinant mouse SP-A and SP-D, respectively. Standard curves using recombinant mouse SP-A and SP-D expressed in Chinese hamster ovary cells (31) were used to determine the linear range of these assays and calculate absolute SP-A and SP-D BALF pool sizes.

Microscopy. The lungs from two mice of each genotype at 4, 8, 12, and 16 wk postnatal age were fixed by intratracheal instillation of 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer for 2 h at room temperature and then were postfixed overnight in 1.5% osmium tetroxide in veronal acetate buffer at 4°C. The lungs were stained en bloc in 1.5% uranyl acetate in maleate buffer and then quickly dehydrated in cold acetone and propylene oxide. The tissue was finally infiltrated and embedded in LX 112 (Ladd Research Industries, Burlington, VT). Semithin sections were stained with toluidine blue, and ultrathin sections were stained with 5% uranyl acetate and 0.8% lead citrate for electron microscopy. Immunochemistry on 2-µm cryosections was performed as previously described (5).

GM-CSF expression and levels. RNase protection was used to detect mRNA specific for GM-CSF, and the alpha - and beta -GM-CSF receptor subunits in the total RNA were isolated from nonlavaged lungs at 8 wk of age (RiboQuant; PharMingen, San Diego, CA). GM-CSF protein levels were measured by enzyme-linked immunoassay (Endogen, Woburn, MA) in cell-free BALF at 8 wk of age.

Statistics. The data are expressed as means ± SE (see Fig. 1) or means ± SD (see Fig. 5). Comparisons between groups were evaluated by Student's t-test, accepting a significance at P < 0.05. 


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Fig. 1.   Cell-free bronchoalveolar lavage fluid (BALF) phospholipid levels. A: total cell-free BALF phospholipid levels expressed as µg phospholipid/mouse at 4, 8, 12, and 16 wk of age. , Wild type for surfactant protein D (SP-D) and granulocyte-macrophage colony-stimulating factor (GM-CSF), simply referred to as D+G+; black-lozenge , null for SP-D alone (D-G+); , null for GM-CSF alone (D+G-); black-triangle, null for both genes (D-G-). Data are means ± SE for 3-6 mice of each genotype at each time point. D+G+ values are significantly different from all other genotypes at all time points by Student's t-test (P < 0.05). D+G- and D-G- are significantly different from D-G+ and each other at all time points except 4 wk. B: same data from A expressed as total cell-free BALF phospholipid/g body wt. Values of each genotype are significantly different from each other at all time points.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic lines. The genotypes of the progeny of double heterozygous mice were consistent with a Mendelian distribution, with 7% of the pups D+G+, 6% D-G+, 7% D+G-, and 5% D-G-. The remaining 75% of the pups were heterozygous at one or both alleles and were not studied further. All mice were maintained in a barrier facility and were indistinguishable by appearance, weight, and activity. No infection was detected at regular autopsy for evidence of pneumonia. An unusual small cell tumor of the lung in a subpleural location was found in one D+G- mouse at routine autopsy.

BALF lipid and protein measurements. As previously reported, cell-free BALF phospholipid accumulated in both the D-G+ and D+G- mice compared with their wild-type littermates (Fig. 1A). The combined deficiency of both SP-D and GM-CSF in the D-G- mice resulted in an approximately additive accumulation of BALF phospholipid over that seen with either deletion alone. The total BALF phospholipid per mouse increased from 4 to 8 wk after birth in each genotype proportional to the increase in body weight during this time. By 4 wk after birth, the total cell-free BALF phospholipid content corrected for body weight was increased 3.5-, 6.6-, and 9.8-fold over control in the D-G+, D+G-, and D-G- mice, respectively. The relative differences between genotypes in BALF phospholipid content were maintained fairly constant from 4 to 16 wk of age, indicating the BALF phospholipid pool size per gram body weight had reached a relatively steady state by 4 wk after birth in each of the four genotypes (Fig. 1B).

Total cell-free BALF protein content was also increased in D-G+, D+G-, and D-G- mice at all ages compared with their wild-type littermates. The increase in protein was approximately proportional to the increase in BALF phospholipid so that the average phospholipid-to-protein ratio of cell-free BALF was not significantly different between D+G+, D-G+, D+G-, and D-G- mice (mean phospholipid-to-protein ratios of 1.45, 1.67, 1.22, and 1.4, respectively).

