Regulation of surfactant proteins by LPS and proinflammatory cytokines in fetal and newborn lung

Outi Väyrynen, Virpi Glumoff, and Mikko Hallman

Department of Pediatrics and Biocenter Oulu, University of Oulu, Oulu FIN-90014, Finland


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Intra-amniotic lipopolysaccharide (LPS) and cytokines may decrease respiratory distress syndrome (RDS) and increase chronic lung disease in the newborn. The aim was to identify the primary inflammatory mediators regulating the expression of surfactant proteins (SP) in explants from immature (22-day-old fetus) and mature (30-day term fetus and 2-day-old newborn) rabbits. In immature lung, interleukin (IL)-1alpha and IL-1beta upregulated the expression of SP-A and SP-B. These effects of IL-1 were diminished, and SP-C mRNA was suppressed additively in the presence of tumor necrosis factor (TNF)-alpha and either LPS or interferon (IFN)-gamma . LPS, TNF-alpha , or IFN-gamma had no effect alone. In explants from the term fetus and the newborn, LPS, IL-1alpha , and TNF-alpha additively suppressed the SPs. LPS acutely induced IL-1alpha in alveolar macrophages in mature lung but not in the immature lung. IFN-gamma that generally has low expression in intrauterine infection decreased the age dependence of the other agonists' effects on SPs. The present study serves to explain the variation of the pulmonary outcome after an inflammatory insult. We propose that IL-1 from extrapulmonary sources induces the SPs in premature lung and is responsible for the decreased risk of RDS in intra-amniotic infection.

lung development; respiratory distress syndrome; chronic lung disease; acute respiratory distress syndrome; rabbit; lipopolysaccharide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DEFICIENCY IN PULMONARY SURFACTANT is the main cause of respiratory distress syndrome (RDS) in the newborn (11). Apart from specific phospholipids, the complex consists of surfactant-specific proteins that are important, if not essential, for its function (22). Surfactant protein (SP)-A binds to surfactant phospholipids and thereby improves surface activity in vitro (4, 48). By binding to specific microbes and alveolar macrophages, it also facilitates the phagocytosis of these microbes (13). The functions of SP-B relate to intracellular processing of the surfactant components, e.g., normal processing of SP-C (58), and to the surface tension-reducing function of the complex (42, 61). SP-C has important roles in the formation and maintenance of the surfactant monolayer (34).

The variety of hormones, growth factors, cytokines, and other agents influences the differentiation and metabolism of the surfactant complex. Glucocorticoid is extensively studied for its roles in the regulation of SPs (39). The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha , which are induced in macrophages as a result of exposure to microbial products, influence the expression of SPs. TNF-alpha inhibits SP-A and SP-B and decreases SP-A and SP-B mRNA in human pulmonary adenocarcinoma cells (60, 63). TNF-alpha also downregulates SP-C mRNA after intratracheal administration to adult mice and inhibits SP-C gene transcription in vitro (1). IL-1alpha enhances the expression of SP-A mRNA in fetal rabbit lung explants in vitro (14). The IL-1-induced effect on the expression of SP mRNA and proteins in vitro was dependent on lung maturity. In immature lung, IL-1alpha upregulated SP-A and SP-B, whereas in mature lung it downregulated SP-B and SP-C (19). Interferon (IFN)-gamma influenced SP-A content in human pulmonary adenocarcinoma cells (63) and SP-A expression in human fetal lung explants, whereas the expression of SP-B and SP-C was unaffected (2).

Intratracheal aerolization or injection of lipopolysaccharide (LPS) in adult rats increases the production of TNF-alpha (38, 55, 56) and IL-1 mRNA in the lung (55). IFN-gamma is induced by LPS in monocytes and lymphocytes (35), whereas IFN-gamma augments LPS-induced TNF-alpha production in monocyte/macrophages, including in alveolar macrophages (62, 64). On the other hand, IFN-gamma did not enhance LPS-induced upregulation of IL-1 mRNA (64). The interactions between IL-1 and TNF-alpha and their capacity to induce and suppress a variety of other cytokines (28, 51, 54) have been extensively studied and are known to possess synergistic or additive effects on many functions (15, 17).

