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PEDIATRICS Vol. 111 No. 3 March 2003, pp. 461-468

Soluble E-Selectin, Soluble L-Selectin and Soluble ICAM-1 in Bronchopulmonary Dysplasia, and Changes With Dexamethasone

Praveen Ballabh, MD*, Jaishree Kumari, MD*,{ddagger},§, Alfred N. Krauss, MD*, Junghee J. Shin, BS{ddagger}, Ajey Jain, MD*, Peter A. M. Auld, MD*, Martin L. Lesser§ and Susanna Cunningham-Rundles, PhD{ddagger}

* Departments of Neonatology
{ddagger} Hematology/Oncology/Immunology
§ Medical Statistics, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York

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   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objective. To evaluate longitudinal change in arterial blood plasma levels of soluble adhesion molecules in infants of <30 weeks’ gestation with respiratory distress syndrome (RDS) and to look for differences in these levels in neonates who subsequently developed bronchopulmonary dysplasia (BPD) compared with those neonates who did not, and also to investigate the effect of dexamethasone treatment on levels of soluble adhesion molecules in plasma.

Methods. We measured plasma concentrations of soluble L-selectin (sL-selectin), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule-1 on days 1, 3, 7, 14, 21, and 28 of life and before and 2 to 3 days after initiating a 6-day course of dexamethasone treatment. Infants with RDS were followed until discharge and were classified as non-BPD and either 1) BPD day 28 reflecting oxygen requirement on day 28 but not at 36 corrected weeks or 2) BPD 36 weeks reflecting oxygen requirement at 36 (corrected) weeks’ gestation. The classification of presence or absence of BPD by oxygen requirement was supported by and was consistent with radiologic findings of BPD for all infants. The difference between BPD day 28 and BPD 36 weeks was supported by more extensive radiologic effects in the latter.

Results. The arterial plasma level of sL-selectin in infants who had RDS and did not develop BPD was significantly decreased compared with term healthy infants, as was the level of sE-selectin. Compared with infants who had RDS and did not develop BPD, sL-selectin levels were even further decreased in infants who had RDS and did develop BPD both at birth and throughout the first 4 weeks of life (day 1 through day 28). Infants with BPD also showed increasing levels of sE-selectin during this period of time, whereas infants without BPD did not. Levels of soluble intercellular adhesion molecule-1 in infants without BPD were not different from infants with BPD initially but increased in infants with BPD compared with infants without BPD, significant on day 28 in both groups. Dexamethasone treatment increased concentration of sL-selectin and decreased concentration of sE-selectin.

Conclusions. Low sL-selectin may be an early indicator of enhanced risk for BPD. Low levels of sL-selectin and increasing levels of sE-selectin may be risk factors for BPD. The effects of dexamethasone treatment include significant modulation of adhesion molecules.

Key Words: soluble E-selectin • soluble L-selectin • soluble ICAM-1 • dexamethasone • bronchopulmonary dysplasia

Abbreviations: BPD, bronchopulmonary dysplasia • RDS, respiratory distress syndrome • sE-selectin, soluble E-selectin • sL-selectin, soluble L-selectin • sICAM-1, soluble intercellular adhesion molecule-1 • CPAP, continuous positive airway pressure • RMANCOVA, repeated measures analysis of covariance • RMANOVA, repeated measures analysis of variance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bronchopulmonary dysplasia (BPD) is a major complication of premature birth associated with younger gestational age that has important long-term consequences for the affected child.1,2 Although the pathogenesis of BPD is not well understood,3,4 a key role for inflammation has been suggested by several studies, indicating that inflammatory changes seem to precede the development of BPD.5–7 Inflammatory mediators and cytokines are released in the neonate by granulocytes and monocytes in response to stimuli such as postnatal ventilation, oxygen exposure, and microbial antigen exposure.6–8 This may lead to accumulation of granulocytes in the lung and subsequent development of BPD. Either infection or inflammation may initiate this process. Inflammation may cause neutrophil activation and transepithelial migration in the absence of clinical sepsis and has been implicated in respiratory distress syndrome (RDS), which precedes the development of BPD in the premature infant.9 The actual movement of neutrophils and monocytes through the vasculature and diapedesis into tissues is mediated by adhesion molecules.10 Therefore, we hypothesized that alteration in adhesion molecules would identify infants with RDS who would develop BPD. Specifically, the study presented here was designed to test 1) whether soluble adhesion molecules including soluble E-selectin (sE-selectin), soluble L-selectin (sL-selectin), and soluble intercellular adhesion molecule-1 (sICAM-1) are increased in infants who have RDS and develop BPD and 2) whether corticosteroid treatment with dexamethasone would decrease these levels.

