Introduction

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The pulmonary surfactant complex in the alveolar lining prevents atelectasis, contributes toward a decrease in alveolar edema and small airway closure, and is involved in pulmonary host defense. Besides lipids, the complex contains specific surfactant proteins (SPs) that are bound to surfactant aggregates, including SP-A, SP-B, and SP-C (1, 2). SP-A, the most abundant SP, a multimeric collagenous glycoprotein with a unit molecular weight (MW) of 28 to 36 kD, contains both carbohydrate- and phospholipid-binding domains. Apart from conferring the unique structural organization of highly surface-active tubular myelin in the presence of calcium, phospholipids and SP-B (1), SP-A affects the secretion and clearance of the surfactant complex in vitro (3). SP-A binds to specific microbes and enhances the phagocytosis and intracellular killing by alveolar macrophages (4, 5). The mature form of SP-B is a hydrophobic protein with apparent MW of 5 to 8 kD, containing intermittent cationic amino acids. SP-B, which is required both for the unique surface properties of the surfactant complex and for the secretory function of type II cells, is essential for postnatal survival (2). The mature SP-C is a uniquely hydrophobic alveolar proteolipid that contains 36 amino acids. It enhances the surface properties of surfactant phospholipids (6). All three SPs undergo specific post-translational modifications and metabolism.

Deficiency or altered function of the pulmonary surfactant is associated with several diseases. The principal cause of respiratory distress syndrome in the newborn (RDS) is a surfactant deficiency due to insufficient differentiation ("immaturity") of type II alveolar cells (7). Surfactant dysfunction and deficiency of specific SPs have been associated with chronic respiratory failure originating from the neonatal period, called bronchopulmonary dysplasia (BPD) (8, 9). Abnormalities in surfactant composition and function are also present in the acute respiratory distress syndrome secondary to severe lung injury (ARDS) due to sepsis, aspiration, shock, or other insult (10, 11).

High interleukin (IL)-1 is associated with serious respiratory morbidity (12). Administration of IL-1 causes lung inflammation and edematous lung injury that resembles changes in the lungs of patients with ARDS. On the other hand, IL-1 pretreatment confers a tolerance to oxidative lung injury and to ischemia-reperfusion insult (13). The production of IL-1 and other proinflammatory cytokines is increased in monocytes/macrophages and other cells in response to microbial toxins, inflammatory agents, and complement and clotting components. The IL-1 gene family is composed of IL-1, IL-1, and IL-1 receptor antagonist that bind to the IL-1 receptors. IL-1 and IL- have very similar effects. For example, they activate nuclear factors destined to regulate a number of genes, including those involved in the synthesis of prostaglandins, proteases, and cytokines in a variety of cells (14, 15).

Spontaneous premature birth caused by intra-amniotic infection (16) is associated with increased concentrations of IL-1 and IL-1 and other cytokines in the amniotic fluid (17) and in the airways after birth (18). In premature infants born due to intrauterine infections, the incidence of RDS is low. On the other hand, the same population of premature infants shows an increased incidence of BPD (19, 20). IL-1 upregulates the expression of SPs in immature fetal lung. When given intra-amniotically, IL-1 increases SP-A and SP-B messenger RNA (mRNA) and decreases the severity of RDS after premature birth (21). In contrast, high activities of IL-1 and other proinflammatory cytokines in airway specimens are associated with surfactant dysfunction (24) and severe lung injury after birth (20).

We hypothesized that the influence of an inflammatory cytokine, IL-1, on the expression of SPs is determined by the degree of differentiation of the surfactant system or by the birth process. In an attempt to better understand this relationship, we studied the IL-1 responsiveness of the SPs in lung explants cultured in serum-free medium. According to the present results, the IL-1-induced increase in SPs in the immature lung is in striking contrast with the IL-1-induced suppression of SP mRNA in transitional and mature lung.

	Materials and Methods

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The Animal Research Committee of the University of Oulu approved the protocol. Timed pregnant New Zealand white rabbits were used. The mating date was defined as Day 0 of gestation. On Day 19, 22, 27, or 30 (± 1 h) of pregnancy (term: 30 to 31 d), hysterotomy was performed, the pups were killed with an intraperitoneal injection of euthanol, and the abdominal aorta was severed. The lungs were recovered under sterile conditions. One-day-old newborns were killed and the lungs were recovered as well.