Responsiveness to microbes has been shown to be deficient in pulmonary macrophages from an early age (49), which is a possible cause of an increased susceptibility to infections, neonatal pneumonia in particular. Infants born very premature because of chorioamnionitis show a decreased incidence of RDS and an increased incidence of chronic lung disease (CLD; see Ref. 59). On the other hand, in intra-amniotic infections, the concentrations of IL-1 (44) and TNF (45) are increased in amniotic fluid and in lung effluent after premature birth. In infants developing CLD, several proinflammatory cytokines and chemokines are increased in the airways, whereas the levels of certain anti-inflammatory cytokines may be decreased (31, 52).

In the present study, we hypothesized that the complex association between the antenatal placental inflammatory disease and the incidence of pulmonary disease in the newborn is in part because of differences in responsiveness to microbial toxins and cytokines between undifferentiated ("immature") and differentiated ("mature") pulmonary alveolar epithelium. We studied the age dependence of the LPS effect on the expression of SP mRNAs, SP, and IL-1alpha in lung explants. Second, we investigated the influence of the individual proinflammatory cytokines (IL-1alpha , TNF-alpha , and IFN-gamma ) on the expression of SPs. According to present results, the modifying effects of proinflammatory signals on the levels of SP expression were both developmentally regulated and reversible. Of the proinflammatory cytokines examined, IL-1 had the greatest effects on SPs in immature lung.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Experimental animals. The animals used were timed pregnant New Zealand White rabbits. The Animal Research Committee of the University of Oulu approved the protocol. The mating date was defined as day 0 of gestation. On days 22 (±1 h) or 30 (±1 h) of pregnancy (term 30-31 days), hysterotomy was performed on animals injected with medetomidine (0.3 mg/kg im) and ketamine (20 mg/kg im). The anesthetized fetuses and 2-day-old newborns were killed by cutting the cervical cord, and the does were killed by intravenous injection of pentobarbital sodium. The lungs were recovered under aseptic conditions. Altogether, 91 fetuses and 18 newborn animals from 30 litters were studied.

LPS and cytokines. The LPS used was LPS Escherichia coli serotype O55:B5 (Sigma-Aldrich). Recombinant human (rh) IL-1alpha , a generous gift of Dr. R. Chizzonite (Hoffmann-LaRoche, Nutley, NJ), has been shown to be biologically active in rabbits (33). The rhTNF-alpha (R&D Systems; see Ref. 43), rhIFN-gamma (Genzyme Diagnostics, Cambridge, MA; see Ref. 53), and rhIL-1beta (R&D Systems; see Ref. 33) used in the study are also biologically active in the rabbit.

Organ culture. The large airways were removed, and the lungs were cut into cubes of ~2 mm3 using sterile scissors. Five pieces were placed in a culture dish on a filter paper placed on a metallic grid. The tissue pieces were partly in contact with the atmosphere, partly with the culture medium. Waymouth's serum-free medium MD 705/1 (GIBCO, Paisley, Scotland) containing penicillin (100 U/ml), streptomycin (100 µg/ml), fungizone (0.25 µg/ml), and glutamine (2 mM) was used in a humidified atmosphere containing 5% CO2 and 95% air. The lung explants were cultured in the presence of LPS, IL-1alpha , TNF-alpha , IFN-gamma , different combinations of these compounds, or vehicle for 12 or 20 h at 37°C. These exposure times were chosen in preliminary experiments evaluating the influence of culture time (4-44 h). The maximum effect of cytokines on the expression of SPs was evident within 12-20 h of incubation time (Ref. 19 and unpublished results). The effect of LPS was also seen after 12 h and did not change after prolonged incubation (data not shown). Different concentrations were also used for IL-1alpha (19), LPS (10-1,000 ng/ml), and TNF-alpha (10-100 ng/ml; data not shown). The most informative data are shown here. After the culture, the explants were harvested in liquid nitrogen and stored at -70°C until used for mRNA or protein analysis. When indicated, the explants were fixed with 4% neutral formaldehyde for immunohistochemistry.

