Allergol et Immunopathol 1999;27:11-17.
Markers of pulmonary inflammation in tracheobronchial fluid of premature infants with respiratory distress syndrome
E. Burón, J. A. Garrote, E. Arranz*, P. Oyágüez, J. L. Fernández Calvo and A. Blanco Quirós
Dpt. of Paediatrics and *Immunology. Hospital Clínico. University of Valladolid, Spain.
SUMMARY
To asses the role of pulmonary inflammation in the outcome of preterm neonates with respiratory distress syndrome (RDS) we measured soluble intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), eosinophil cationic protein (ECP) and transforming growth factor beta-1 (TGFß1) in tracheobronquial lavage (TBL) fluid of 15 preterm infants; 9/15 completely recovered from RDS and 6/15 developed chronic lung disease (CLD).
ICAM-1 (p: 0.001) and TGFß1 (p: 0.04) levels increased in TBL fluid during the first days of life. The values of ICAM-1 were correlated to the days of 02 and mechanical ventilation dependency. At 3 days of age, ICAM-1 levels in TBL fluid were higher in infants who later developed CLD compared to infants without CLD (24.5 vs 8.3 µg/ml; p: 0.02). Thereafter no significant differences were found although the CLD group had higher values. IL-8 levels showed a fall, specially from 1 to 3 days of age in children without CLD (77.0 to 41.7 ng/ml) although not significant. No differences in TGFß1 values were found between both groups, but the TGFß1 levels were lower in patients with CLD and they showed undetectable values in 8 samples.
ICAM-1 is a major factor associated with airways inflammation whereas IL-8 is not a good marker during the first days of life to predict the RDS outcome. A defect of TGFß1 in the smallest premature infants may delay the lung repair process which occurs after tissue injury. High ICAM-1 levels and low TGFß1 levels in lung fluid are related to oxygen dependency at 28 days of age.
Key words: Respiratory distress syndrome. Chronic lung disease. Prematurity. ICAM-1. IL-8. ECP. TGFß1.
RESUMEN
Para valorar el papel de la inflamación pulmonar en la evolución de neonatos pretérmino con síndrome de insuficiencia respiratoria aguda (IRA) medimos los niveles de la molécula de adhesión intercelular soluble 1 (ICAM-1), interleucina-8 (IL-8), proteína catiónica de los eosinófilos (ECP) y factor transformado de crecimiento beta-1 (TGFß1) en el fluido de lavado traqueobronquial (LTB) de 15 recién nacidos pretérmino. Nueve de los 15 neonatos se recuperaron completamente de la IRA y 6 de los 15 neonatos desarrollaron neumopatía crónica (NPC).
Los niveles de ICAM-1 (p: 0,001) y TGFß1 (p: 0,04) en el líquido del LTB aumentaron en los primeros días de la vida. Los valores del ICAM-1 se correlacionaron con el día 2 y con la dependencia de la ventilación mecánica. A los tres días de edad, los niveles de ICAM-1 en el líquido TB fueron mayores en los recién nacidos que evolucionaron a NPC comparado con los recién nacidos sin NPC (24,5 frente a 8,3 µg/ml; p: 0,02). A partir de entonces no se encontraron diferencias significativas, aunque el grupo de NPC tuvo valores más elevados. Los niveles de IL-8 evidenciaron una caída, sobre todo entre los días 1 y 3 de la vida, en los recién nacidos que no evolucionaron a NPC (de 77,0 a 41,7 ng/ml), aunque este cambio no fue significativo. No se encontraron diferencias en los valores de TGFß1 entre los dos grupos, pero los niveles de TGFß1 fueron más bajos en los pacientes que evolucionaron a NPC no se detectó en ocho muestras.
La ICAM-1 es un factor importante que se asocia con inflamación de las vías aéreas, pero la IL-8 no es un buen marcador en los primeros días de la vida para predecir la evolución de una IRA. Un defecto del TGFß1 en los recién nacidos prematuros más jóvenes puede atrasar el proceso de reparación pulmonar que se produce en respuesta a las lesiones hísticas. Los niveles altos de ICAM-1 y niveles bajos de TGFß1 en el líquido pulmonar se relacionaron con la dependencia de oxígeno a los 28 días de vida.
Palabras clave: Síndrome de insuficiencia respiratoria aguda. Neumopatía crónica. Pretérmino. ICAM-1. IL-8. ECP. TGFß1.