SP-A and SP-D mRNA and BALF protein measurements. As expected, SP-D mRNA or protein was not detectable in either D-G+ or D-G- mice. Both SP-A mRNA (1.5-fold) and SP-D mRNA (2.4-fold) were increased significantly in D+G- mice compared with wild-type littermates (Fig. 2A). SP-A mRNA levels were decreased to 50% of wild-type levels in the D-G+ mice (Fig. 2A). There was ~30-fold more SP-A than SP-D in cell-free BALF of wild-type mice (10 ± 3 µg SP-A/ml compared with 0.3 ± 0.08 µg SP-D/ml). SP-D protein was increased in D+G- mice 50-fold relative to wild-type littermates (15.3 ± 1.8 compared with 0.3 ± 0.08 µg/ml; Figs. 2B and 3). Similar to the mRNA levels, cell-free BALF SP-A protein levels were approximately one-half the levels of the wild type in D-G+ mice (5.4 ± 1.2 compared with 10 ± 3 µg/ml). SP-A protein levels in cell-free BALF were increased approximately twofold in both D+G- (18.6 ± 3.1 µg/ml) and D-G- (24 ± 4.3 µg/ml) mice (Fig. 2B).


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Fig. 2.   Whole lung SP-A and SP-D mRNA and cell-free BALF SP-A and SP-D protein levels. A: mRNA levels normalized to D+G+ levels. B: cell-free BALF protein levels normalized to D+G+ levels. Note the degree of change is expressed on a log scale in A and B. *Significantly different from wild type, P < 0.05; n = 4 for each genotype.



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Fig. 3.   SP-D immunohistochemistry in the GM-CSF-deficient mouse. a: Staining for SP-D in type II cells and alveolar macrophages in wild type is only weakly detected at this magnification. b: Phase-contrast micrograph of the wild-type lung. c: Intense staining for SP-D in the airspaces of a GM-CSF-deficient mouse. d: Phase-contrast micrograph of the GM-CSF-deficient lung. Red background reflects tissue autofluorescence; a-d are the same magnification.

GM-CSF mRNA and BALF protein measurements. GM-CSF and GM-CSF receptor subunit mRNA levels in whole lung of D+G+ and D-G+ mice were analyzed by RNase protection assay. A protected GM-CSF fragment was detected at low levels in both wild-type and D-G+ lungs. When corrected for equal loading by normalizing to the ribosomal protein L32 mRNA content, GM-CSF mRNA was increased 1.4-fold in D-G+ lungs compared with wild-type littermates (P < 0.05). There was no statistical difference in GM-CSF receptor subunit mRNA levels in wild-type and D-G+ lungs. GM-CSF protein was measured in cell-free BALF by an ELISA with a lower limit of detection of 5 pg/ml. GM-CSF was not detected in any wild-type BALF (n = 6) but was consistently detectable, albeit at low and quite variable levels, in D-G+ BALF (6.6 ± 7.8 pg/ml).

Alveolar macrophages. Accurate cell counts from the BALF of the double gene-targeted mice using a hemocytometer were not possible because of contaminating noncellular material. To obtain an estimate of alveolar macrophage abundance by the same technique in all genotypes, alveolar macrophages were counted in randomly selected fields from toluidine blue-stained semithin sections of two mice per genotype by an investigator blind to the genotype. Consistent with our previous findings using direct cell counts on BALF, the number of alveolar macrophages per high-power field was sevenfold greater in the D-G+ mice than in the wild type. The numbers of macrophages per high-power field in the D+G- mice and the D-G- mice were not significantly different from the wild type (Fig. 4).


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Fig. 4.   Alveolar macrophage numbers at 16 wk of age. The number of alveolar macrophages per high-power field (hpf) were counted (25-92 high-power fields/genotype) and are expressed as means ± SD. *Significantly different from D+G+, P < 0.05.

Microscopy. There were striking, progressive histological changes in the lungs of the D-G+, D+G-, and D-G- mice. By 4 wk of age, there was a scattered accumulation of material in the alveolar lumen, most notably in the subpleural regions of the lung in all three genotypes. At all ages, the alveolar accumulation of secretions was greatest in D-G- mice. An increase in size of the type II cells and lamellar bodies was present in D-G+ mice but not in D+G- or D-G- mice. By 16 wk of age, these changes had progressed to involve more of the lung (Fig. 5). Some alveoli in each of the genotypes appeared to be completely full of secretions, but many alveoli still appeared to be relatively unaffected. At all ages, type II cell changes were only observed in D-G+ mice.