Analysis of mRNA. Total RNA was isolated, and SP-A, -B, and -C mRNAs were quantified using Northern blot analysis, as described previously (19).

Immunohistochemistry of IL-1alpha . The explants for immunohistochemistry were fixed with 4% formaldehyde in PBS overnight and embedded in paraffin. The 5-µm sections were cut on Super Frost Plus microscopic slides (Menzel-Gläser) for immunodetection. 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 H2O for 15 min at room temperature. After being washed with PBS, the sections were incubated with anti-rabbit IL-1alpha antibody (Endogen, Woburn, MA) at a dilution of 1:250 for 1 h. The following steps were accomplished using the Strept ABComplexes kit from Dako (Glostrup, Denmark). Detection was done with the Liquid DAB substrate of ZYMED Laboratories (San Francisco, CA), and the sections were counterstained with hematoxylin.

Western blot analysis of SP-B. Proteins from the explants were isolated essentially according to Clark et al. (10). The lung explants were homogenized in 10 mM Tris (pH 7.5), 0.25 M sucrose, 1 mM EDTA, 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). Quantitation of the protein content of the supernatant was done by the Bio-Rad DC Protein Assay. Altogether, 200 µg of proteins from the supernatant were centrifuged at 22,500 g for 30 min (4°C). The pellet containing 10 µg protein was suspended 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 to a Protran BA85 (Schleicher & Schuell, Dassel, Germany) nitrocellulose filter. Blocking and antibody incubations were made according to ECL-Plus (Amersham, Buckinghamshire, UK). A 1:10,000 dilution of mouse antiporcine SP-B antibody (a kind gift from Dr. Y. Suzuki, Kyoto University, Kyoto, Japan) and a 1:10,000 dilution of horseradish peroxidase-conjugated goat antimouse immunoglobulin G (Bio-Rad) served as primary and secondary antibodies, respectively. The bound antibody was visualized using the ECL-Plus Detection kit (Amersham), and the bands were analyzed by video imaging and densitometry.

Expression of the results and statistics. The mRNA and protein levels of SP-A, SP-B, and SP-C are presented as means ± SE for convenience. To compensate for gel-loading artifacts, the Northern blot membranes were probed with a 32P-radiolabeled 28S RNA-specific cDNA clone. mRNA or protein in the presence of IL-1alpha , TNF-alpha , LPS, or IFN-gamma was expressed on the basis of the mRNA or protein present in the vehicle-treated controls. Statistical significance was analyzed using Student's t-test for nonpaired data. When indicated, one-way ANOVA followed by post hoc analysis using the Fisher test was performed. A P value < 0.05 was considered significant.


    RESULTS
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INTRODUCTION
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DISCUSSION
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LPS and cytokine effects on SP mRNA. LPS at two different concentrations (100 ng/ml and 1 µg/ml) did not significantly affect the SP-A, -B, or -C expression in lung explants from 22-day-old fetal rabbits. In LPS-treated explants from 30-day-old fetal and 2-day-old newborn rabbits, SP-B and SP-C mRNAs were suppressed in a concentration-dependent manner. SP-A mRNA was suppressed only at 1 µg/ml (Fig. 1 and see Fig. 4).


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Fig. 1.   Effect of lipopolysaccharide (LPS; 100 ng/ml and 1 µg/ml) on surfactant protein (SP)-A, -B, and -C mRNA expression in 22 (n = 27-30)- and 30 (n = 7-13)-fetal-day lung explants and 2-day-old newborns (n = 13-14). Incubation time in the presence of agonist or vehicle was 12 h, and the bars show SP expression levels relative to the expression in the presence of vehicle (shown as line). Data are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