Recombinant human (rh) IL-1 (endotoxin content < 0.01% of dry weight) was a generous gift of Dr. R. Chizzonite (Hoffmann LaRoche, Nutley, NJ). rhIL-1 was purchased from Genzyme Co. (Cambridge, MA). Both rhIL-1 and rhIL-1 have been shown to be biologically active in the rabbit (25, 26).

Organ Culture

After removal of the large airways, lung tissue was cut into pieces of approximately 2 mm³ using sterile scissors. Five such pieces of lung tissue were placed on a filter paper that was on a metallic grid in a culture dish. The tissue pieces were partly in contact with the atmosphere and partly with the culture medium. The medium used was serum-free Waymouth's MD 705/1 (GIBCO, Paisley, Scotland) containing penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml). The tissue was maintained in culture at 37°C in a humidified atmosphere of 5% CO₂ and 95% air for 20 h in the presence of rhIL-1 or vehicle, unless otherwise indicated. After the culture, the explants were harvested, frozen in liquid nitrogen, and stored at 70°C until processed for mRNA analysis.

Analysis of mRNA

Approximately 15 mg of the explant tissue was pulverized with a pellet pestle in a microcentrifuge tube and homogenized in 0.5 ml RNA STAT-60 solution (Tel-Test, Inc., Friendswood, TX). Isolation was carried out as indicated by the manufacturer. The RNA pellet was dried, then resuspended in diethyl pyrocarbonate-treated water and stored at 70°C. RNA was quantitated by determining absorbance at 260 nm. A total of 10 µg of RNA was size-separated in a 1% agarose, 6.5% formaldehyde gel, and transferred by capillary blotting onto a nylon hybridization membrane (BIODYNE B; Pall Gelman Sciences, Northampton, UK). After the transfer, the membrane was baked at 80°C for 2 h. The membranes were hybridized using probes of 1.9-kb rabbit SP-A complementary DNA (cDNA), 0.6-kb rabbit SP-B cDNA, or 0.5-kb rabbit SP-C cDNA (21). The purified inserts were labeled with ³²P using the Oligolabeling Kit from Pharmacia (Uppsala, Sweden). To compensate for gel-loading artifacts, the membranes were probed with [³²P]-radiolabeled 28S RNA-specific cDNA clone. The bands were quantitated using a PhosphorImager.

Western Blot Analysis of SP-A and SP-B

For analysis of SP-A, the culture medium was concentrated 50-fold with a Microcon 10 (Amicon, Inc., Beverly, MA). After 20 µl of sodium dodecyl sulfate (SDS) loading buffer (Bio-Rad, San Diego, CA) had been added to 10 µl of the concentrate, the mixture was boiled for 5 min and the samples were loaded on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel under reducing conditions. For analysis of SP-B, proteins from the explants were isolated essentially according to Clark and colleagues (27). Briefly, the lung explants were homogenized in 10 mM Tris (pH 7.5), 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin A, aprotinin, leupeptin, and chymostatin, followed by centrifugation at 140 × g for 10 min (4°C). The protein content of the supernatant was quantitated by Bio-Rad DC Protein Assay. Altogether, 200 µg of proteins from supernatant were centrifuged at 22,500 × g for 30 min (4°C). The resulting pellet was mixed in 10 µl of loading buffer, boiled for 5 min, and loaded on a 15% SDS-Tricine PAGE gel under nonreducing conditions. The gels were electrotransferred onto a Protran BA85 (Schleicher & Schuell, Dassel, Germany) nitrocellulose filter. Blocking and antibody incubations were made according to ECL-Plus (Amersham, Buckinghamshire, UK). A 1:1,000 dilution of guinea-pig antirabbit SP-A antibody (a kind gift from Dr. J. M. Snyder [University of Iowa Hospitals, and Clinics, Iowa City, IA]) and a 1:10,000 dilution of mouse antiporcine SP-B antibody (a kind gift from Dr. Y. Suzuki [Department of Molecular Pathology, Kyoto University, Kyoto, Japan]) were used as the primary antibodies. The secondary antibody consisted of a 1:3,000 dilution of horseradish peroxidase-conjugated goat antimouse immunoglobulin G (Bio-Rad). The bound antibody was visualized using ECL-Plus Detection kit, and the bands were analyzed by video imaging and densitometry.

