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GM-CSF is a cytokine that promotes the proliferation and differentiation of hematopoietic stem cells to neutrophils and monocyte/macrophages (1). Studies on GM-CSF–deficient mice generated by the knockout technique reveal no apparent defect in steady state hematopoiesis. Instead, they show that GM-CSF has a critical role in pulmonary surfactant homeostasis. Adult GM-CSF–deficient animals develop alveolar proteinosis characterized by progressive accumulation of surfactant lipids and proteins in the alveolar space (2, 3) and recurrent infections (3). Pulmonary alveolar macrophages and type II alveolar cells of GM-CSF– or GM-CSF receptor–deficient mutant mice revealed an excess of material that immunostains with SF (2–4).

According to Ikegami et al. (5), GM-CSF knockout mice had 6- to 8-fold levels of pulmonary DPC and no detectable catabolism of DPC in vivo. There was no detectable degradation of labeled DPC or SP-A given by tracheal instillation. Reversal of GM-CSF receptor–deficient pulmonary alveolar proteinosis in mice was accomplished by bone marrow transplantation, suggesting the importance of pulmonary alveolar macrophages (6). Overexpression of mouse GM-CSF, directed by the human SP-C gene promoter, was associated with type II cell and alveolar macrophage hyperplasia and an increase in lung size without evidence of fibrosis or lung damage (7, 8). Overexpression of GM-CSF in GM-CSF knockout mice normalized the abnormally large alveolar DPC pool, increased the incorporation of precursors to DPC, and increased the clearance rates of DPC and SP-B from the alveolar space (9).

Surfactant phospholipids are synthesized in the smooth endoplasmic reticulum of the type II alveolar epithelial cells and subsequently transported into the lamellar bodies, which are then released by exocytosis into the alveolar lining. According to current evidence, the clearance of surfactant from adult lung is mediated primarily by type II cells that take up 70–80% of the surfactant phospholipids and proteins for recycling or degradation, whereas approximately 20% is cleared by alveolar macrophages (10). However, in term newborn rabbit or sheep, the catabolism rate of surfactant phospholipid is very slow, and the pool size of surfactant assessed per kilogram of body weight is approximately 1 order of magnitude higher than in adult or young animals (10). In preterm and term rabbits, the intracellular transport of surfactant phospholipids from their site of synthesis is delayed (3 to 24 h) compared with adult rabbits (1–4 h) (11, 12).

It has been shown that the concentrations of GM-CSF are low in amniotic fluid from preterm pregnancies without chorioamnionitis and in airway specimens from premature infants with respiratory distress syndrome (13). GM-CSF in lung effluent increases as a function of normal gestation (13). A similar increase in plasma GM-CSF at birth was not evident (14). Synthesis of GM-CSF in alveolar and bronchial cells has been shown (15, 16).

GM-CSF, which is developmentally regulated in lung effluent during fetal development, has roles in the metabolism of surfactant in the adult. According to the present hypothesis, GM-CSF is involved in the regulation of surfactant phospholipids during the perinatal development. The aim of the present study was to investigate whether the administration of rhGM-CSF to either preterm or full-term rabbits acutely influences surfactant DPC metabolism.

METHODS

The present study was approved by the Institutional Review Board at the University of California, Irvine. The New Zealand White rabbits were from Irish Farms (Orange County, CA). The natural surfactant was isolated from BAL of adult rabbits by differential and density gradient centrifugation (17). The natural surfactant containing labeled dipalmitoyl phosphatidylcholine was prepared. Briefly, a trace of [2-palmitoyl-9,10-3H]-dipalmitoyl phosphatidylcholine (40 Ci/mmol; final radioactivity, 8 μCi/mL; New England Nuclear, Boston, MA) was evaporated under N2, followed by an addition of natural surfactant suspension (final phospholipid concentration, 0.5 μmol/mL) and a trace of [1-14C]-acetate (60 mCi/mmol; final radioactivity, 10 μCi/mL; New England Nuclear) in PBS. The mixture was sonicated intermittently (5 s followed by 20 s intermission) for 5 min at 0°C using a sonicator with a microtip (Sonic Dismembrator model 300, Fisher Scientific). When indicated, rhGM-CSF (final concentration, 10 or 50 μg/mL) or placebo was added. RhGM-CSF was a kind gift of Dr. Tony Troutt, Immunex Co. (Seattle, WA).