The migration of neutrophils from capillaries to the alveoli, producing inflammation, occurs in several steps that may vary according to the setting but involve transient adhesion and rolling, followed by firm adhesion, and then transendothelial migration. Transient adhesion and rolling are mediated by E-selectin, P-selectin, L-selectin, and intercellular adhesion molecule, which have somewhat overlapping roles in neutrophil recruitment.11 Selectin-deficient mice show impaired leukocyte recruitment into inflammatory sites.12 A recent study reported increased levels of sE-selectin and sICAM-1 in cord blood and sera of premature infants who subsequently developed BPD.13 Decreased sL-selectin has been observed in the neonate, and even lower levels were found in the premature infant.14 Although the significance of increased levels of circulating soluble adhesion molecules has been evaluated in a wide range of infections and inflammatory conditions,8,15–21 possible relevance for the cause of BPD has not been systematically studied. Related studies in lung diseases in which inflammation has been shown to contribute to the development of lung injury have shown that injury is associated with elevated levels of soluble adhesion molecules, sE-selectin and sICAM-1, that were detectable in the circulating blood.22–24 Dexamethasone is widely used in the management of evolving BPD. Its anti-inflammatory effects are thought to alter the course of BPD by 1) maintaining capillary and alveolar membrane stability, thus preventing accumulation of fluid in the lung; 2) affecting cytokine release and subsequent influx of leukocytes into the alveoli; and 3) inhibiting fibrosis during the repair process.25,26 Dexamethasone administration has been reported to decrease sE-selectin and sICAM-1 concentrations in healthy adults,27but this has not been examined in the neonatal setting or even in inflammatory disease.

This investigation was based on the premise that the level of soluble adhesion molecules in plasma might be used as a kinetic indicator of this dynamic process providing a biological index of whether the infant with RDS would be more or less likely to develop BPD. To test this, we undertook a longitudinal study to obtain serial measurements of plasma sL-selectin, sE-selectin, and sICAM-1 during the first 4 weeks of life. In addition, we assessed the levels of adhesion molecules before and 2 to 3 days after initiating dexamethasone treatment. In these studies, we categorized infants as having BPD according to oxygen dependence at 28 days’ and 36 weeks’ corrected gestational age. The definition of BPD continues to be controversial. BPD has been defined as oxygen dependence beyond 28 days (BPD d28) with radiographic anomalies28 and also as oxygen dependence at 36 weeks’ (BPD 36 weeks) corrected gestational age.29 We evaluated longitudinal changes in soluble adhesion molecules comparing premature infants who had RDS and did not develop BPD with those who did according to both criteria.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The Institutional Review Board at Weill Medical College of Cornell University, New York, approved this study. Parental informed consent was obtained using an institutional review board-approved consent form. The study population was a sample of 57 neonates who were admitted to the neonatal intensive care unit at New York Presbyterian Hospital-Weill Medical College of Cornell University from April 1999 to July 2000. Patients were essentially enrolled consecutively except for those who could not be enrolled for logistic reasons or when the mother did not consent. Patients were preterm neonates who had RDS, weighed <1200 g, and were <30 weeks’ gestation. Infants were considered to have RDS when they required continuous positive airway pressure (CPAP) >1 day, mechanical ventilation >1 day, or supplemental oxygen >3 days with chest radiograph suggestive of RDS. Our practice is to use mechanical ventilation when oxygen requirement is >40% to 50% for infants on CPAP of 5 cm of water or when there is a significant respiratory acidosis. All infants who were placed on a ventilator received surfactant. Infants with culture-proven sepsis in the first 2 weeks or major congenital anomalies or death within 1 month were excluded. Of 57 infants enrolled, only 44 were eligible. Thirteen infants were excluded from the study: 7 because of death within 1 month and 6 for early neonatal sepsis. None of our infants had maternal history of documented chorioamnionitis. Infection is considered to be a contributory factor to the development of BPD. However, we excluded infants with culture-proven sepsis because alteration in adhesion molecules as a result of sepsis may confound our results on BPD.