INTRODUCTION
Respiratory distress syndrome (RDS) is a severe disturb of preterm newborns and in spite of new mechanical ventilation techniques have improved their survival an incresing number of infants are later complicated with a chronic lung disease (CLD) (1). The pathogenesis of RDS and CLD remains partially unclear. The surfactant deficiency is the initial cause, but the airways inflammation is very likely implicated in the process (2) because tumor necrosis factor alpha and interleukin-6 are present in secretions (3).
Even in absence of bacterial colonisation, bronchial fluid of prematures contains enough inflammatory cells for developing a local inflammation (4-8). Migration of these cells in tissues occurs under the influence of local chemoattractants (9), such as interleukin-8 (IL-8) which has been detected in sputum and bronchoalveolar lavage fluid of patients with diseases characterised by neutrophilic infiltration as it happens in cystic fibrosis (10), adult respiratory distress syndrome (11) and bacterial pneumonia (12). The cell migration across endothelial cells is dependent on the intercellular adhesion molecule (ICAM-1), extensively upregulated in inflammatory disorders (13). The role of IL-8 has been studied in preterm babies with respect to the pathogenesis of CLD (14-16) and raised levels of soluble ICAM-1 and eosinophil cationic protein (ECP) have been found in the tracheal aspirate of infants with CDL (14, 17, 18). Pulmonary fibrosis is prominent in infants who die of CLD (19). Several lines of evidence point to transforming growth factor beta 1 (TGFß1) as a key cytokine in tissue repair, and the sustained production of TGFß1 underlies the development of tissue fibrosis (20). Broncheoalveolar cells obtained by lavage from patients with autoimmune diseases and lung fibrosis contained 10 times more TGFß1 mRNA than similar cells from normal subjects or patients with asthma (21). High concentrations of TGFß1 from TBL fluid were reported in infants with CLD (22).
A major difficulty for research and clinical practice is the absence of a standard definition of CLD. The diagnostic criteria established by Northway (23) were later reviewed (24) and it has become apparent that in extremely low birth weight infants the length of oxygen requirements is a function of immaturity (25). We believe that cytokine study in lung fluid may be useful to identify the infants prone to CLD. The aim of this study was to evaluate whether levels of some inflammatory factors in tracheobronchial lavage (TBL) fluid of preterm newborns are related to: a) Infant immaturity. b) Severity of RDS. c) Evolution of RDS to CLD.
PATIENTS
Fifteen preterm infants, from 25-35 gestational weeks, all requiring assisted ventilation for RDS, were consecutively recruited during the study period. Microbiological studies were weekly performed in all infants. Patients who developed clinical, bacteriological or radiological pulmonary infection evidence were excluded. Intravenous antibiotic therapy was given to all infants during the first 10 days. Prenatal maternal steroids were administered in 4 infants (table I). Before the first sample of TBL fluid, all preterm babies included in the study received one o two doses of natural surfactant. None infant was treated with dexamethasone before the 10th day of age and we did not analyse samples after the steroid therapy was started. After discharge from hospial, infants included in the study were followed-up for 2 years and none of them have to be admitted to the hospital for respiratory causes. The diagnosis of CLD was retrospectively made on the basis of oxygen dependency by 28th day of life and clinical and X-ray abnormalities (25, 26). Patients were divided in 2 groups:
Table I Clinical characteristics of studied premature infants | |||
| RDS with CLD | RDS without CLD | p | |
| Number | 6 | 9 | NS |
| Gestational age (w.)* | 27 (26-30) | 31 (27-32) | < 0.01 |
| Birth weight (g)* | 993 (650-1440) | 1465 (960-2021) | < 0.05 |
| Mechanical ventilation (days)** | 24.7 ± 11.7 | 6.6 ± 3.8 | < 0.0001 |
| O2 dependency (days)** | 51.8 ± 12.3 | 12.6 ± 10.5 | < 0.0001 |
| O2 at corrected age (w.)** | 33 ± 2 | 31 ± 2 | NS |
| Antenatal steroids | 2/6 | 2/9 | NS |
| Postnatal steroids | 5/6 | 2/9 | NS |
| *mean and range. | |||
| **mean ± SD. | |||
a) RDS groups (n: 9). Infants who received mechanical ventilation for RDS, subsequently recovered without CLD and they were nursed in air before 28 days of age.
b) CLD group (n: 6). Infants who required mechanical ventilation for RDS and subsequently developed CLD.
The study was approved by the local Hospital Ethics Committee.