Treatment of animals.

On d 29 (±1 h) of pregnancy, 16 does were anesthetized by intramuscular ketamine (35 mg/kg) and xylazine (5 mg/kg). Shortly after the induction of anesthesia, 134 pups were delivered by hysterotomy. The newborn pups were dried, stimulated to breathe, and weighed. Altogether, 126 animals survived until the experiment that was started before the age of 2 h. The second group of animals consisted of 119 term rabbits from 16 litters. These animals were kept with their does until the experiment at the age of 3 d. Before the intratracheal treatment, all animals were given antibiotics (penicillin 100 000 IU/kg, streptomycin 10 mg/kg i.m.). During the experiment, all animals were gavage fed using an Esbilac formula fortified with lactalbumin (5 g/100 mL). Seventy to 90 mL·kg−1·d−1 were given to the preterm and 120–140 mL·kg−1·d−1 to the term animals. The ambient temperature was adjusted to 29–30°C (preterm animals) or to 24–26°C (term animals) using a radiant heater connected to a thermostat. This maintained the rectal temperature close to 37°C.

The newborn rabbits were anesthetized with 10 mg/kg ketamine and 1 mg/kg xylazine intraperitoneally. Lidocaine was given for local anesthesia. A 26- (premature) or 24-gauge (term) angiocatheter was introduced into the trachea via skin incision under aseptic conditions. The mixture containing rabbit surfactant (0.5 μmol phospholipid per milliliter), rhGM-CSF (50, 10, or 0 μg/mL), and [3H-palmitate-2]-dipalmitoyl phosphatidylcholine (8 μCi/mL), 14C-acetate (10 μCi/mL) was given intratracheally in two boluses while the animal was in a supine reversed Trendelenburg position. During the administration, the animal was tilted to the left and the right flank, respectively. The volume instilled was 0.1 (premature) or 0.2 mL (term). The catheter was then withdrawn and the skin incision was sutured. When indicated, 24 h after the administration of rhGM-CSF/placebo, an identical dose of GM-CSF/placebo was given intraperitoneally. The animals were killed 1 to 48 h after the isotope administration. When indicated, rhGM-CSF/placebo was given intraperitoneally 0–10 min before administration of the isotopes without rhGM-CSF intratracheally.

Isolation of lung fractions and organs.

The animals were killed by injecting Euthanol intraperitoneally. Intracardiac blood was collected into an EDTA tube. BAL was performed through a catheter in the trachea (18). A volume of 35–40 mL/kg sterile PBS was instilled, and the lavageate was recovered by gentle suctioning. This procedure was repeated six times. The isolation of surfactant fractions was performed at 0–4°C. The combined BAL return was centrifuged at 150 ×g for 10 min to recover the cells. The cell fraction was washed by suspension in PBS, and the cells were concentrated by centrifugation. Large surfactant aggregates were isolated from cell-free BAL by centrifugation at 27 000 ×g for 10 min. Both the supernatant (i.e. the small aggregate fraction) and the pellet (the large aggregate fraction) were recovered. The residual lung after BAL was weighed and homogenized in isotonic sucrose solution, and the lamellar body fraction was isolated as described (17). An estimate of the recovery of lamellar bodies was performed by adding freshly isolated labeled lamellar bodies to the lung homogenates of two preterm and two term animals that did not receive any radioactive material. The mean recovery of radioactivity in the lamellar body fraction was 83 ± 8% (±SEM).

The liver and intestines were recovered, weighed, homogenized in PBS by use of a Potter homogenizer, and processed for radioactivity measurement.

Isolation of lipids and analysis of radioactivity.