Immunohistochemistry of SP-A and SP-B

The explants were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) overnight and embedded in paraffin. The 5-µm sections were cut on Super Frost Plus microscopic slides (Menzel-Gläser, Braunschweig, Germany) for immunodetection. For immunostaining of SP-A, deparaffinized sections were incubated for 10 min in boiling 10 mM sodium citrate (pH 6.0), washed in PBS, and treated with 3% H₂O₂ in methanol for 15 min at room temperature. After washing with PBS, the sections were incubated with antirabbit SP-A antibody at a dilution of 1:1,000 for 1 h. The subsequent steps were made using the broad-spectrum Histostain-Plus kit (Zymed Laboratories, San Francisco, CA). Detection was made with Liquid DAB substrate (Zymed), and the sections were counterstained with hematoxylin.

The method of immunostaining of explants for SP-B has been described previously (21).

Other Analytical Methods

Lactate dehydrogenase (LDH) activity and the concentration of total phospholipid (21) in the culture medium were analyzed using standard methods.

Expression of the Results and Statistics

The SP-A, SP-B, and SP-C mRNA levels are presented as means ± standard error of the mean (SEM) for convenience. Unless otherwise indicated, the mRNA in the presence of IL-1 was expressed on the basis of the mRNA present in the vehicle-treated controls. Statistical significance was analyzed using Student's t test. When indicated, one-way analysis of variance followed by post hoc analysis using the Fisher test was performed. The differences in the rates of degradation of mRNA were analyzed using multiple regression analysis. A P value of < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Age Dependence of the Effect of IL-1 on the Expression of mRNA

rhIL-1 at 57 or 570 ng/ml for 20 h increased the expression of SP-A and SP-B mRNA in lung explants from 19-d-old fetal rabbits. In contrast, rhIL-1 in lung explants from term rabbit fetuses suppressed SP-C and SP-B mRNA within 20 h. Figure 1 shows the representative Northern blots for SPs and 28S RNA. Figure 2 shows the quantitative effects of 570 ng/ml rhIL-1 on SP mRNA in lung explants from a number of litters. Explants from 19-, 22-, 27-, and 30-d-old fetuses, and 1-d-old term newborns were studied. For each gestational age, the results were expressed on the basis of 28S RNA. In the explants from Day 19 and 22 fetuses, IL-1 increased SP-A and -B mRNA. However, in the explants from transitional and mature lungs (fetal Days 27 and 30, and 1 d after birth) IL-1 suppressed SP-C and -B. On Day 19, rhIL-1 increased SP-C mRNA by 64 ± 16%. In explants from 27- and 30-d-old fetuses and from 1-d-old newborns, IL-1 tended to decrease SP-A by 14 ± 8% (P = 0.09), 12 ± 6% (P = 0.05), and 15 ± 4% (P < 0.05), respectively. rhIL-1 and rhIL-1 had very similar effects on the expression of SP mRNA (data not shown).

View larger version (78K): [in this window] [in a new window]   Figure 1.   Effect of IL-1 on SP mRNA and 28S RNA expression. Representative Northern blots show the expression in explants cultured for 20 h in the presence of vehicle (C) or 57 or 570 ng/ml rhIL-1. (A) Explants from 19-d-old fetuses. (B) Explants from 30-d-old fetuses.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Effect of IL-1 on the expression of SP-A, -B, and -C mRNA at 19, 22, 27, and 30 d of gestation, and 1-d-old newborn (nb). The bars show the SP expression levels in explants 20 h after the addition of 570 ng/ml rhIL-1, relative to the expression in the presence of vehicle, shown as dashed line. Gel-loading artifacts have been corrected. Data are means ± SEM and the number of independent experiments is shown for each bar.

Expression of SP-A and SP-B Proteins

In lung explants from 19-d-old fetuses, the increase in SP-A mRNA within 20 h after the addition of rhIL-1 was associated with an induction in the immunoreactivity of SP-A protein in the epithelial lining of the future air spaces (Figure 3). The concentrate of the culture medium contained trace of SP-A immunoreactivity after IL-1. In explants from 22-d-old fetuses, an IL-1-induced increase in the SP-A immunoreactivity was also evident. Moreover, 20 h after the addition of IL-1 to the explants from 22-d-old fetuses, SP-A was detected in the concentrate of the culture medium, whereas the control medium contained traces of immunoreactivity (Figure 3). There was no detectable increase in total phospholipid (< 1 nmol/ml), or LDH (14 to 18 U/ml) in the culture medium after the addition of rhIL-1.