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Fig. 5.   Light micrographs of lungs at 16 wk of age. a: Normal alveolar architecture surrounds a small blood vessel in the lung of a D+G+ mouse. A single small alveolar macrophage is visible in the center of the field (arrow). b: Increased secretions, numerous giant foamy alveolar macrophages (arrows), and abnormally enlarged lamellar bodies in the lung of a D-G+ mouse. c: Increased secretions in the airspaces but no significant increase in the number of alveolar macrophages (arrow) in the lung of a D+G- mouse. d: Marked increase in secretions and several intraluminal macrophages (arrows) in the lung of a D-G- mouse. At this magnification, densely packed secretions are difficult to distinguish from alveolar macrophages. Although the alveolar walls appear more cellular, enlarged lamellar bodies are not seen; a-d are the same magnification.

There was a significant difference in the appearance of alveolar macrophages between genotypes. Most macrophages in the D-G+ mice had a pronounced foamy appearance with numerous extracted cytoplasmic inclusions. In contrast, the macrophages in the D+G- and D-G- mice were less numerous and did not contain the clear cytoplasmic inclusions. By 16 wk, a prominent cellular infiltrate around blood vessels associated with small airways resembling bronchial-associated lymphoid tissue was present in all three null genotypes (data not shown).

The material accumulating in the alveolar space was better defined under the electron microscope at 16 wk of age. Alveolar surfactant forms were rarely seen in situ in wild-type lungs, but abundant secretions were always present in all three null genotypes. Although normal-appearing tubular myelin and other vesicular structures were all present in each of the null genotypes, tubular myelin forms seemed more abundant in D+G- and D-G- mice compared with D-G+ animals. The alveolar secretions in the D-G+ mice were predominantly the giant lamellar body-like forms. Electron microscopy confirmed the presence of numerous giant lamellar bodies within enlarged type II cells in D-G+ mice. Enlarged lamellar bodies were extremely rare in any of the other genotypes (Fig. 6). Alveolar macrophages were grossly enlarged and stuffed with both membrane-bound phospholipid inclusions and cytoplasmic oil droplets, characteristic of foamy macrophages in D-G+. Although most alveoli contained several macrophages in the D-G+ mice, macrophages were sparser and contained only occasional membrane-bound phospholipid inclusions in D+G- and D-G- mice (Fig. 7).


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Fig. 6.   Representative type II cell morphology at 16 wk of age. a: Type II cell in the lung of a D+G+ mouse with several lamellar bodies and the absence of visible secretions in the airspaces. b: Enlarged type II cells with giant lamellar bodies and increased airspace secretions in the lung of a D-G+ mouse. Adjacent type II cells are common in D-G+ mice but rare in D+G+ mice. c: Two type II cells with normal-appearing lamellar bodies and increased airspace secretions in the lung of a D+G- mouse. d: Single type II cell with normal-appearing lamellar bodies but markedly increased airspace secretions in the lung of a D-G- mouse. a-d are the same magnification.



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Fig. 7.   Representative alveolar macrophage morphology at 16 wk of age. a: Alveolar macrophage in the airspace with a few intracellular phagolysosomes in the lung of a D+G+ mouse. b: Enlarged alveolar macrophage with intracytoplasmic neutral lipid inclusions and crystalline rods in the lung of a D-G+ mouse. Giant lamellar bodies within the type II cells and increased airspace secretions are also visible. c: Alveolar macrophage with phagolysosomes containing phospholipid whorls and increased airspace secretions in the lung of a D+G- mouse. d: Alveolar macrophage in the lung of a D-G- mouse. There are numerous phagolysosomes containing phospholipid whorls but none of the intracytoplasmic neutral lipid inclusions that characterize the D-G+ mouse; a-d are the same magnification.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, the combined deficiency of GM-CSF and SP-D in mice led to an additive accumulation of phospholipid in the airspaces, suggesting that the underlying metabolic disturbances leading to pulmonary lipidosis in the two genotypes are distinct. In addition, selective GM-CSF deficiency prevented the markedly abnormal type II cell and alveolar macrophage phenotype seen in SP-D gene-targeted mice, supporting the hypothesis that GM-CSF-mediated signaling is required for the proliferation and activation of macrophages and hypertrophy of type II cells in SP-D-deficient mice.

The absolute BALF surfactant accumulation was progressive in all genotypes, reaching a plateau between 8 and 12 wk after birth. When corrected for body weight, the BALF surfactant levels were surprisingly constant in all genotypes from 4 to 16 wk of age. We have not been able to consistently perform lavage on mice pups <4 wk of age, and we do not know how quickly after birth the increased surfactant pool sizes in each null genotype are reached. In other species, early neonatal BALF surfactant levels are transiently increased by up to 10-fold due to delay in establishing surfactant clearance pathways (21, 35). Type II cells and alveolar macrophages both contribute to surfactant phospholipid clearance from the airspaces (13, 26). Although type II cells recycle intact most of the cleared surfactant (13, 26) in vivo, alveolar macrophages rapidly degrade surfactant phospholipids (27) in vitro. Recent studies, reported in preliminary form, by Ikegami and colleagues (18) suggest that the expected postnatal fall in BALF surfactant pool size does not occur in SP-D-deficient mice despite an increased influx of alveolar macrophages. The ability of these macrophages to degrade surfactant is not known.