rhIL-1alpha at 57 ng/ml for 12 h increased the expression of SP-A and SP-B mRNA in lung explants from 22-day-old fetal rabbits. In immature lung, TNF-alpha (100 ng/ml) affected neither SP expression nor IL-1alpha -induced upregulation of SP-A and SP-B mRNA. In explants from 2-day-old newborn rabbits, IL-1alpha suppressed SP-B mRNA within 12 h (Fig. 2) and SP-C mRNA within 20 h (data not shown), whereas it had no detectable effect on SP-A expression. Similar to IL-1alpha , IL-1beta (25 ng/ml) increased the expression of SP-A in immature lung and suppressed the expression of SP-B in explants from newborn animals (data not shown). In lung explants from newborn rabbits, TNF-alpha suppressed the expression of all three SPs and potentiated the IL-1-induced suppression of SP-C mRNA. In near-term fetal rabbits, IL-1alpha at 57 ng/ml did not significantly downregulate any of these SPs, although TNF-alpha at 100 ng/ml suppressed SP-B mRNA. IL-1alpha and TNF-alpha combined decreased SP-A, SP-B, and SP-C mRNAs (Fig. 2 and see Fig. 4).


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Fig. 2.   Effects of interleukin (IL)-1alpha (57 ng/ml), tumor necrosis factor (TNF)-alpha (100 ng/ml), and the combination of these two cytokines on SP-A, -B, and -C mRNA expression in 22 (n = 21-25)- and 30 (n = 8)-day fetal lung explants and 2-day-old newborns (n = 5-11). Incubation time was 12 h, and the bars show SP expression levels relative to the expression in the presence of vehicle (shown as line). Data are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

IFN-gamma at 10 or 100 ng/ml had little, if any, detectable effect on SP expression at any age (Table 1). However, IFN-gamma added in combination with LPS (100 ng/ml) suppressed SP-B and SP-C mRNAs in explants from 22-day-old fetuses. In explants from 30-day-old fetal rabbits, IFN-gamma (100 ng/ml) tended to potentiate the LPS-induced downregulation of SP-B and SP-C mRNAs, and the effect on SP-C was significant when IFN-gamma was added together with LPS at 100 ng/ml (P = 0.02). In contrast, in explants from 2-day-old newborn rabbits, IFN-gamma (100 ng/ml) tended to decrease the suppressive effect of LPS (1 µg/ml) on SP expression.

                              
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Table 1.   Effect of IFN-gamma on expression of SP-A, SP-B, and SP-C

In explants from 22-day-old fetuses, the addition of IL-1alpha and TNF-alpha together with LPS had a different effect on SP mRNA expression compared with the cytokines (Fig. 2) or LPS (Fig. 1) alone. For SP-A mRNA, the IL-1alpha -induced upregulation persisted, whereas for SP-B the IL-1alpha -induced effect disappeared and SP-C became suppressed. In mature lung (30-day-old fetus and 2-day-old newborn), the combination (IL-1alpha  + TNF-alpha  + LPS) suppressed all three SPs, similar to the cytokines (Fig. 2) or LPS (Fig. 1) added alone. In explants from 30-day-old fetuses, IL-1, TNF, and LPS together tended to be more suppressive (SP-A: P = 0.15; SP-B: P = 0.46; SP-C: P = 0.059) than the cytokines or LPS alone. In immature lung, IFN-gamma added together with IL-1alpha and TNF-alpha abolished the IL-1-induced upregulation of SP-A and SP-B and induced the suppression of SP-C. In lungs from postnatal animals, on the other hand, IFN-gamma tended to decrease the IL-1- and TNF-induced suppression of SP-B (P = 0.21-0.27) and SP-C (P = 0.15-0.20) mRNA (Figs. 3 and 4).


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Fig. 3.   Effects of the combinations of LPS (100 ng/ml or 1 µg/ml) and interferon (IFN)-gamma (100 ng/ml); LPS (1 µg/ml), IL-1alpha (57 ng/ml), and TNF-alpha (100 ng/ml); and IL-1alpha (57 ng/ml), TNF-alpha (100 ng/ml), and IFN-gamma (10 or 100 ng/ml) on SP-A, -B, and -C mRNA expression in lung explants from 22 (n = 4-12)- and 30 (n = 8-14)-fetal-day lung explants and 2-day-old newborns (n = 8-12). Incubation time was 12 h, and the bars show SP expression levels relative to the expression in the presence of vehicle (shown as line). Data are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.