METHODS
Sampling of TBL fluid
Tracheobronquial lavage was performed once a day, at the time of clinically indicated tracheal suctioning. A standardise procedure for tracheobronchial aspiration was performed by instillation of sterile 0.9% saline (0.5 ml/kg) into the endotracheal tube, while the infants were supine. Then the infant was hand-ventilated and a soft catheter was advanced through the end porthole of the endotracheal tube and gently inserted beyond the distal tip of the tube. Airway secretions were immediately aspirated with a negative pressure no greather than 60-100 Hg mm. The lavage fluid was collected in sterile specimen traps. Samples were centrifuged at 4,000 rpm for 10 minutes and the supernatant storet at -20° C until further analysis. The first sample was taken 24 hours after birth. Subsequent samples of intubated babies were taken at 3, 7 and 10 days of age.
TBL fluid dilution factor
The dilution of the epithelial lining fluid in lavage medium was calculated as the ratio of TBL fluid urea concentration to plasma urea concentration (27). Serum urea was measured within 4 h of the collection of lavage fluid by a ureasa kinetic method, using a Hitachi 747 analyser. Urea concentration in lavage fluid was measured on the same analyser at an increased sensitivity whereby a linear dynamic range was achieved over 0.04-6.0 mmol/L. Concentrations of ICAM-1, IL-8 and TGFß1 in TBL fluid were adjusted using this ratio.
Measurements of factor levels
Commercial enzyme linked immunosorbent assay was used to measured levels of ICAM-1, IL-8 (Bender Med Systems, Austria), TGFß1 (Amersham Int. England). To measure total TGFß1, latent TGFß1 was previously activated by acidification of samples at room temperature, and neutralised to a pH 7.4 by the addition of alkali. All samples were measured in duplicate and the results averaged. ECP was determined by the CAP system (Pharmacia) using an automatic assay (Auto-CAP) and measuring the fluorescent reaction by a FluoroCount 96.
Statistical analysis
Difference between RDS and CLD groups were compared by using the Man-Whitney U test and the correlation value with the non parametric Spearman''s test. The unpaired t-test was applied to assess differences in clinical data. Results were expressed as median and 25th%-75th% interquartiles. A p value < 0.05 was considered statistically significant.
RESULTS
Clinical differences were found between groups with and without CLD when the gestational age (p < 0.01), birth weight (p < 0.05), days of mechanical ventilation (p < 0.0001) and days of oxygen requirements (p < 0.0001) were compared, although no difference was observed between both groups when oxygen dependency at a post-conceptional age was compared (table I).
TGFß1 and ICAM-1 values increased along the first days of life, showing a significant correlation with the age (p: 0.04 and p: 0.001, respectively) (Fig. 1). On the contrary, any factor was correlated to the gestational age although there was a negative trend of TGFß1 (p: 0.08) and IL-8 levels (p: 0.16) in the first day of life values (table II). The total requirement days of mechanical ventilation and the O2 administration only show significant correlation with ICAM-1 levels in TBL fluid (Fig. 2).
Table II Correlation of inflammatory factors with several characteristics | ||||
| ICAM-1 | IL-8 | ECP | TGFß | |
| Gestational age | NS | NS | NS | NS |
| Days of postnatal life | p: 0.001 | NS | NS | p: 0.04 |
| Days of mechanical ventilation | p: 0.01 | NS | NS | NS |
| Days of O2 | p: 0.009 | NS | NS | NS |
| NS: not significant; p calculated by Spearman test. | ||||
Figure 1.--A positive correlation was present between the days of post-natal age and the tracheobronchial transforming growth factor beta-1 (TGFß1) and the intercellular adhesion molecule-1 (ICAM-1) levels in samples from tracheobronquial lavage fluid (Spearman''s test).
Figure 2.--The values of intercellular adhesion molecule-1 determined in tracheobronchial fluid showed a positive correlation with respect to the number of requiered days of O2 therapy (p: 0.009) and mechanical ventilation (p: 0.01).
At 3th day of age, ICAM-1 levels in TBL fluid were higher in the group with CLD than in the group without CLD (24.5 vs 8.3 pg/ml; p: 0.02), these differences were not statistically significant at days 1, 7 and 10, although ICAM-1 levels were higher at anytime in the CLD group (Fig. 3). IL-8 levels were not different in RDS and CLD groups at any time of the study. In infants with CLD, IL-8 concentrations decreased from 34.2 ng/ml on day 1 to 21.0 ng/ml on day 10. In the group without CLD and IL-8 decrease was also noted from 77.0 ng/mL on day 1 to 41.7 ng/mL on day 3, with a mild ulterior trend towards increase. Transforming growth factor beta-1 (TGFß1) levels in TBL fluid were lower in the CLD group as compared to the RDS group without CLD but no significant differences were found, and levels were undetectable in 8 samples of lavage fluid taken at various times only from the CLD group (table III).