DPC was isolated and quantified according to Mason et al. (19). A fraction of DPC was processed for the measurement of 3H and 14C radioactivity by use of a liquid scintillation system. The variation in channel overlap was corrected using external standardization. Total radioactivity in the lung fractions, liver, intestines, and blood was measured. When necessary, the specimens were made colorless by using sodium hydroxide and H2O2.

The radioactivity associated with the 2-position of DPC was measured. Briefly, the specimen of DPC was stirred to a slurry containing phospholipase A2 from Naja Naja venom (Sigma Chemical Co. Chemicals) in PBS-ether (1:1 vol/vol) for 1 h. Thereafter, the phases were separated by sedimentation, and the radioactivity of the ether (FFA) and aqueous (lysophosphatidylcholine) phases was quantified. Subsequently, the FFA, phosphatidylcholine, and lysophosphatidylcholine fractions were isolated using one-dimensional thin-layer chromatography. The respective areas were scraped from the plate and analyzed for radioactivity. Phosphatidylcholine contained no radioactivity, indicating that the reaction catalyzed by phospholipase A2 was complete.

Analysis of SP-B.

To study whether the membranous SP-B was affected, premature animals from three litters were euthanized 24 h after intratracheal GM-CSF (50 μg/mL) (n= 7) or placebo (n= 7). The animals used in the experiment did not receive the radioactive isotopes. Immediately after the animals were killed, pieces of the upper and lower lobes of the left lung were immersed in liquid nitrogen. Thereafter, the total RNA was isolated using the single-step acidic guanidinium isothiocyanate method. The RNA was processed for quantification of SP-B mRNA by Northern blot analysis. The membranes were hybridized with random primer 32P-labeled 1.7-kb rabbit SP-B and cytochrome oxidase subunit II probes (20). The bands were analyzed using video imaging densitometry. Densitometric measurements for SP-B were normalized to cytochrome oxidase subunit II. The content of SP-B was expressed on the basis of SP-B content after placebo (100%). The right lung was recovered and fixed in 4% neutral buffered formaldehyde for 24 h and embedded with paraffin. Five-micrometer sagittal sections on glass slides were immunostained with MAb against SP-B as described (20).

Quantification of SP-B in BAL was performed according to Krämer, as described previously (20, 21).

Analysis of cell counts and DNA.

For white blood cell counts, blood was recovered by intracardiac aspiration into EDTA tubes. Total and differential white cell counts were obtained by a single medical technologist blinded to the treatment allocation. Total lung DNA was measured as described by Shapiro and Schrier (22). The number of cells in BAL was counted using the Buerger chamber.

The alveolar tissues of the sagittal sections of the right lung, immunostained for SP-B, were viewed by a single investigator blinded to the treatment allocation. Four consecutive fields from both lobes were counted for the number of alveolar cells. The regions were selected at random to represent the lung parenchyma. The SP-B–positive alveolar cells were considered to be the type II alveolar cells. The total number of type II alveolar cells in the lung fields and the percentage of type II alveolar cells out of the total alveolar epithelial cells were recorded.

Statistics.

The mean results for each litter were analyzed. The combined results were expressed as mean ± SEM. The differences between the individual group means were calculated using ANOVA or the t test. A p value of < 0.05 was considered significant. The dose-dependency of the effect of rhGM-CSF was evaluated using regression analysis.

RESULTS

Evidence of biologic efficacy.

High dose of intratracheal rhGM-CSF did not change the number or distribution of white cells in peripheral blood. However, 24 h after intraperitoneal rhGM-CSF (5 μg) to premature animals, there were significant increases in eosinophil (GM-CSF, 565 ± 49 cells/mm3; placebo, 114 ± 54 cells/mm3) and in granulocyte counts (GM-CSF, 1885 ± 118 cells/mm3; placebo, 1170 ± 92 cells/mm3) in peripheral blood. The effect of GM-CSF was similar in term animals (data not shown). This indicates the biologic efficacy of heterologous GM-CSF.

Dose of GM-CSF, outcome, and number of animals studied.