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Fig. 4.   Effect of LPS, combination of IL-1alpha and TNF-alpha , or IFN-gamma on SP-A, SP-B, and SP-C mRNA and 28S RNA expression in lung explants. Representative Northern blots show the expression after culture for 12 h in the presence of vehicle (CTR), 1 µg/ml LPS, the combination of recombinant human (rh) IL-1alpha 57 ng/ml and rhTNF-alpha 100 ng/ml, or 10 ng/ml rhIFN-gamma . A: explants from 22-day-old fetal lung. B: explants from 30-day-old fetal lung. C: explants from 2-day-old newborn lung.

Expression of IL-1alpha . In lung explants from 30-day-old fetuses and 2-day-old newborns, LPS increased the immunoreactivity of IL-1alpha in future airspaces. In lung explants from 22-day-old fetuses, after the addition of LPS, there was some IL-1 immunoreactivity associated with macrophages from pleura and interstitium (data not shown), but no immunoreactivity was evident in airspaces (Fig. 5).


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Fig. 5.   Immunohistochemical detection of IL-1alpha . Explants from 22-day-old (A-D) or 30-day-old (E-H) fetal rabbits were cultured for 12 h in the presence of vehicle (A, B, E, and F) or 1 µg/ml LPS (C, D, G, and H). Sections in A, C, E, and G were incubated with anti-rabbit IL-1alpha antibody; sections in B, D, F, and H were incubated without the primary antibody. IL-alpha immunoreactivity was induced (dark brown) in the alveolar explants of 30-day-old rabbit fetus when incubated with LPS (G).

SP-B. In mature lung (30-day-old fetal, 2-day-old newborn) within 12 or 20 h (n = 7), IL-1alpha had no detectable effect on the SP-B protein content compared with vehicle-treated controls (1.13 ± 0.23, P = 0.52). In explants from mature lung, TNF-alpha (0.77 ± 0.15, P = 0.16) and the combination of IL-1alpha and TNF-alpha (0.73 ± 0.17, P = 0.14) tended to decrease SP-B (Fig. 6).


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Fig. 6.   Detection of SP-B on Western blots. Lung explants from 30-day-old fetuses were cultured for 20 h in the presence of vehicle (C), 57 ng/ml rhIL-1alpha (IL-1), 100 ng/ml rhTNF-alpha (TNF), or a combination of 57 ng/ml IL-1alpha and 100 ng/ml TNF-alpha (IL-1 + TNF). SP-B was suppressed by TNF-alpha and by the combination of IL-1alpha and TNF-alpha .


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

LPS binds to its receptors on inflammatory cells and induces the expression of proinflammatory cytokines, principally IL-1 and TNF-alpha , which bind to their receptors in macrophages and to other cells, resulting in further propagation of the inflammatory cascade (3, 55). In the present study, we have shown that the influence of LPS from E. coli and the cytokines on SP expression was dependent on the age of the animals. In immature rabbit lung (day 22; term 30-31 days), LPS had no influence on the expression of SP mRNA, whereas, at term and after birth, LPS downregulated SP-A, SP-B, and SP-C in a concentration-dependent manner. As shown in the present and previous studies (19), IL-1alpha and IL-1beta increased SP-A and SP-B mRNA and protein in the immature alveolar tissue from glandular or canalicular lung. In contrast, in the saccular or alveolar stage of lung development, IL-1 suppressed or had no effect on SP mRNA. We further found that, when administered together with IL-1 to transitional or mature lung, TNF-alpha additively suppressed the expression of SPs. In contrast, IFN-gamma served as an effective modifier for the other inflammatory mediators. In the canalicular lung, IFN-gamma decreased the expression of the SPs when LPS or the cytokines were present, whereas after birth IFN-gamma tended to moderate the suppression of SP mRNA by LPS or the cytokines. The present study serves to explain the variation of the pulmonary outcome after an inflammatory insult.