Table III Tracheobronchial values of inflammatory factors in preterm infants with and without later chronic lung diseases (CLD) | ||||||||||
| ICAM (µg/ml) | IL-8 (ng/ml) | TGFß1 (ng/ml) | ECP (ng/ml) | |||||||
| age (days) | CLD | no CDL | CLD | no CDL | CLD | no CDL | CLD | no CDL | ||
| 1 | n = 6 | n = 9 | n = 4 | n = 9 | n = 2 | n = 6 | n = 2 | n = 3 | ||
| 4.17 | 4.22 | 34.27 | 77.04 | 0.7 | 2.28 | 4,21 | 1,05 | |||
| (1.28-5.97) | (2.25-5.88) | (18.9-63.6) | (37.9-131) | (1.01-2.38) | (0.36-4.11) | |||||
| 3 | n = 5 | n = 8 | n = 5 | n = 8 | n = 5 | n = 8 | n = 5 | n = 2 | ||
| 24.58 | 8.34* | 35.64 | 41.78 | 0.2 | 1.95 | 2.89 | 5.11 | |||
| (11.2-35.6) | (4.99-9.96) | (30.6-37.2) | (21.6-72.5) | (0-1.32) | (0.56-2.90) | (1.74-12.6) | ||||
| 7 | (n = 6 | n = 8 | n = 6 | n = 8 | n = 6 | n = 8 | n = 4 | n =1 | ||
| 22.20 | 7.95 | 30.70 | 47.96 | 0.4 | 3.09 | 13.8 | 373 | |||
| (7.8-31.5) | (5.14-11.4) | (17.1-44.3) | (22.9-77.1) | (0.1-2.59) | (1.16-3.22) | (5.75-17.4) | ||||
| 10 | n = 4 | n= 2 | n = 4 | n = 2 | n = 4 | n = 2 | n = 2 | n = 2 | ||
| 21.58 | 14.05 | 21.06 | 57.33 | 0.05 | 2.27 | 4.73 | 1.95 | |||
| (16.6-36.8) | (9.8-22.8) | (0-0.55) | ||||||||
| Values expressed as median and 25-75 interquartiles (in brackets). | ||||||||||
| *p: 0.002. | ||||||||||
Figure 3.--Concentrations of ICAM-1 in tracheobronchial lavage fluid from prematures with non complicated respiratory distress syndrome (RDS) and complicated with later chronic lung disease (CLD). Values are expressed in median and interquartiles. (*p: 0.02).
The studied factors on TBL fluid from prematures infants show a very independent behaviour among them and only TGFß1 and ECP levels show each other a mild correlation (p: 0.04) (table IV).
Table IV Correlation among inflammatory factors obtained from tracheal fluid samples | ||||
| ICAM-1 | IL-8 | ECP | TGFß | |
| ICAM-1 | XXX | NS | NS | NS |
| IL-8 | XXX | NS | NS | |
| ECP | XXX | 0.04 | ||
| TGFß | XXX | |||
| NS: not significant; p calculated by Spearman test. | ||||
DISCUSSION
Neonatal bronchoalveolar lavage has not been standardised in premature babies but several workers have adapted methods for intubated infants using non-bronchoscopic techniques. Saline instillation may be done with a fixed volume or adjusted according to body weight, may be injected directly into the endotracheal tube or via a catheter located through the right main bronchus. It is likely that the same caveats that apply to bronchoscopic lavage will operate in these situations and care should be taken when interpreting results obtained from different techniques. Selective "suction bronchoalveolar lavage" of intubated infants appears to sample both the proximal and distal airways (28), but we used a non-selective suctioning because it was part of the normal nursing care of intubated babies. Further work is required to determine whether levels of cytokines in lung fluid depend on the method of sampling.
The effect of steroids on CLD is controversial but positive results were recently published and it supports the active role of the inflammation (29, 30). All our determinations were done before postnatal steroids were administered to infants and the prenatal steroids was used only in 4 cases, 2 mothers of children with CLD and 2 without CLD.
Gestational age did not influence the respiratory levels of inflammatory factors, neither on day 1 nor on following days. Nevertheless a trend to decreased values was found in TGFß1 and IL-8 determinations.