The weights of preterm and 3-d-old term animals were 41.6 ± 0.8 and 79.6 ± 0.9 g, respectively; there were no detectable differences between the subgroups. The mean doses of intratracheal rhGM-CSF given to the preterm and term animals were similar: low dose 24.9 ± 1.2 (1 μg per animal) and 23.9 ± 0.8 μg·kg−1·d−1 (2 μg per animal), respectively; high dose 120.0 ± 4.8 (5 μg per animal) and 134 ± 5.3 μg·kg−1·d−1 (10 μg per animal), respectively. A group of preterm (four litters) and term (five litters) animals received placebo or the high dose of rhGM-CSF intraperitoneally: 129.3 ± 5.5 (5 μg per animal) and 126.0 ± 4.7·kg−1·d−1 (10 μg per animal), respectively.

Twelve preterm and eleven term litters were treated with intratracheal GM-CSF or placebo. Of the 94 preterm animals treated with intratracheal rhGM-CSF/placebo, 16 (17%) died before the end of experiment. Of those that died, eight were among 35 placebo-treated, four received the low dose of rhGM-CSF (n= 25), and four were treated with the high dose of GM-CSF (n= 34) (NS). Of the 32 preterm animals treated with intraperitoneal rhGM-CSF or placebo, six (19%) died. Of the 119 term animals, 5 (4%) died during treatment.

Altogether, 78 surviving premature pups received intratracheal GM-CSF/placebo. The recovery of total radioactivity was studied in eight pups, the dose response of rhGM-CSF in 36, the rate of incorporation of radioactivity to DPC in 38, the pool size of SP-B in 14, and the lung histology in six pups. The total number of studies exceeded the total number of animals, because a single pup was often used for more than one measurement. Altogether, 68 surviving term pups received intratracheal rhGM-CSF or placebo. The recovery of total radioactivity was studied in six animals, dose response of rhGM-CSF on DPC pools and on distribution of radioactivity in 34, the rate of incorporation of radioactivity into DPC in 40, and the lung histology in four pups.

Recovery of total radioactivity.

The distribution of the intratracheal radioactivity was studied. Twenty-four hours after the administration of the isotope, 78.9 ± 10.5% of 3H was recovered in the lung, 11.3 ± 3.3% in the intestinal tract, 3.9 ± 0.7% in the blood (assuming a blood volume of 80 mL/kg), and 5.9 ± 2.0% in the liver; the recovery was 82 ± 11% of total intratracheal 3H. Compared with term animals, the recovery of 3H radioactivity in the intestine in preterm animals was higher (preterm 14.1 ± 2.2%versus term 7.4 ± 1.9%). This suggest that the preterm animals regurgitated more material than the term animals despite the smaller volumes instilled (0.1 versus 0.2 mL). GM-CSF did not affect the recovery of total radioactivity. The recoveries of 14C radioactivity were similar to those of 3H (data not shown).

Pool sizes of DPC in the lung 24 h after GM-CSF.

In the premature animals, total DPC in the lung tended to increase 24 h after intratracheal rhGM-CSF (5 μg) (p= 0.08). Administration of 5 μg of GM-CSF significantly increased DPC in the surfactant fractions, i.e. the lamellar bodies, BAL, and the large aggregate fraction of BAL. This effect was dose-dependent (p< 0.05) (Fig. 1). GM-CSF had no effect on the distribution of DPC between extracellular (BAL) and intracellular (lamellar bodies) surfactant or between the large aggregate surfactant fraction and the nonsedimentable BAL. As shown in Table 1, administration of 5 μg of rhGM-CSF intraperitoneally did not increase DPC either in the lung or in the surfactant fractions.

Figure 1
figure 1

Pool sizes of DPC in the cell-free fraction of BAL, the large aggregate fraction of BAL, the intracellular lamellar bodies, and in the total lung (residual lung plus total BAL). Results are from 12 litters of preterm rabbits (gestation 29 d) 24 h after intratracheal injection of rhGM-CSF (1 or 5 μg) or placebo and from 11 litters of 4-d-old term rabbits 24 h after intratracheal injection of rhGM-CSF (2 or 10 μg) or placebo (mean ± SEM). Significant differences are indicated (★★p< 0.01; ★p< 0.05).

