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distress syndrome by elevating fetal interleukin-6 serum concentration. Koichiro ... that H441-4, a human pulmonary adenocarcinoma cell line,. 1993). In contrast ...
Human Reproduction vol.15 no.10 pp.2234–2240, 2000

Chorioamnionitis decreased incidence of respiratory distress syndrome by elevating fetal interleukin-6 serum concentration

Koichiro Shimoya1,2,7, Takeshi Taniguchi3, Noboru Matsuzaki4, Akihiro Moriyama1, Yuji Murata1, Hiroyuki Kitajima5, Masanori Fujimura5 and Masahiro Nakayama6 1Department

of Obstetrics and Gynecology, Faculty of Medicine Osaka University, 2Department of Obstetrics and Gynecology, Osaka Police Hospital, 3Department of Obstetrics and Gynecology, Taniguchi Hospital, 4Department of Obstetrics and Gynecology, Ikeda City Hospital, 5Department of Neonatal Medicine and 6Pathology, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan 7To

whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University, 2–2 Yamada-oka, Suita City, Osaka 565-0871, Japan. E-mail: [email protected]

Respiratory distress syndrome (RDS) of newborns is one of the most important factors determining neonatal morbidity and mortality. The interleukin-6 (IL-6) titre in cord sera of RDS-free neonates born to mothers with histological chorioamnionitis was significantly higher than that in RDScomplicated neonates without chorioamnionitis. Maternal administration of glucocorticoid suppressed the IL-6 concentrations in the cord sera of fetuses with chorioamnionitis. The fetuses without chorioamnionitis who suffered from RDS even after maternal glucocorticoid administration showed a similar IL-6 titre to that of RDS-affected neonates without chorioamnionitis. Examination of the mechanism by which IL-6 decreased the incidence of fetal RDS revealed that H441-4, a human pulmonary adenocarcinoma cell line, stimulated with recombinant (r)-IL-6 started the synthesis of mRNA and protein of pulmonary surfactant protein (SP)-A. The present study shows that IL-6 elevation in fetuses with chorioamnionitis promotes fetal lung maturation by inducing SP-A synthesis, thereby decreasing the incidence of RDS in the preterm neonates. Key words: chorioamnionitis/interleukin-6/respiratory distress syndrome/surfactant protein

Introduction Preterm delivery remains the most important cause of perinatal morbidity and mortality despite recent advances in tocolytic therapy and neonatal intensive care. Lettieri et al. reported that maternal conditions, such as placenta previa, abruption, intrauterine infection, immunological abnormalities, cervical incompetence, and uterine abnormalities, caused preterm delivery (Lettieri et al., 1993). Neonatal complications of the premature infants include intraventricular haemorrhage, bron2234

chopulmonary dysplasia, sepsis, and necrotizing enterocolitis (Cox, 1997). The major problems associated with preterm delivery are intrauterine infections such as chorioamnionitis (Hiller et al., 1988) and respiratory distress syndrome (RDS) of preterm neonates (Wegman, 1991). Perinatal factors which relate to modulating the incidence of RDS have been reported (Bryan et al., 1990). The factors which decrease the incidence of RDS include higher gestational age, premature rupture of the membranes (PROM), vaginal delivery and corticosteroid therapy, whereas the factors increasing the incidence of RDS include lower gestational age and Caesarean section with or without labour. An especially low incidence of RDS has been reported in babies delivered to mothers with PROM of over 24 h duration in the absence of overt maternal infection (Bryan et al., 1990), although the exact mechanism underlying PROMinduced decrease of the incidence of RDS remains to be determined. However, Hallak and Bottoms reported no effect of PROM on the incidence of RDS (Hallak and Bottoms, 1993). RDS accounts for significant neonatal morbidity and mortality of preterm infants. RDS is induced by a paucity of pulmonary surfactant proteins produced by the fetal lung (Jobe, 1993). To reduce the incidence and severity of RDS, administration of glucocorticoid to the mother (Liggins and Howie, 1972) or surfactant to the neonates (Jobe et al., 1993) is clinically performed. An additive or synergistic effect of maternal corticosteroid treatment followed by postnatal surfactant treatment on the incidence of RDS has been reported for preterm infants without inflammatory diseases (Jobe et al., 1993). In contrast, the incidence of RDS has been reported to be low in mothers with prolonged PROM irrespective of glucocorticoid administration (Garite et al., 1981). It was reported that the incidence of RDS was increased with clinical chorioamnionitis (Alexander et al., 1998). However, it was also reported that histological chorioamnionitis might accelerate lung maturation (Watterberg et al., 1996). A characteristic feature of histological chorioamnionitis is infiltration of polymorphonuclear leukocytes (PMN) into the placenta (Hollander, 1986). In chorioamnionitis, placental cells are activated by infectious stimuli such as bacterial endotoxins to carry out increased production of interleukin (IL)-1α (Taniguchi et al., 1991), IL-1β (Taniguchi et al., 1991), IL-6 (Matsuzaki et al., 1993), IL-8 (Shimoya et al., 1992a, 1999) and monocyte chemotactic and activating factor (Shimoya et al., 1998). Such elevated placental cytokines result in elevation of the fetal cytokine concentrations in the fetal circulation (Shimoya et al., 1992b). Amniochorionic membranes are also the source of cytokines, such as IL-6 and IL-8 (Fortunato et al., 1995, 1996). Activated fetal immunocompetent cells also contribute to elevation of the cytokine © European Society of Human Reproduction and Embryology