View larger version (165K): [in this window] [in a new window]   Figure 3.   Detection of SP-A in lung explants and in the culture medium. For immunohistochemistry the explants from 19-d-old fetal rabbits were cultured during 20 h in the presence of vehicle (A and B), 570 ng/ml rhIL-1 (C and D), or 57 ng/ml rhIL-1 (E). The sections shown in A, C, and E were incubated with antirabbit SP-A antibody, and those in B and D without the primary antibody. Immunopositive cells are detected in the periphery of epithelium in C and E. (F ) A Western blot of culture medium of lung explants from 22-d-old fetuses, exposed to vehicle (C), 570 ng/ml rhIL-1 (IL-H), or 57 ng/ml rhIL-1 (IL-L) for 20 h.

Although IL-1 tended to increase SP-B immunoreactivity in the epithelium of the future air spaces in explants from 19- or 22-d-old fetuses, there was no detectable increase in SP-B immunoreactivity in the culture medium. In contrast, in lung explants from 27-d fetuses, IL-1 added to the culture medium decreased SP-B protein in explants by 45 to 60% (Figure 4). A similar trend toward a decrease of SP-B immunoreactivity in the epithelium peripheral air spaces was evident (Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 4.   Detection of SP-B on Western blots. Lung explants from 27-d fetuses were cultured in the presence of vehicle (C), 570 ng/ml rhIL-1 (IL-H), or 57 ng/ml rhIL-1 (IL-L) for 20 h. IL-1 suppresses the expression of SP-B.

View larger version (119K): [in this window] [in a new window]   Figure 5.   Detection of SP-B in lung explants. For immunohistochemistry the explants from 27-d fetal rabbits were cultured for 20 h in the presence of vehicle (A and B) or 570 ng/ml rhIL-1 (C and D). The sections shown in A and C were incubated with monoclonal SP-B antibody; those in B and D were without the primary antibody. Strong signals are detected in the epithelium in vehicle-treated explants, whereas the signals appear less pronounced in explants exposed to IL-1. Bar: 50 µm.

Concentration and Time of Exposure to IL-1

The effect of IL-1 concentrations ranging from 5.7 to 2,850 ng/ml was studied in explants from three different age groups (Figure 6). On fetal Days 19 and 22, the upregulation of SP-A and -B mRNA was constant between 57 and 2,850 ng/ml. On Days 22 (SP-C) and 30 (SP-C and -B), the mRNA expression of the hydrophobic SPs decreased as a function of the concentration of IL-1.

View larger version (16K): [in this window] [in a new window]   Figure 6.   Effect of rhIL-1 concentration on SP mRNA expression in lung explants from 19 (a), 22 (b), and 30 (c) d of gestation. The explants were exposed to 5.7, 57, 570, and 2,850 ng/ml of IL-1 for 20 h. The results, corrected for gel-loading artifacts, are presented as means ± SEM (n = 4). The dashed lines show the line of identity.

The time in culture and the time of exposure to IL-1 affected the mRNA contents. However, there was no detectable change in the tissue content of 28S RNA. In 22-d fetal lung, the effect of IL-1 on SP-A became evident within 4 to 6 h (Figure 7). The upregulation of SP-A by IL-1 decreased when the incubation time exceeded 20 h. This was principally due to the increase in SP-A mRNA in vehicle-treated lung (28) (data not shown). In the explants from 27-d fetal lung, only a transient increase in SP-A mRNA was evident between 4 and 12 h after the addition of IL-1 (Figure 8). In the explants from 30-d fetal lung, the downregulation of SP-B mRNA by IL-1 was first evident after 12 h (Figure 7).

View larger version (13K): [in this window] [in a new window]   Figure 7.   Effect of time of rhIL-1 exposure on the expression of SP-A and SP-B mRNA. (a) SP-A expression in explants from 22-d fetal lung explants; (b) SP-B expression in 30-d fetal explants. Solid symbols: 570 ng/ml rhIL-1; open symbols: vehicle. The results, corrected for gel-loading artifacts, are presented as the means ± SEM. The expression at 0 h is given the value of 1.