We found ICAM-1 and Il-8 detectable levels in TBL fluid from all preterm infants with RDS. In agreement with previous reports, it suggests the existence of inflammation in the airways of infants with RDS requiring mechanical ventilation after birth (2, 14-17). Our results show that ICAM-1 levels were higher in infants who developed CLD, but no differences were found in IL-8 concentrations between the two groups at any time of the study. We also found that TGFß1 levels had a trend to be lower in infants with CLD than infants without CLD.
In our study, infants with CLD showed considerable increase of ICAM-1 values obtained on 3th day of age, although thereafter not overall changes were found in this group (Fig. 1). Kotecha et al (14) reported that high concentrations of ICAM-1 in tracheal aspirates from premature infants at 10th day of age predict the development of CLD. Our increase was more precociously patent. Only two infants remained intubated at 10 th day in the group without CLD and they had elevated values of ICAM-1.
ICAM-1 was the only factor shich show a positive correlation with the number of days that infants needed be treated with mechanical ventilation or oxygen therapy. Both variables are related to the severity of the RDS and its evolution to a CLD. These results support the hypothesis that high ICAM-1 levels in the first days of age or their increase from previous values cold identify the premature infants who are prone to develop CLD. Increased levels of ICAM-1 were found also in blood at 3-7 days of life by Ramsay et al (31) although the raise was not present at birth, on the contrary, there was elevated E-selectin levels in cord blood before starting the RDS (31), suggesting that this adhesion molecule favours the respiratory inflammation.
Interleukin-8 has been proposed as a major neutrophil chemoattractant in lung (32). High IL-8 levels were reported by day 10-15 in TBL fluid from premature infants who developed CLD (1, 10). Some authors have suggested that an IL-8 raise in the first hours after birth is a good predictor of CLD in premature babies (14). We found the highest IL-8 levels during the first days of life in all infants without differences between groups with and without CLD. However, it is noteworthy to mention than in the first hours of life, part of IL-8 may be of maternal origin via aspiration of amniotic fluid (33, 34) and tis prognostic value for detecting CLD risk could be affected.
The absence of a significant difference may be related to the wide variability of Il-8 values as it was published in other studies (35). On the other hand, IL-8 appears to be a marker of disease progression (16) and it is possible to speculate that at the time of finishing our study differences between groups had not been yet established. It would be necessary to study infants after the 10th day of life to set the role of this cytokine up in the pathogenesis of
CLD.
The sequence of events leading to tissue fibrosis involves the inflammation and the repair process (36). The release of cytokines in response to injury is a central event in tissue repair. At physiological concentrations, TGFß1 is involved in normal remodelling (37, 38), but after repeated injury, the TGFß1 production is sustained, leading to the a progressive deposition of extracellular matrix and fibrosis (39). In preterm infants, TGFß1 concentrations were increased at 4th day of age in infants who developed CLD, suggesting that early perinatal factors had a pivotal role in the pathogenesis of CLD (22). Other authors reported that TGFß1 levels were low in the first 24 hours of life and increased 3-6 fold increase with a TGFß1 bioactivity peaking at 20-25 day (40). These findings implicate TGFß1 in tissue injury and repair but its role in the premature human lung remains unclear because it is also had potent antiinflammatory activity (41). Although no statistically significant, we have found lower TGFß1 concentrations in less mature infants and cases who later developed CLD. It is possible to speculate that a low production of TGFß1 may signal a defect to control tissue remodelling which can prolong the lung injury and triggering subsequent CLD.
In our study infants with CLD requiered oxygen at 28 days of age, but they were all on room air at 36 weeks'' postconceptional age, pointing a less severe process. Nevertheless, it has been shown that the ned for oxygen at 28 days is only a good predictor for CLD in infants of >= 30 weeks of gestational age but become progressively useless as gestational age decreased (25). The question whether oxygen dependency at 28 days is associated to the lung immaturity or lung inflammation and fibrosis, remains open.
In summary, ICAM-1 was higher in TBL fluid from premature infants who later suffered CLD and it may be an early inflammation marker of premature infants prone to develop CLD. By contrast, measurement of IL-8 during the dirst days of age were useless and low TGFß1 levels in the first days of age were found in infants with CLD.
Acknowledgments
This work was supported by a grant from Neonatal Spanish Society. We thant the Nursing Staff of Neonatal Unit at University Hospital of Valladolid for their help in obtaining TAF samples.
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Correspondence:
Prof. A. Blanco Quirós
Facultad de Medicina. Pediatría
Ramón y Cajal, 5
47005 Valladolid. Spain.