Decrease of respiratory distress syndrome by chorioamnionitis

concentrations in the cord serum (Matsuzaki et al., 1990; Taniguchi et al., 1993). These cytokines participate in potentiation of the fetoplacental defence mechanism in histological chorioamnionitis. Early detection and proper obstetric management of chorioamnionitis may promote such defence mechanisms, thereby decreasing serious sequels in both mothers and neonates. Pulmonary surfactants are composed of a lipid–protein complex. The complex is 90% phospholipid and 10% pulmonary apoproteins including surfactant protein (SP)-A, SP-B, SPC and SP-D (Mendelson and Goggaram, 1991). The pulmonary surfactant proteins play an important role in the development of pulmonary maturity (Mendelson and Goggaram, 1991). There is increasing evidence of the biological functions of SPA in the fetal lung. SP-A has an important function in the assembly and maintenance of the alveolar surfactant monolayer (Mendelson and Goggaram, 1991). SP-A maintains an appropriate alveolar amount of surfactant by controlling its secretion from and uptake into alveolar type II epithelial cells. SP-A also possesses such activities that increase the bactericidal capacity of immunoreactive cells by modulating and increasing their migration and phagocytic activity of alveolar macrophages (Manz-Keinke et al., 1992); thus, SP-A is actively involved in the potentiation of the host defence mechanism. Indeed, a clinical study reported that a higher SP-A content in tracheal aspirates reduced the incidence of neonatal RDS (Steven et al., 1992). The important immune defence function of pulmonary surfactant is being increasingly recognized (Tino and Wright, 1996). SP-A promotes phagocytosis of bacteria by alveolar macrophages and is chemotactic for these phagocytes. An increase in SP-A concentrations and associated events might be critical for fetal lung maturation (Tino and Wright, 1996). Moreover, a recent study on SP-A deficient mice showed that SP-A mediates mycoplasmacidal activity of alveolar macrophages and plays an important role in group B Streptococcus clearance (Hickman-Davis et al., 1999; LeVine et al., 1999). Various factors are associated with surfactant protein production. Tumour necrosis factor α (TNFα) inhibits SP-A mRNA and protein synthesis but not those of SP-B (Wispe et al., 1990). Prenatal exposure to TNFα might be a risk factor for RDS (Hitti et al., 1997). Recently, several studies reported that IL-1 (Bry et al., 1997; Dhar et al., 1997) accelerated surfactant protein synthesis and that epidermal growth factor (EGF) stimulated fetal lung maturation (Gross et al., 1986; Goetzman et al., 1994). In the present study, clinical profiles of preterm neonates were compared, to examine the correlation between RDS and chorioamnionitis which was histopathologically diagnosed from the placenta after the delivery (Blanc, 1981). To examine whether a low incidence of RDS showed any correlation with the fetal cytokine concentration, the cytokine concentrations in the cord sera of newborns with or without chorioamnionitis were determined and compared with those who suffered from RDS. The IL-6 concentration in neonates whose mothers had been administered glucocorticoid was similarly compared with those whose mothers had not been administered glucocorticoid. The cell line studies were performed to investigate the direct