View larger version (22K): [in this window] [in a new window]   Figure 8.   Effects of actinomycin D and cycloheximide on SP-A and -B mRNA expression. Explants from 27-d fetal lung were cultured in the presence (solid symbols) or absence (open symbols) of 570 ng/ml rhIL-1. IL-1 was added at 0 h, actinomycin D and cycloheximide at 4 h. Squares: IL-1 or vehicle; inverted triangles: IL-1 or vehicle with actinomycin D (5 or 50 µg/ml); circles: IL-1 or vehicle with actinomycin D (5 or 50 µg/ml) and cycloheximide (2.5 µg/ml). The results, corrected for gel-loading artifacts, are presented as means ± SEM. The expression at 0 h is given the value of 1.

Stability of mRNA

To study the stability of mRNA, explants exposed to either IL-1 or vehicle were cultured in the presence of actinomycin D. The two different concentrations of actinomycin D (5 or 50 µg/ml) gave identical results. In immature lung (Day 22), IL-1 did not affect the actinomycin D- induced rate of SP-A mRNA degradation (data not shown). Due to the low SP-B mRNA content, the degradation of mRNA could not be studied. In the lungs from 27-d-old fetuses, IL-1 increased the degradation of SP-B mRNA in the presence of actinomycin D or in the presence of both actinomycin D and cycloheximide (Figure 8). IL-1 increased the degradation of SP-A when both actinomycin D and cycloheximide were present. However, in the presence of actinomycin D only, IL-1 had no effect on the degradation of SP-A mRNA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates for the first time that the expression of lung SPs (SP-A, -B, and -C) controlled by the primary proinflammatory cytokine IL-1 is dependent on the degree of differentiation. In the glandular and canalicular stages of fetal rabbit lung development (19 to 22 d from conception), IL-1 upregulated SP mRNA, whereas in the saccular and alveolar stages (27 to 30 postconceptional d and 1 d after birth), it suppressed mRNA. The IL-1-induced changes in SP-A and SP-B protein levels were similar to those of mRNA. In intrauterine infections characterized by high intra-amniotic cytokines (16), the incidence of RDS is low (19). Intra-amniotic IL-1 accelerates surfactant maturity (21). On the contrary, high proinflammatory cytokines in the airways associate with BPD and surfactant dysfunction (8, 20). The effective concentrations of IL-1 in vitro (Figure 6) were similar to those present in transcellular spaces in vivo in accelerated lung maturity (16, 17, 19) and in lung injury (18, 19, 20). Thus, the present in vitro findings may explain the contrasting associations between inflammatory cytokines and diseases during the perinatal period.

The present experimental setup has certain limitations. In lung explants consisting of a number of different cell types, the availability of oxygen, nutrients, growth factors, and drugs to individual cells is variable. According to immunohistochemistry, IL-1 caused homogenous changes in the expression of SP-A and SP-B in alveolar epithelium. Studies using type II alveolar cells in culture have limitations as well, including a lack of interaction with other cells, spontaneous dedifferentiation of primary cultures, and transformation of continuous cell lines. Short culture periods were used to avoid changes induced during culture in vitro. In the preliminary study of lung explants from 22-d fetuses, only SP-A was induced (22). This was due to a decrease in the inducibility by IL-1 during prolonged culture of immature lung explants (28). The pattern of SP mRNA induced by IL-1 either in vitro or in immature lungs after intra-amniotic IL-1 in vivo (21) was similar. On the basis of the similarity between the findings in vivo and in vitro, we propose that the upregulation of SPs after intra-amniotic IL-1 (21) is due to the direct effect of the cytokine on the lung. Apart from IL-1, other inflammatory mediators are likely to affect the lung maturity in intrauterine infections (29). The impact of the IL-1 system on the spectrum of pulmonary outcomes of fetuses prematurely born due to intrauterine infections (19, 30) remains to be elucidated.