IL-1 and LPS accelerate fetal lung maturity in vivo. Both IL-1 and LPS, given intra-amniotically to 25-day-old rabbit fetuses, increased SP-A and SP-B mRNA and improved lung stability, increasing saturated phosphatidylcholine and SP-B in bronchoalveolar lavage (6, 7). Likewise, intra-amniotic IL-1 or LPS also increased SP-A, SP-B, and saturated phosphatidylcholine concentrations in alveolar space of premature lambs and had favorable effects on lung function after premature birth (18, 30).

There was no detectable IL-1 in amniotic fluid in normal human pregnancy and only a small increase in IL-1 levels in term labor (44), whereas IL-1 receptor antagonist (IL-1ra) levels have been reported to be high (8, 46). Because, additionally, intra-amniotic IL-1ra failed to suppress the expression of SPs, IL-1 appears to serve as a salvage pathway rather than obligatory signaling of lung maturation (7). In premature labor resulting from intrauterine infection, the concentrations of IL-1 and TNF-alpha increase in the amniotic fluid toward levels used in the present in vitro study (44, 45). However, the concentrations of LPS in the amniotic fluid were generally two orders of magnitude lower than those of IL-1alpha , IL-beta , or TNF-alpha (20). This is consistent with the hypothesis that cytokines accelerate maturation of the fetal lung. Within 2-4 h after the addition of IL-1alpha to lung explants from 19- to 22-day-old rabbit fetuses, IL-1 increased the expression of SP-A and SP-B (14, 19). This acute IL-1 effect was not affected when the explants were preincubated in the presence of indomethacin, prostaglandin E2, or dibutyryl-cAMP (data not shown). However, in the immature lung, LPS neither influenced the expression of SPs nor induced IL-1alpha in the terminal airspaces. On the basis of current evidence, we propose that, in intrauterine infection or after intra-amniotic injection of LPS (6, 30), the generation of IL-1 and as yet undefined downstream mediators are responsible for the increase in the expression of SPs (7, 18, 19) and for the decrease in the incidence of RDS (59). The lack of IL-1alpha induction in the airways after the addition of LPS to immature lung in vitro implies that extra-alveolar macrophages mediate the acceleration of fetal lung maturation. Macrophages from the fetal membranes are a likely source of cytokines since they produce IL-1 as a response to LPS (47), and the fetal breathing movements enable the contact of amniotic fluid with the future respiratory tract (24).

Toll-like type-1 transmembrane receptor 4 (TLR4) together with MD2 and CD14 mediates the LPS signal that leads to the activation of transcription factor nuclear factor (NF)-kappa B (9, 27, 50). TLR4 may also activate the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) pathway (36). The NF-kappa B and SAPK/JNK pathways upregulate the expression and synthesis of proinflammatory cytokines IL-1 and TNF-alpha . It has been recently found that, in fetal murine lung, the expression of both TLR4 and TLR2 (putative receptor of gram-positive and fungal toxins) was much lower than in the lung of newborn or adult mice (26). The absence of the monocyte/macrophage TLR4-signaling pathway resulting from paucity of macrophages within the terminal airways is a possible cause for the observed failure of LPS to induce IL-1alpha in the immature alveolar tissue.

IL-1 binds to IL-1 receptor 1. This type-1 transmembrane protein of the IL-1/TLR superfamily (40) is likely to be present on the surface of alveolar epithelial cells (23). IL-1 also induces transcription factor C/EBPdelta (CCAAT/enhancer binding protein-delta ; see Ref. 32). In fetal rabbit lung, the expression of C/EBPdelta is developmentally regulated and reaches its peak level parallel to SP-A on day 28 of gestation (5). However, there is no direct evidence of the role of the IL-1R/TLR superfamily members or the transcription factor C/EBPdelta controlling the responsiveness of alveolar cells to inflammatory mediators.