effect of cytokine on synthesis of pulmonary surfactant proteins. Materials and methods Reagents and cell line Recombinant (r)- IL-6 was from Dr T.Kishimoto (Osaka University, Osaka, Japan) and rIL-8 was from Dr K.Matsushima (Tokyo University, Tokyo, Japan). cDNA for surfactant protein-A (SP-A) and SP-B were kindly provided by Dr J.A.Whitsett (University of Cincinnati, Cincinnati, OH, USA). A human pulmonary adenocarcinoma cell line, H441-4, was purchased from the American Type Culture Collection (Rockville, MD, USA). Criteria for chorioamnionitis and RDS Microscopic histopathological analysis of the placenta was performed according to Blanc’s criteria (Blanc, 1981) and the severity of chorioamnionitis, i.e. inflammation of the placental surface, was determined by the degree of maternal polymorphonuclear lymphocyte (PMNL) infiltration into either the subchorionic space (stage 1), the intervillous space (stage 2) or amniotic cavity (stage 3). The criteria for RDS required clinical signs such as tachypnoea, cyanosis, grunting, retraction, supplemental oxygen therapy for at least 48 h and a chest X-ray consistent with generalized atelectasis. The histological diagnosis of chorioamnionitis and clinical diagnosis of RDS were made blind to other findings. Infant profile For analysis of cytokine concentrations in cord serum, 60 preterm infants who were born to mothers admitted to Osaka Medical Center between 1983 and 1990 were included in this study. All clinical findings including the time and duration of PROM, the indication for Caesarean section, placental histopathology and neonatal outcome were available for these infants and mothers. Fourteen infants were complicated with RDS, whereas 46 infants were completely free of RDS. Seventeen infants were born to mothers who received an i.m. injection of 12 mg of glucocorticoid twice, 24 h apart. Twentytwo infants were delivered with placentae showing histological chorioamnionitis, while 38 infants were delivered with placentae showing the chorioamnionitis. All cord sera of the infants were stored at –80°C until titration. All infants who suffered from severe forms of transient tachypnoea of the newborn, asphyxial lung disease or congenital pneumonia were excluded from the present study. In addition, the infants who developed pneumonia or septicaemia after delivery, or those with either chromosomal or congenital anomalies or hydrops fetalis were also excluded. No infants received thyrotrophin-releasing hormone (TRH) treatment (to those mothers) before delivery or surfactant therapy after birth. No infants were born to mothers who suffered from diseases such as systemic lupus erythematosis (SLE), hyperthyroidism or (gestational) diabetes mellitus which might potentially modulate the incidence of RDS or IL-6 concentrations in the cord sera. Gestational age of the neonates was based on the last menstrual period and ultrasonographic examination of the fetal crown–rump length in the first trimester. Informed consent to use the cord sera was obtained from all mothers in this study. Assay for IL-6, IL-8, IL-1α, IL-1β and TNFα in cord sera The IL-6 titre in cord serum obtained at delivery was measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The detection range of the kit was 3.13– 300 pg/ml. The intra- and interassay variabilities of the kit were

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Table I. Clinical profile of preterm neonates with or without RDS and maternal steroid treatment RDS (⫹)

Gestational week Body weight (g) Male/total Placenta previa Eclampsia PROM Caesarean section Chorioamnionitis

RDS (–)

Steroid (–)

Steroid (⫹)

Steroid (–)

Steroid (⫹)

27.5 ⫾ 1.9 980 ⫾ 275 5/10 3/10 4/10 2/10 6/10 0/10

27.6 ⫾ 3.2 990 ⫾ 410 2/4 0/4 1/4 1/4 2/4 0/4

28.1 ⫾ 2.2 1090 ⫾ 469 20/33 2/33 8/33 22/33a 13/33 25/33b

27.0 ⫾ 2.8 1007 ⫾ 361 9/13 1/13 2/13 5/13 5/13 13/13

⬍ 0.01 versus the RDS (⫹) and steroid (–) neonates. ⬍ 0.0001 versus the RDS (⫹) and steroid (–) neonates. RDS ⫽ respiratory distress syndrome. PROM ⫽ premature rupture of the membranes.

aP

bP

Table II. Patient matrix of this study RDS (⫹)

Steroid (⫹) Steroid (–)

RDS (–)

CA (–)

CA (⫹)

CA (–)

CA (⫹)

4 Group IV 10 Group I

0

0

0

8 Group III

13 Group V 25 Group II

CA ⫽ chorioamnionitis.