In explants from immature fetal rabbit, IL-1 increased both SP-A mRNA and immunoreactive SP-A in epithelial cells. As shown by the Western blot analysis, SP-A increased in the concentrates of the culture medium within 20 h. The lack of increase of LDH does not support the possibility that intracellular proteins were released into the medium as a result of IL-1-induced cell damage. In contrast to SP-A, IL-1 caused no detectable increase in phospholipids or SP-B in the culture medium. This was expected because in premature fetal lung in vivo, the secretory pathway of surfactant phospholipids from endoplasmic reticulum via intracellular lamellar bodies to the alveolar space takes place first within 24 to 48 h (31). Unlike surfactant phospholipids, SP-A is rapidly secreted into the alveolar space without involvement of lamellar bodies (32). On the basis of present findings, SP-A induced by IL-1 in the immature lung is rapidly secreted from the alveolar cells.

High IL-1 activity has been associated with serious inflammatory lung damage, including BPD (18) and ARDS (12). In both conditions, surfactant dysfunction and decrease in alveolar SP-A and SP-B (8) have been documented. In the present study, IL-1 decreased the expression of SPs during the saccular and alveolar stages. A modest decrease in the expression of SP-B, similar to that found here, caused some pulmonary dysfunction in newborn mice (27). The suppression of SP by tumor necrosis factor (TNF)- (33) and the additive suppression of SP by IL-1 and TNF- (34) further imply that the proinflammatory cytokines decrease surfactant components in severe inflammatory lung injury.

Specific surfactant genes showed individual patterns of IL-1 responsiveness. In immature lung on Day 19, IL-1 increased SP-A and SP-B mRNA severalfold, whereas SP-C increased modestly. On the other hand, in lung explants from more advanced gestation (Days 27 to 30, and first postnatal day), IL-1 decreased SP-C and -B mRNA and caused a barely detectable decrease in SP-A mRNA (Figure 2). Despite remarkable differences in IL-1 responsiveness, all three SPs demonstrated a similar age-dependent switch in the expression of mRNA.

The IL-1-induced increase in SP-A mRNA was detectable within 4 to 6 h, suggesting the increase in the transcription rate. On the other hand, the suppression of SP-B mRNA became evident first after 12 h, and was concentration-dependent (Figures 6 and 7). In explants from 27-d fetal lung, between 8 and 12 h after the addition of actinomycin D, IL-1 accelerated the degradation of SP-B mRNA. This suggests that the downregulation of SP-B mRNA by IL-1 is due at least in part to the decrease in the stability of mRNA. In contrast, IL-1 had no effect on actinomycin D-induced degradation of SP-A mRNA (Figure 8). The degradation of SP-A mRNA became dependent on IL-1 first in the presence of cycloheximide. This supports the possibility that the stability of mRNA was dependent on labile protein(s) (35).

Several mediators that are known to interact with IL-1 also influence the expression of SPs. These include transforming growth factor (TGF)- and TNF- that suppress, and glucocorticoid, cyclic adenosine monophosphate, epidermal growth factor, and interferon- that increase the expression of specific SPs (29). TGF- acts as a biologic switch, antagonizing or modifying biologic action of other growth factors and cytokines, including IL-1 (36). TNF- that, together with IL-1, has additive or synergistic effects on several genes (15), suppressed SP-A and -B gene expression in pulmonary adenocarcinoma cell lines in culture (37). The glucocorticoid-induced increase or decrease in the expression of SP-B has been shown to be dependent on the concentration, the time of exposure in vitro (41, 42), and the stage of lung development (29, 43, 44).

Besides the SPs, IL-1 influences the expression of a number of specific proteins, and promotes differentiation of hematopoietic cells and keratinocytes (14, 45). By separate signaling pathways, IL-1 may activate nuclear transcription factors, nuclear factor-B and c-Jun/activating protein (AP)-1 (46) that subsequently drive the transcriptional regulation of many genes (15, 47). Putative binding sites for AP-1, a collective term referring to dimeric transcription factors (48), have been identified among the 5'-flanking sequences of SP-A, SP-B, and SP-C genes. Roles of IL-1-inducible transcription factors in the regulation of the expression of SP genes are unknown at present.

We have identified a single agonist that affects the expression of specific surfactant genes in both directions. The direction of gene expression was shown to be associated with conceptional age and stage of prenatal differentiation. That SP-B is critically required for the survival of the neonate, and that SP-A has distinct roles in the innate immunity, give an added significance to this finding. The identity of the selectors that determine the direction of the IL-1 effect on the expression of SPs remains to be identified.