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Table 1 Pool sizes of DPC (μmol) in preterm and term lungs 24 h after intraperitoneal rhGM-CSFResults are expressed as the litter mean ± SEM. Altogether, four litters of premature animals and five litters of term animals were studied. There were no statistically significant differences between the treatment groups.
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In the term animals, the administration of rhGM-CSF was not associated with any significant changes in the pool sizes of DPC (Fig. 1 and Table 1).

Pulmonary distribution of DPC radioactivity.

14C-acetate and 3H-dipalmitoyl phosphatidylcholine were given intratracheally. Thereafter, the intrapulmonary distribution of radioactivity associated with DPC was studied. The DPC radioactivity associated with total lung, intracellular lamellar bodies, BAL, large aggregate fraction of BAL, the nonsedimentable fraction of BAL, and the cell fraction of BAL was studied. The results were expressed as the total DPC radioactivity in each fraction.

To facilitate comparison between the different groups, the mean radioactivity recovered in the total lung of the placebo-treated animals was adjusted to 100. Figure 2 illustrates the DPC radioactivity in whole lung, intracellular lamellar bodies, and BAL 24 h after the isotopes to premature rabbits. Intratracheal rhGM-CSF (5 μg) increased the incorporation of 14C-acetate into DPC associated with lamellar bodies and BAL. This effect was dose-dependent (p= 0.05). The incorporation of 14C-acetate into DPC of the whole lung tended to increase but not significantly. GM-CSF significantly increased 3H-DPC in lamellar bodies and in lamellar bodies and BAL combined. Less than 3% of total 14C- and 3H-DPC radioactivities was associated with the cell fraction of BAL. There were no significant differences between GM-CSF and placebo-treated animals in the distribution of DPC radioactivity between the large surfactant aggregates and the nonsedimentable BAL (data not shown).

Figure 2
figure 2

Distribution of radioactivity in DPC 24 h after intratracheal injection of rhGM-CSF (1 or 5 μg)/placebo, 14C-acetate, and 3H-dipalmitoyl phosphatidylcholine to 12 litters of premature rabbits. Results have been normalized and presented as mean ± SEM. Mean total radioactivity in the total lung after the placebo treatment is adjusted to 100. Significant differences are indicated (★★p< 0.01; ★p< 0.05).

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In the term animals, rhGM-CSF given intratracheally decreased the recovery of 3H-DPC radioactivity in the whole lung 24 h after the isotope (Fig. 3). There was, additionally, a trend toward an increase in 14C-acetate incorporation into DPC in lamellar bodies (p= 0.06) and in BAL (p= 0.15). There were no differences in the distribution of DPC radioactivity between the two fractions of BAL.

Figure 3
figure 3

Distribution of radioactivity in DPC 24 h after intratracheal injection of rhGM-CSF (2 or 10 μg)/placebo, 14C-acetate, and 3H-dipalmitoyl phosphatidylcholine to 11 litters of 3-d-old term rabbits. Results have been presented as mean ± SEM. Mean total radioactivity of the total lung after the placebo treatment is adjusted to 100%. Significant difference is indicated (★p< 0.05).

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In both preterm and term animals, the rhGM-CSF that was given intraperitoneally had no detectable effect on the incorporation of the isotopes into DPC (data not shown).

Rate of incorporation of radioactivity into lung DCP.

Table 2 shows the pool sizes and specific radioactivities of lung DPC 1, 6, 24, and 48 h after the intratracheal GM-CSF in the premature lung. The pool sizes of lamellar bodies and BAL increased first 24 h after GM-CSF. There were no differences in the specific radioactivities of 3H-DPC or 14C-DPC.

Table 2 Pool sizes and specific activities of DPC in the lung of premature rabbits after intratracheal injection of 5 μg of rhGM-CSF or placebo, 14C-acetate, and 3H-dipalmitoyl phosphatidylcholine Results are expressed as mean ± SEM (n = 5; * n = 4). † Significant difference (p < 0.05) compared with the placebo-treated animals of the same age (ANOVA).
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Table 3 shows the pool sizes and specific radioactivities of DPC in the term lung. No changes in the pool sizes of DPC were evident. However, 48 h after intratracheal GM-CSF, the specific activities of 3H-DPC and 14C-DPC were decreased.