2.7–4.1% and 4.1–7.8% respectively. The kit detected no crossreactivity with other cytokines such as IL-1, TNFα and IL-8. To detect the IL-8 concentration in the cord serum, an ELISA kit specific for human IL-8 (R&D Systems) was used. This assay used a quantitative immunometric ‘sandwich’ technique. The kit consistently detected serum IL-8 concentrations over 4.7 pg/ml. The intra- and interassay variabilities of the kit were 5.4–9.2% and 7.3– 12.2% respectively. The kit was incapable of detecting other cytokines. The concentrations of IL-1α, IL-1β and TNFα in the cord sera were determined by ELISA specific for each cytokine (R&D Systems). Serum concentration which each kit detected covered ⬎0.2 pg/ml of IL-1α, ⬎0.3 pg/ml of IL-1β and ⬎0.75 pg/ml of TNFα respectively. No mutual cross-reactivity could be found. Intra- and interassay variabilities were within 9.5%. Measurement of mRNA expression of pulmonary surfactant protein and their protein synthesis H441-4 cells were maintained and passaged, as described previously (Wispe et al., 1990). Briefly, 1⫻106 H441-4 cells, seeded into a 100 mm tissue culture plate, were exposed to various concentrations of either rIL-6 or rIL-8 for 12 h, 24 h, 48 h and 72 h for preparation of mRNA expression and measurement of surfactant proteins. The total cellular RNA was recovered from unstimulated or rIL-6- or rIL8-stimulated H441-4 cells using the acid–guanidium thiocyanate– phenol–chloroform extraction method, as described previously (Chomczynski and Sacchi, 1987). SP-A and SP-B mRNA were detected using the 32P-labelled human cDNA. Detection of SP-A protein was performed by enzyme immunoassay (EIA) specific for SP-A (Teijin Co. Ltd, Tokyo, Japan) (Sato et al., 1992). The

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Figure 1. Interleukin (IL)-6 and IL-8 titres in the cord sera of neonates with or without either chorioamnionitis (CA), respiratory distress syndrome (RDS) or steroid treatment. (A) The IL-6 titres in cord sera of group I–V neonates were determined by enzyme immunoassay (EIA). (B) The IL-8 titres were simultaneously determined by EIA. CA and steroid treatment represent histological chorioamnionitis and glucocorticoid administration to mothers respectively. Each dot represents (A) IL-6 and (B) IL-8 titres in the cord sera. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

experiments to demonstrate the effects of rIL-6 and rIL-8 on SP-A and SP-B mRNA and the protein synthesis of SP-A were repeated at least three times.

Decrease of respiratory distress syndrome by chorioamnionitis

Statistical analysis Comparison of cytokine concentrations and clinical profiles including gestational weeks of patient groups were performed by non-parametric methods and χ2 analysis. Statistical analysis of rIL-6-induced surfactant synthesis experiments was conducted using Student’s t-test and analysis of variance followed by Duncan’s multiple range test. Values were expressed as the mean ⫾ SEM. The correlation between IL-6 and IL-8 in cord sera were examined by a simple linear analysis. P ⬍ 0.05 was considered statistically significant.