In addition, 15 healthy adults and 15 healthy term neonates (<24 hours old) were enrolled in the study to compare their plasma adhesion molecule concentrations with premature neonates without BPD. The care of these children and their treatment were not affected by participation in this protocol. To assess the initial neonatal risk of the infants, we computed a clinical risk index for babies score30 for each neonate. This score is based on factors such as gestational age, birth weight, maximum and minimum inspired oxygen concentration and maximum base deficit in the first 12 hours of life, and presence of major congenital malformations.

Design
Arterial blood samples were collected on premature infants on days 1, 3, 7, 14, 21, and 28 of life. Nineteen infants received dexamethasone after 2 weeks of age for the purpose of weaning them off the ventilator or off oxygen, depending on their clinical status. This was done at the discretion of their attending neonatologist. Arterial blood samples were also taken before and 2 to 3 days after initiation and 2 to 3 days after discontinuing dexamethasone therapy. One blood sample was drawn from each of the healthy adults and term neonates who consented to participate. Volume of the blood drawn on preterm infants for the study was 2 mL on each occasion. Part of the blood sample was used for flow cytometry to study surface adhesion molecules on neutrophils and monocytes.31 Blood for the usual laboratory tests for premature infants such as complete blood count, electrolytes, and arterial blood gases were also drawn at the same time so that this did not cause an additional stick. We used arterial blood source because it was easier to draw 2 mL or more blood from an artery compared with a vein. The blood samples were collected in sodium heparin tubes. Spinning the blood sample at 5000 rpm for 5 minutes was used to separate plasma, which was aspirated. All plasma samples were stored at -70°C until assayed. The premature infants were followed until discharge and were classified into the following 3 groups: 1) non-BPD: these infants did not require oxygen at day 28; 2) BPD 28 days: these infants received oxygen at day 28 and not at 36 weeks’ (corrected) gestational age; our practice is to administer oxygen to infants to keep oxygen saturation between 90% and 95%; radiographic changes were consistent with BPD; 3) BPD 36 weeks: these infants received oxygen at day 28, and oxygen requirement was continued at 36 weeks’ (corrected) gestational age. Radiographic changes were compatible with BPD. Chest radiographs at 25 to 30 days of age of all neonates who were oxygen dependent on day 28 were reviewed by an experienced pediatric radiologist for the radiologic signs of BPD.32 The radiologist was blinded to the study groups.

Dexamethasone
Nineteen infants received a 6-day course of dexamethasone after 2 weeks of life. The initial dose was 0.5 mg/kg/d in 2 divided doses for 2 days, tapered to 0.25 mg/kg/d in 2 divided doses for next 2 days and then 0.125 mg/kg/d in 2 divided doses for the last 2 days. No infant received dexamethasone in the first 2 weeks of life, 15 infants received it in the third or fourth week of life, and 4 infants received it in the fifth or sixth week of life. Blood samples were drawn before initiation, 2 to 3 days after initiation, and 2 to 3 days after discontinuing dexamethasone therapy and at the time of their routine blood draw on 1, 3, 7, 14, 21, and 28 days of life. The blood levels of sE-selectin, sL-selectin, and sICAM-1 before (presteroid), 48 to 72 hours after (on-steroid), and after discontinuation of dexamethasone (off-steroid) were compared.

Measurement of Plasma Concentration of Adhesion Molecules
Measurement of plasma concentration of sE-selectin, sL-selectin, and sICAM-1 was conducted by quantitative sandwich enzyme immunoassay kits (R& D Systems, Minneapolis, MN). This method uses a micro plate with 96 wells precoated with monoclonal antibody specific for selected adhesion molecules. Standard samples and control were pipetted into these wells followed by polyclonal antibody conjugated with horseradish peroxidase in buffer. After a wash to remove any unbound antibody enzyme reagent, substrates for horseradish peroxidase were added. The intensity of color was measured by means of a microplate reader at 450 nm, with use of a correction wavelength set at 640 nm.

Analysis and Statistics
Plasma concentration of soluble adhesion molecules was studied as a function of postnatal age. Adhesion molecule levels in the BPD d28 and BPD 36 weeks infants were compared with infants without BPD (control). Analysis of variance for repeated measures (RMANOVA) was used to examine the changes in the adhesion molecule concentrations with postnatal days (day effect) and to compare infants without BPD with BPD d28 and BPD 36 weeks infants. On finding significant differences, Bonferroni corrected pair-wise comparisons using Mann-Whitney U test were conducted to determine which postnatal days differed from one another.