IFN-gamma is not detectable in amniotic fluid at any stage of human pregnancy (57), not even in preterm labor with histologically diagnosed chorioaminionitis (37). This reflects the paucity of the Th1 cytokines in the tissues of both the pregnant mother and the fetus (41). Addition of IFN-gamma to explants from immature rabbit lung did not acutely influence the expression of SPs. Instead, IFN-gamma served as a modifier of the effects of the cytokines on the expression of SPs. In explants from the immature lung, cultured in the presence of IL-1alpha and TNF-alpha , addition of IFN-gamma suppressed SP-C mRNA. Similar suppression of SP-C was evident when IFN-gamma was replaced by LPS. In contrast, IFN-gamma increased the expression of SP-A in human fetal lung explants from 16 to 18 wk of human pregnancy after prolonged culture (4 days; see Ref. 2). The observed difference in the IFN-gamma response between the two studies may be because of the difference in the duration of culture (19) or because of species difference. IFN-gamma is derived mainly from CD4-positive T cells, where its synthesis is activated by IL-18 in the presence of a costimulant, such as LPS (16). IL-1 and IL-18 are structurally related, and their receptor complexes, as members of the IL-1/TLR superfamily (16, 40), have a cytoplasmic domain that is very similar to that of TLR4 (40). IFN-gamma and LPS added to explants from the immature lung downregulated both SP-B and SP-C mRNA. The present results demonstrate that, although IL-1 is associated with the inflammatory induction of SPs in immature lung, other inflammatory mediators may prevent or reverse the induction (Fig. 3). The latter includes IFN-gamma , which, however, has been found to have low expression levels in fetal and placental tissues (41).

The downregulation of SP mRNAs by the proinflammatory cytokines and LPS may play an important role in the pathogenesis of life-threatening respiratory failure. As shown here and by others (19, 63), cytokine-induced changes in SP mRNA are reflected in the levels of the proteins. In small premature infants developing CLD (31, 52, 59), and in acute respiratory distress syndrome (ARDS; see Ref. 29), the proinflammatory cytokines are increased in the airways. The surfactant defects evident in CLD and in ARDS include deficiencies in SP-B (21) and SP-A (25). As shown here and previously (1, 60, 63), the suppression of SPs by microbial proteins is mediated by IL-1 and TNF-alpha . Here, we have provided evidence of the role of IFN-gamma in moderating the effects of LPS and cytokines on the expression of SP-A, SP-B, and SP-C.

According to current evidence, both the quality of the inflammatory challenge and the degree of differentiation of the alveolar cells determine the expression levels of the SPs. The functional consequences of the inflammatory response range from induction of SPs, leading to protection against RDS, to profound suppression of the surfactant, resulting in predisposition to CLD or ARDS. The induction of SPs takes place in rapidly growing immature lung that lacks terminal differentiation and possesses a deficient innate immune response. Individual components SP-A and SP-D bind to microbial toxins, influencing the inflammatory response and the elimination of microbes in a variety of ways (12). According to present evidence, by inducing the surfactant complex in alveolar epithelial cells, IL-1 decreases the risk of RDS in premature fetuses and contributes to activation of the pulmonary defense against microbes.


    ACKNOWLEDGEMENTS

We thank Dr. Tiina Kangas for help in the project. We thank Maarit Hännikäinen, Elsi Jokelainen, and Mirkka Parviainen for excellent technical assistance.


    FOOTNOTES

This work was supported by the Academy of Finland, Biocenter Oulu (M. Hallman), and the Foundation for Pediatric Research in Finland (O. Väyrynen).

Preliminary results were presented as an abstract at the annual meeting of the European Society of Pediatric Research in Copenhagen, Denmark, in 1999 (Pediatric Res 45: 896A, 1999).

Address for reprint requests and other correspondence: O. Väyrynen, Dept. of Pediatrics and Biocenter Oulu, Univ. of Oulu, P.O. Box 5000, Univ. of Oulu, FIN-90014 Oulu, Finland (E-mail: ovayryne{at}paju.oulu.fi).

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.

10.1152/ajplung.00274.2001

Received 19 July 2001; accepted in final form 20 November 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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