Results Table I shows the clinical profiles of the preterm neonates assayed for their IL-6 titres. Ten RDS (⫹) neonates without steroid treatment [steroid (–)], 4 RDS (⫹) and steroid (⫹) neonates, 33 RDS-free [RDS (–)] and steroid (–) preterm neonates and 13 RDS (–) and steroid (⫹) neonates showed no statistical differences in their gestational weeks, body weight at birth, the ratio of male/female and the incidence of placenta previa, eclampsia or Caesarean section. However, RDS (–) and steroid (–) neonates did show a significant difference in the incidence of preterm rupture of membrane (PROM) [P ⬍ 0.01 versus the RDS (⫹) and steroid (–) neonates]. As shown in Table I, both RDS (–) neonates with steroid (–) and steroid (⫹) showed a much higher incidence of histological chorioamnionitis [P ⬍ 0.0001 versus the RDS (⫹) and steroid (–) neonates]. The 60 neonates were divided into five groups in terms of the presence or absence of chorioamnionitis (CA), RDS and maternal steroid treatment (steroid) (Table II). Determination of the IL-6 titre in the cord sera of the steroid (–) neonates showed a significant difference between CA (⫹) and RDS (–) neonates (group II) and CA (–) and RDS (⫹) neonates (group I) (P ⬍ 0.001) (Figure 1A), suggesting that CA increased IL-6 in the cord sera. Figure 1A also shows that CA (–) and RDS (–) neonates (group III) showed a higher titre than the CA (–) and RDS (⫹) neonates (group I) (P ⬍ 0.01), indicating IL-6 in cord sera reduced the incidence of RDS. In contrast to these findings of IL-6 concentrations, fetal IL-8 concentrations failed to show significant differences in the presence or absence of RDS (Figure 1B). Although group II neonates showed a significantly higher IL-8 value than group I neonates (P ⬍ 0.01), group III neonates were not significantly different from group I neonates. The effects of glucocorticoid administration on the IL-6 and the IL-8 concentrations were simultaneously examined (Figure 1A). CA (–), RDS (⫹) and steroid (⫹) neonates (group IV) showed no difference from group I neonates in the IL-6 titre. However, the IL-6 titre of CA (⫹), RDS (–) and steroid (⫹) neonates (group V) was significantly lower than the titre of group II neonates (P ⬍ 0.01), suggesting that the steroid treatment of CA (⫹) neonates reduced IL-6 concentrations in the cord sera. Although group V neonates showed a significantly reduced IL-8 value compared with group II neonates (P ⬍ 0.01), they did not show any difference from group IV neonates (Figure 1B). No significant difference was observed between the titres of IL-1α, IL-1β and TNFα in cord sera of group I–V. The relationship between cord serum IL-6 titres and stages of chorioamnionitis was then examined after exclusion of

Figure 2. IL-6 titres in the cord sera of preterm neonates with and without chorioamnionitis and titration of IL-6 concentrations by grade of histological chorioamnionitis. IL-6 titres in cord sera of preterm neonates with (n ⫽ 25) and without chorioamnionitis (n ⫽ 18) were assayed by EIA. All these neonates were born to mothers who had not received steroid treatment. All sera from groups I ⫹ III neonates were classified as the normal group (n ⫽ 18), while group II neonates were devised into three groups (stage 1–3) by grade of chorioamnionitis: stage 1 (n ⫽ 4), stage 2 (n ⫽ 10) and stage 3 (n ⫽ 11).

group IV and group V neonates because of steroid-mediated suppression of IL-6 titres. As shown in Figure 2, although neonates with stage 1 chorioamnionitis (n ⫽ 4, median pg/ml, range 160–2000 pg/ml) showed a higher titre than the normal neonates (n ⫽ 18, median 170 pg/ml, range 160– 480 pg/ml) (P ⬍ 0.01), they did not show a significant difference from neonates with stage 2 chorioamnionitis (n ⫽ 10, median 693 pg/ml, range 205–1820 pg/ml, P ⬍ 0.001 versus the normal neonates) or neonates with stage 3 chorioamnionitis (n ⫽ 11, median 810 pg/ml, range 160–2130 pg/ml, P ⬍ 0.001 versus the normal neonates). The IL-6 titres in RDSfree neonates with histological chorioamnionitis, therefore, did not show a linear increase in parallel with the severity of the chorioamnionitis in contrast to the cord serum IL-8 concentrations in those neonates (Shimoya et al., 1992b). Measurement of cord serum IL-6 titre showed 79% of sensitivity and 79% of specificity, whereas that of IL-8 titre showed 89% of sensitivity and 97% of specificity for prenatal prediction of chorioamnionitis (Shimoya et al., 1992b). To study the mechanism by which IL-6 reduces the incidence of RDS, the effect of rIL-6 on the pulmonary surfactant protein metabolism was examined. Figure 3A and B show that rIL-6 had a time dependent effect on SP-A mRNA and SP-A protein synthesis, respectively. As shown in Figure 3C, rIL-6 induced a dose-dependent increase in the amount of SP-A mRNA in H441-4 cells. Figure 3D shows the dose-dependent effect of rIL-6 on SP-A production by H441-4 cells. The rIL-6-mediated increases in mRNA and protein synthesis became apparent from 0.2 ng/ml of rIL-6 (P ⬍ 0.05 versus the control) and reached a plateau at 2.0 ng/ml of rIL-6 (P ⬍ 0.01 versus the control). Addition of rIL-8 failed to show any effects on the expression of mRNA for SP-A, or on SP-A protein production by H441-4 cells (data not shown). IL-6 and a high amount of IL-8 slightly induced mRNA for SP-B (Figure 4). 2237