Table 3 Pool sizes and specific activities of DPC in the lung of term rabbits after intratracheal injection of 10 μg of rhGM-CSF or placebo, 14C-acetate, and 3H-dipalmitoyl phosphatidylcholine Results are expressed as mean ± SEM (n = 5). * Significant difference (p < 0.05) compared with the placebo-treated animals of the same age.
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To determine whether rhGM-CSF affects the reutilization of 3H that was attached to palmitate moiety in the 2-position of dipalmitoyl phosphatidylcholine, surfactant DPC was analyzed for the distribution of 3H radioactivity at 24 h. In the premature lung, 5 μg of intratracheal rhGM-CSF had no effect on the distribution of the radioactivity (fatty acid in 2-position/total radioactivity of DPC: GM-CSF 98.5 ± 2.4%; placebo 98.8 ± 2.5%). In the lungs from term animals, 10 μg of intratracheal rhGM-CSF caused only a nonsignificant trend toward a decreased distribution of 3H radioactivity in the 2-position of DPC (radioactivity in 2-position/total radioactivity of DPC: GM-CSF 94.6 ± 2.0%, controls 98.3 ± 1.7%).

Expression of SP-B.

Intratracheal rhGM-CSF did not affect the content of SP-B mRNA in the premature lung 24 h later (5 μg of GM-CSF, 114 ± 12%, n= 7; placebo, 100%, n= 7). There was no difference in the degree of SP-B immunostaining between the GM-CSF– (5 μg) and placebo-treated animals (data not shown). However, intratracheal GM-CSF increased the amount of SP-B in cell-free BAL (GM-CSF, 3.2 ± 0.4 μg; placebo, 1.5 ± 0.3 μg, p< 0.05).

Lung cells.

The cells recovered by BAL, the total lung cells (total lung DNA), and the number of type II alveolar cells (number of SP-B–positive alveolar epithelial cells microscopic field and per total alveolar epithelial cells) were counted 24 h after administration of intratracheal rhGM-CSF or placebo, as described in “Methods.” GM-CSF had no effect on the number of lung cells in preterm or term animals.

DISCUSSION

In the premature lung, pulmonary clearance rates of surfactant components are slow (23, 24). In addition, the synthesis, intracellular transport, and secretion rates of DPC are low, resulting in deficient pool sizes of alveolar surfactant (25). On the other hand, in term newborn, the pool size of surfactant phospholipid, expressed on the body weight basis, is approximately 1 order of magnitude higher than in adult (10). The cause of the high surfactant phospholipid pool at term is due to a very slow clearance and catabolism rate and efficient reutilization rate rather than to a high phospholipid synthesis rate compared with adult. Similar to term newborns, GM-CSF knockout adult mice have an excessive alveolar pool of surfactant. This is due to a virtually absent clearance rate rather than to active synthesis of surfactant phospholipid (5). The surfactant system of term newborn superficially resembles that of GM-CSF–deficient adults, raising the possibility that GM-CSF, which is known to be developmentally regulated in lung effluent (13), may affect the turnover of the surfactant during the perinatal period.

According to present results, intratracheal GM-CSF increased the surfactant DPC within 24 h in premature rabbits The effects of GM-CSF both on the quantity and the incorporation of acetate into surfactant DPC were detectable after 6 h. In premature rabbits, intratracheal GM-CSF did not change the clearance rate of DPC from the whole lung, which was very slow regardless of treatment. GM-CSF did not decrease the clearance of DPC from the alveolar space, either. Therefore, GM-CSF was likely to increase the formation of surfactant DPC. The increase in surfactant DPC during the 24 h was not associated with an increase in the specific activity of DPC from 14C-acetate. Acetate pulse labels DPC (present study) (26). The rate of DPC synthesis cannot be quantified using acetate as the precursor without knowledge of the specific activity of the precursor pool. Therefore, present results do not exclude the possibility that GM-CSF increased the synthesis of surfactant DPC in the premature. However, an increase in the pulmonary synthesis of DPC does not solely explain the increase in surfactant DPC.