Additional analysis was performed by combining the 2 groups of infants with BPD and comparing adhesion markers to the group without BPD. This was done to obtain a larger sample size and to look for general relationships between adhesion markers and BPD. Repeated measures analysis of covariance (RMANCOVA) was used to adjust for the covariates, gestational age, and birth weight and to determine whether a particular outcome variable (sE-selectin, sL-selectin, sICAM-1) varied by group (patients without BPD versus patients with combined BPD) and whether a group-by-day interaction (ie, the difference between groups varied according to day) existed. Both BPD groups had chest radiograph changes consistent with BPD, and all of the infants in both groups were oxygen dependent on day 28. Analysis of residuals suggested that the normality assumptions required for application of RMANCOVA were tenable. An RMANCOVA result was considered significant at P < .05. On finding a significant group-by-day interaction, individual ANCOVA analyses with Bonferroni adjustment were conducted for each of the 6 days to determine whether the 2 groups differed on a specific day. Also, RMANCOVA was conducted to determine the presence of a day effect, separately for each of the 2 groups. It should be noted that in the absence of a group-by-day interaction, no separate analyses were performed for each day because lack of interaction indicates that the group effect was uniform across all days. Analysis of variance for repeated measures was also used to compare presteroid, on-steroid, and off-steroid levels. Statistical significance was attributed to P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The characteristics of the 44 infants with RDS who completed the study are shown in Table 1. Infants in the non-BPD group were significantly larger in weight and more advanced in gestational age at birth compared with infants in the BPD d28 and the BPD 36 weeks groups. Infants with more severe BPD (BPD 36 weeks) had significantly lower Apgar scores than the infants without BPD at 1 and 5 minutes (P < .01 each), but these values were not different in the less severe BPD group (BPD d28) of infants. None of the infants had 5-minute Apgar score <6. Mean number of days of mechanical ventilation, length of oxygen treatment, length of hospital stay, and clinical risk index for babies score were significantly greater in the 2 groups of infants with BPD compared with infants without BPD (P < .01 each). Fifty percent of infants without BPD did not require mechanical ventilation and were treated with CPAP, whereas all of the infants in the 2 BPD groups received mechanical ventilation. Use of prenatal steroid was similar among all 3 groups. In contrast, postnatal dexamethasone treatment was very different among the 3 groups. Only 14.3% of the infants in non-BPD group were treated with steroids during the third and fourth weeks of life compared with 41.2% in BPD d28 and 46.2% in BPD 36 weeks infants. Blood cultures were positive for the bacterial growth in 23.5% of infants in the BPD d28 group and 46.2% in the BPD 36 weeks group during the third and fourth weeks of life. Because it seemed possible that either the steroid or the presence of an infection might have effects on adhesion molecule levels, all of the data from time points during which infants were on dexamethasone treatment and for 3 days after discontinuing dexamethasone were excluded from the general analysis of longitudinal effects. Similarly, all of the data from time points when blood cultures were positive and until a negative culture was obtained were not included in this part of the analysis. Furthermore, none of the infants who completed the study were culture positive or received dexamethasone during the first 2 weeks, so no data from this period of study were removed from the analysis.


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  		TABLE 1. Characteristics of Infants

 
Soluble Adhesion Molecule Levels in Infants With RDS but Not BPD Versus Term Infants Versus Adults
We first examined the difference in soluble adhesion molecule levels among the groups of infants who initially presented with RDS but did not develop BPD compared with term infants and adults. Figure 1 presents the mean concentrations of the soluble adhesion molecules in premature neonates without BPD on day 1 compared with healthy term neonates on day 1 and that of healthy adults. The results showed that mean sL-selectin concentrations in infants with RDS who did not have BPD as neonates were significantly reduced compared with term neonates (P < .02) and healthy adults (P < .001). Mean sE-selectin level was also decreased in premature neonates without BPD compared with healthy term neonates (P < .004) but was significantly increased compared with adults (P < .01). sICAM-1 concentrations in premature infants without BPD were not significantly different compared with term infants and adults.



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  		Fig 1. sE-selectin, sICAM-1, and sL-selectin in adults (n = 15), healthy term infants day 1 (n = 15), and premature infants without BPD day 1 (n = 14). Data are expressed as means ± standard deviation (SD). sL-selectin concentrations were decreased in preterm infants without BPD compared with term infants (P < .02) and healthy adults (P < .001). sE-selectin levels were decreased in preterm infants without BPD compared with term infants (P < .004) but were increased compared with adults (P < .01). sICAM-1 levels were similar among the 3 groups (Mann-Whitney U test used).