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Figure 3. Time-dependent and dose-dependent effects of recombinant (r)IL-6 on surfactant protein (SP)-A mRNA and its protein synthesis by H441 cells. (A) H441 cells were cultured for different time periods with 2.0 ng/ml of rIL-6 to observe the effect on the amount of mRNA and (B) protein synthesis of SP-A and then cultured for 48 h with various concentrations of rIL-6 to observe the effects on (C) amounts of mRNA and (D) protein synthesis of SP-A. *P ⬍ 0.05, **P ⬍ 0.01.

Figure 4. The effects of rIL-6 and rIL-8 on SP-B mRNA by H441 cells. H441 cells were cultured for 48 h with various concentrations of rIL-8 and rIL-6 to observe the effect on amount of mRNA. Lane 1: H441 cells were cultured without rIL-8 and rIL-6 (medium only, control). Lane 2: H441 cells were cultured with 1.0 ng/ml rIL-8. Lane 3: 10 ng/ml rIL-8. Lane 4: 100 ng/ml rIL-8. Lane 5: 1.0 ng/ml rIL-6. Lane 6: 10 ng/ml rIL-6. Lane 7: 100 ng/ml rIL-6.

Discussion The significant effect of chorioamnionitis on the incidence of RDS has not yet been adequately addressed. However, it was 2238

reported that histological chorioamnionitis elevated the fetal pulmonary surfactant concentration in the amniotic fluid, although they did not clarify the relationship between chorioamnionitis and the incidence of RDS (Higuchi et al., 1992). In the present study, chorioamnionitis showed a strong impact on decrease of the incidence of RDS despite the relatively small sample size. It was also reported that histological chorioamnionitis might accelerate lung maturation (Watterberg et al., 1996). However, it was recently reported that the incidence of RDS was increased with clinical chorioamnionitis (Alexander et al., 1998). The reason for the discrepancy between the present findings and Alexander’s study is unknown. The different method used to diagnose chorioamnionitis might be the reason for this inconsistency. The IL-6 concentration in cord sera of neonates with chorioamnionitis was significantly elevated compared with that of neonates without chorioamnionitis. Since a significant amount of endotoxin (lipopolysaccharide) is present in chorioamnionitis, fetal IL-6 might be derived from fetal immuno-