In other situations that lead to alveolar macrophage foam cell transformation, the presence of numerous cytoplasmic neutral lipid deposits correlates with active phospholipid hydrolysis and triglyceride storage (34), suggesting that the macrophages in SP-D-deficient mice are actively degrading surfactant phospholipids. In contrast, the loss of GM-CSF activity in the lung appears to almost completely block surfactant lipid degradation without significantly changing surfactant synthesis or secretion by type II cells (17). The macrophages in the D+G- or D-G- mice had phospholipids and SP-A in phagolysosome compartments but did not develop the neutral lipid intracytoplasmic deposits suggestive of increased phospholipid degradation. In our study, the loss of GM-CSF activity in SP-D-deficient mice was associated with an additive accumulation of phospholipids in the BALF at all time points studied. This result and the appearance of the alveolar macrophages suggest that macrophages contribute significantly to surfactant clearance in D-G+ mice throughout the first 4 mo of life. It is unknown whether the accumulation of intracellular lipids by alveolar macrophages can eventually lead to decreased surfactant uptake. Other factors, not regulated by GM-CSF, must play a role in the alveolar and tissue surfactant accumulation in SP-D-deficient mice.

Surfactant protein levels were also variably affected by GM-CSF and SP-D deficiency. There is a 2-fold increase in SP-A levels and a 50-fold increase in SP-D levels in GM-CSF-deficient mice. When corrected for the ~30-fold difference in absolute SP-A and SP-D pool sizes and for the difference in molecular weight, the absolute molar accumulations of SP-A and SP-D in the GM-CSF-deficient mice were similar. Although there is a modest increase in SP-A and SP-D mRNA content in D+G- mice, delayed clearance of SP-A and SP-D probably contributes significantly to the elevated protein levels (17). In this study, we found a significant decrease in SP-A mRNA levels and cell-free BALF SP-A levels in SP-D-deficient mice. We previously reported elevated SP-A levels in the BALF of SP-D-deficient mice (5). In that initial report, SP-A levels were measured in whole BALF before the macrophages were removed. The difference between the whole BALF and the cell-free BALF SP-A levels suggests significant association or uptake of SP-A into the macrophages from SP-D-deficient mice. Consistent with this speculation, macrophages in SP-D-deficient lungs react strongly with antibodies to SP-A (Fig. 8).


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Fig. 8.   SP-A immunohistochemistry in SP-D-deficient mice. a: Type II cells, wild-type macrophages, and airspace secretions stain for SP-A. b: Phase-contrast micrograph of the wild-type lung with an alveolar macrophage indicated by the arrow. c: Alveolar macrophages in the lung of an SP-D-deficient mouse stains strongly for SP-A, with minimal SP-A-positive secretions. d: Phase-contrast micrograph of the SP-D-deficient lung with several alveolar macrophages indicated by arrows. Red background reflects tissue autofluorescence; a-d are the same magnification.

Similar to the additive effect that double-gene ablation has on BALF phospholipid content, we found it had a similar additive effect on cell-free BALF SP-A content, again suggesting that macrophage clearance of SP-A is probably intact in SP-D-deficient mice. We do not know the reason for the decreased SP-A mRNA in SP-D-deficient mice. SP-B and SP-C mRNA levels are unchanged in SP-D-deficient mice (5, 22), suggesting that there is not a global downregulation of surfactant protein expression. It is also unlikely that there is direct feedback between SP-D levels and SP-A expression because SP-A levels are normal in mice significantly overexpressing SP-D (11). Specific proinflammatory cytokines, such as TNF-alpha and IL-1 (12, 41), which downregulate SP-A expression in mature lung, are modestly elevated in SP-D-deficient mice (data not shown). Perhaps the particular cytokine milieu in the SP-D-deficient mice results in a downregulation of SP-A expression. Given the close linkage between the SP-A and SP-D genes on mouse chromosome 14 (1), it is also possible that contiguous gene effects from the targeting construct have perturbed SP-A expression.