In premature lung after intratracheal rhGM-CSF, there was a significant increase in distribution of DPC toward the surfactant fractions (i.e. intracellular lamellar bodies and BAL combined). Intratracheal GM-CSF may thus improve the pathways that maintain DPC in the surfactant fractions. They may include both the intracellular transport of DPC to lamellar bodies after de novo synthesis and the transport of alveolar surfactant phospholipid (3H-dipalmitoyl phosphatidylcholine) to intracellular lamellar bodies (reutilization pathway) (18, 27). There was no significant change in the distribution of DPC within the surfactant fractions (intracellular lamellar bodies, total BAL, large aggregate fraction of BAL). Further studies are required to define the GM-CSF–sensitive pathways that promote the accumulation of surfactant phospholipid after premature birth.

Overexpression of GM-CSF in type II alveolar cells in adult mice causes hyperplasia of alveolar cells (8). The present results do not exclude the possibility that GM-CSF increases the number of alveolar cells in the newborn, However, in the present study, 24 h after rhGM-CSF, there was no acute increase in the number of type II cells, lavageable alveolar macrophages, or in the amount of total lung DNA. Thus, the increase in surfactant DPC in the premature lung was not associated with an increase in the number of alveolar cells. Intratracheal GM-CSF increased neither SP-B mRNA nor SP-B immunostaining of type II alveolar cells. However, the quantity of SP-B in BAL was increased. This raises the possibility that GM-CSF affects the intracellular transport of both surfactant phospholipid and SP-B in the premature lung.

In rabbits after term birth, rhGM-CSF increased the pulmonary clearance of intratracheal dipalmitoyl phosphatidylcholine. Although GM-CSF acutely increased the 14C-acetate labeling of surfactant DPC, the pool sizes of surfactant DPC were not affected, suggesting that GM-CSF increased both the formation and the clearance rates of surfactant phospholipids. The distribution of DPC between the surfactant and nonsurfactant fractions was unaffected, too. These findings are consistent with previous evidence showing that overexpression of GM-CSF in type II alveolar cells of adult mice increased the turnover rate of surfactant phospholipids, whereas the alveolar phospholipid pool was not affected (9).

RhGM-CSF given intraperitoneally had no effect on surfactant phospholipids. Instead, the cytokine increased blood eosinophil and granulocyte counts, indicating the biologic activity of rhGM-CSF (28, 29). Peripheral white blood cell counts were not affected after intratracheal rhGM-CSF. The different effects of systemic and intratracheal GM-CSF are likely to be due to deficient availability of the systemic cytokine to alveolar cells and to deficient systemic bioavailability of the intratracheal cytokine. This supports the possibility that GM-CSF, found in the airways (15, 16) and in alveolar macrophages (27), has specific local effects on the alveolar cells.

We have shown for the first time that rhGM-CSF given to the airways accelerates the spontaneous increase in surfactant phospholipid pool soon after premature birth. In respiratory distress syndrome shortly after birth, the lung effluent tends to have a lower concentration of GM-CSF than the controls of same gestation (13), consistent with the possibility that this cytokine is involved in regulation of surfactant pool size in the premature lung. Exogenous GM-CSF given after term birth stimulated the pulmonary clearance of surfactant DPC without affecting the pool sizes of surfactant DPC. These data are consistent with the possibility that GM-CSF has multiple age-dependent effects on metabolism of the surfactant system. GM-CSF profoundly affects the function of alveolar macrophages (2, 6, 27). Similarly, the surfactant components modify the microbicidal function of alveolar macrophages (30). Very high levels of proinflammatory cytokines, including GM-CSF, are associated with inflammatory lung damage in infants developing chronic lung disease (13, 31). Intravenous rhGM-CSF given to very low birth weight neonates may reduce the incidence of nosocomial infection (32). The ontogeny and the roles of GM-CSF in alveoli remain to be further defined before the intrapulmonary cytokine therapy may be further considered.