Decrease of respiratory distress syndrome by chorioamnionitis

competent cells, alveolar macrophages, type II alveolar cells and placental cells which had been activated by infectious stimuli, including LPS, in chorioamnionitis. Yoon et al. previously reported IL-6 concentrations in umbilical cord plasma in preterm neonates (Yoon et al., 1996). There were discrepancies between the present study and the Yoon study. One possible reason for this might be the differences of the assay systems. Another possible reason might be the process of clotting which could affect the cytokine concentrations. We previously reported that the IL-8 concentrations in cord sera of preterm neonates were enhanced in direct proportion to the severity of histological chorioamnionitis (Shimoya et al., 1992b). Measurement of IL-8, therefore, can be used as a marker to prenatally diagnose or predict preterm chorioamnionitis because of its high sensitivity and specificity (Shimoya et al., 1992b). In contrast to IL-8, cord IL-6 did not show such a linear increase in response to the severity of chorioamnionitis (Figure 2) and showed less accuracy in the diagnosis of chorioamnionitis. However, group III neonates showed a significantly higher IL-6 titre than group I neonates even in the absence of clear histological chorioamnionitis. The significant difference in IL-6 titres became larger in the presence of active chorioamnionitis, as shown in group II neonates. Moreover, IL-6 measurement of group V neonates resulted in a clear difference from group IV neonates. In contrast, measurement of cord serum IL-8 concentration failed to discriminate group III neonates from group I neonates and group V neonates from group IV neonates, although the patient number of group IV neonates should be increased for further detailed analysis. This correlation between the IL-6 titre and the incidence of RDS suggests that IL-6 may be deeply involved in the decrease of the incidence of RDS and has potential use in prenatal diagnosis of RDS in preterm infants, although IL-6 is less useful for prenatal diagnosis of chorioamnionitis than IL-8. Pulmonary surfactants are composed of a lipid–protein complex. Since the pulmonary surfactant proteins play an important role in the development of pulmonary maturity (Mendelson and Goggaram, 1991), the effect of fetal IL-6 on the synthesis of the major pulmonary surfactant proteins, i.e., SP-A and SP-B, was focused on. We examined the effect of IL-6 concentrations within cord serum range of preterm neonates. The present study showed that IL-6 promoted mRNA expression and protein synthesis for SP-A and that IL-6 slightly induced mRNA for SP-B. Human experimental model showed that TNFα, in contrast, inhibits SP-A mRNA and protein synthesis but not those of SP-B (Wispe et al., 1990). Animal models, such as rabbit, rat, and non-human primate, demonstrated that IL-1 accelerated surfactant protein synthesis (Bry et al., 1997; Dhar et al., 1997) and that EGF stimulated fetal lung maturation (Gross et al., 1986; Goetzman et al., 1994). Taken together, a network of cytokines appears to regulate fetal lung maturation. There is increasing evidence of the biological functions of SP-A in the fetal lung. The important immune defence function of pulmonary surfactant is being increasingly recognized (Tino and Wright, 1996). SP-A promotes phagocytosis of bacteria by alveolar macrophages and is chemotactic for these phagocytes. An increase in SP-A concentrations and associated events might be critical for fetal

lung maturation (Tino and Wright, 1996). These findings suggest that IL-6 in cord serum is a major cytokine acting to locally stimulate pulmonary SP-A synthesis, thereby promoting pulmonary maturation and resulting in decrease of the incidence of RDS. Glucocorticoid has clinically been used in an attempt to accelerate fetal lung differentiation (Liggins and Howie, 1972; Mendelson and Goggaram, 1991). Glucocorticoid, after binding to its receptor, increases SP-A and SP-B mRNA and protein synthesis as well as the contents of fatty acid synthetase, collagen and elastin in fetal lung (Mendelson and Goggaram, 1991). In addition, there is a general agreement that glucocorticoid increases the synthesis of major surfactant phospholipids in many species. Such combined glucocorticoid-mediated effects result in a reduced incidence of RDS in chorioamnionitis-free neonates at certain gestational weeks (Liggins and Howie, 1972). However, maternal administration of glucocorticoid induced a striking depression in fetal IL-6 concentrations (group V versus group II) as well as IL-8 concentrations (Shimoya et al., 1992b), which implies that glucocorticoid inhibits IL-6-induced promotion of fetal lung maturity. In contrast, the group V neonates remained RDS-free even after glucocorticoid administration to the mothers, while group IV neonates showed RDS even after glucocorticoid administration. Further studies are required with increased numbers of group IV neonates. The present findings suggest that glucocorticoid exerts no influence on lung maturity of neonates without chorioamnionitis at certain gestational weeks, as shown in group IV. They also suggest that glucocorticoid suppresses the IL-6 concentration in CA (⫹) fetuses but the reduced IL-6 retains the capacity to induce lung maturity in neonates, as shown in group V. Such glucocorticoid-mediated failure to bring about any improvement in the incidence of RDS is consistent with the report, especially for neonates of less than 28 gestational weeks (Garite et al., 1992), although Papageorgiou et al. showed significant benefits of glucocorticoid with ritodorine to reduce the RDS incidence in babies at similar gestational age (Papageorgiou et al., 1989). In contrast to exogenously administered glucocorticoid, fetal IL-6 is an endogenous regulatory cytokine of pulmonary surfactant proteins and plays an especially important role in lung maturity of extremely premature babies. For clinical use of rIL-6 in the prevention of RDS, further studies will be required to examine whether IL-6 shows other multiple effects on fetal lung differentiation in addition to its effect on SP-A synthesis.

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