We found a modest increase in GM-CSF mRNA and protein in the lungs of SP-D-deficient mice, with no change in GM-CSF receptor mRNA levels compared with the wild type. Consistent with previous reports, we did not detect GM-CSF in the BALF of wild-type mice. The biological significance of the low but consistently detectable GM-CSF levels we measured in the BALF of SP-D-deficient mice is hard to determine. After tracheal instillation of endotoxin, GM-CSF levels rise acutely to levels 100-fold higher than the levels we measured in nonchallenged SP-D-deficient mice (15). Similarly, high BALF levels were achieved by the ectopic overexpression of GM-CSF in type II cells using the human SP-C promoter (15). Both GM-CSF and the GM-CSF receptor are expressed in several cell types in the lung, including type II cells and alveolar macrophages (3, 15). The cellular source of the increased GM-CSF in SP-D-deficient mice is unknown. The lung abnormalities in these mice are patchy, so it is possible that significantly higher GM-CSF levels are present in local alveolar microenvironments, as recently shown for matrix protease expression (39). Perhaps the best indication that GM-CSF signaling is relevant to the SP-D-deficient phenotype comes from the normalization of type II cell and macrophage appearance in SP-D null mice made selectively GM-CSF deficient. Further studies will be needed to address cell-specific GM-CSF expression and cell-specific signaling in the SP-D-deficient animals.

Two striking consequences on alveolar cell morphology of selective GM-CSF deficiency in the SP-D-deficient background were the almost complete absence of the giant lamellar bodies and hypertrophied type II cells and the absence of hugely enlarged foamy macrophages that characterize the SP-D-deficient lung. Littermates were used for this study to control for the possible compounding effects of different genetic background. The SP-D-deficient line used was not fully backcrossed, so some genetic variability was still possible. We have subsequently analyzed SP-D-deficient mice backcrossed 10 generations into a C57BL6 background and have found the same characteristic surfactant accumulation, type II cell hyperplasia, and foamy macrophages (data not shown). It is likely therefore that the normalization of the type II cell and macrophage phenotypes was a direct consequence of selective GM-CSF deficiency rather than some subtle effect of genetic background. Our results suggest that GM-CSF directly or a molecule dependent on GM-CSF mediates the type II cell hyperplasia and is necessary for recruitment and/or proliferation and the foam cell-like transformation of the alveolar macrophages seen with SP-D deficiency. Marked chronic overexpression of GM-CSF in the type II cell results in type II cell hyperplasia and a dramatic increase in alveolar macrophage number (15). Our results suggest that GM-CSF levels well below the high levels achieved with transgenic overexpression can also modulate the alveolar cell phenotype.

In summary, GM-CSF levels are modestly elevated in the lungs of SP-D gene-targeted mice. GM-CSF deficiency in the SP-D null background further increased the accumulation of phospholipid in the airspaces but prevented the hypertrophic type II cell response and the accumulation of giant foamy alveolar macrophages. These findings suggest a model in which in the absence of SP-D there is increased GM-CSF expression in the lung, with subsequent autocrine or paracrine signaling to type II cells and alveolar macrophages, resulting in hypertrophy and hyperplasia of both cell populations and foamy transformation of the macrophages. At present, the stimulus for or site of increased GM-CSF expression in the setting of SP-D deficiency remains unknown. We speculate that SP-D might normally facilitate the removal of inhaled foreign particles such as allergens (37) or endotoxin (36), or possibly endogenous alveolar subphase components such as oxidized surfactant lipids or denatured proteins, without induction of an inflammatory response. SP-D might achieve this "housekeeping function" by directly opsonizing and aggregating foreign material (7) or by generally dampening the immune responses of immune-competent cells in the lung to exogenous stimuli (4, 38). In the absence of SP-D, such stimuli might be free to trigger increased cytokine and chemokine expression from alveolar macrophages, type II cells, or pulmonary lymphocytes and initiate the complex changes seen in SP-D-deficient mice.


    ACKNOWLEDGEMENTS

We thank Dr. Glenn Dranoff for permission to use granulocyte-macrophage colony-stimulating factor-deficient mice for this study, Dr. Jo Rae Wright for the kind gift of anti-mouse surfactant protein D antibody, Erin Collins for excellent assistance with the immunohistochemistry, and Dr. John Clements for critical review of the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-24075 and HL-58047 and by a Transgenic Animal Grant from the Howard Hughes Medical Institute.

Address for reprint requests and other correspondence: S. Hawgood, Suite 150, Laurel Heights Campus, Univ. of California San Francisco, 3333 California St., San Francisco, CA 94118-1245 (E-mail: hawgood{at}itsa.ucsf.edu).

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.

Received 3 November 2000; accepted in final form 22 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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