 
Longitudinal Changes in Soluble Adhesion Molecules
sL-Selectin
Plasma sL-selectin levels were measured in serial arterial blood samples in premature infants with RDS and were compared among non-BPD, BPD d28, BPD 36 weeks, and combined BPD infants (Fig 2). sL-selectin concentrations increased with postnatal age in the 3 study groups of infants (P < .0001 each, RMANOVA model). However, infants who developed BPD showed plasma sL-selectin levels that were strikingly decreased despite this increasing trend. Levels of sL-selectin were lower in both groups of infants who developed BPD compared with the infants who had RDS and did not develop BPD. Levels were significantly decreased on days 1, 14, and 21 for the group of RDS infants classified as BPD d28 infants compared with infants without BPD (P < .006, .001, and .012, Mann-Whitney U test used, and Bonferroni correction made). Comparison of sL-selectin levels between BPD 36 weeks and infants without BPD were not significant after making the Bonferroni correction.



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  		Fig 2. sL-selectin concentrations in non-BPD, BPD d28, BPD 36 weeks, and combined BPD neonates in first 4 weeks of life. Data are expressed as means ± SD (unadjusted for weight and gestational age). sL-selectin concentrations increased with postnatal age in all groups of infants (P < .001 each, RMANOVA used). sL-selectin levels were significantly decreased in BPD d28 infants on days 1, 14, and 21 compared with non-BPD (P < .01, .001, and .012 respectively, Mann-Whitney U test, Bonferroni corrections made). Comparisons were not significant between non-BPD and BPD 36 weeks infants after making Bonferroni corrections. On adjusting the data for weight and gestational age, sL-selectin levels were significantly decreased in combined BPD infants compared with infants without BPD across all postnatal days (P < .023, RMANCOVA used).

 
For evaluating the possibility of a basic relationship between development of BPD and changes in sL-selectin, the 2 groups of infants who had RDS and developed BPD were combined into a single group (combined BPD) and compared with infants who had RDS and did not develop BPD. After weight and gestational age were adjusted for, sL-selectin levels were found to be significantly decreased in the infants with BPD compared with the infants without BPD (RMANCOVA, P < .023). This difference was significant across all days. As found for the analysis of separate BPD infant groups, sL-selectin level did increase significantly in the combined BPD group as well as in the non-BPD group as a function of postnatal age (P < .0001 for each). Despite this, sL-selectin in the combined BPD infants was again significantly lower compared with sL-selectin levels in the infants without BPD.

sE-Selectin
In contrast to the results described above for sL-selectin, sE-selectin plasma levels increased with postnatal age only in the 2 BPD groups of infants from day 1 through day 14 (P < .0001 each group studied separately, RMANOVA) but not in infants without BPD. Data are shown Fig 3. This difference was unrelated to gestational age and birth weight. Although sE-selectin levels were significantly increased on days 21 and 28 for both BPD d28 infants (P < .005 and .001) and BPD 36 weeks infants (P < .01 and .001) compared with infants without BPD, these differences proved to be attributable to lower birth weight and gestational age in the infants who developed BPD. When both groups of infants who had RDS and developed BPD were evaluated as a single group and compared with infants who had RDS and did not develop BPD, significant increase in level of sE-selectin was again seen over time for infants with BPD (P < .0001, RMANCOVA) but not for infants without BPD as observed before.



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  		Fig 3. sE-selectin concentrations in non-BPD, BPD d28, BPD 36 weeks, and combined BPD neonates in first 4 weeks of life. Data are means ± SD (unadjusted data shown). sE-selectin concentrations increased with postnatal age from day 1 through day 14 in all BPD groups of infants (P < .004 and .001, RMANOVA model) but not in infants without BPD. sE-selectin levels were significantly increased on days 21 and 28 for BPD d28 (P < .01 and .001) as well as BPD 36 weeks infants (0.001 each, Mann-Whitney U test used and Bonferroni corrections made) compared with infants without BPD. After adjusting the data for weight and gestational age, there was no significant difference in the sE-selectin levels between infants with and without BPD. However, there was significant increase in level of sE-selectin over time for combined BPD infants (P < .001) but not in infants without BPD (RMANCOVA used).

 
sICAM-1
sICAM-1 levels increased as a function of postnatal age in all 3 groups of neonates (P < .001 each). However, sICAM-1 was significantly increased in infants with BPD compared with infants without BPD. sICAM-1 level was significantly increased on day 28 for BPD d28 infants (P < .01) and on days 3, 14, 21, and 28 for BPD 36 weeks infants compared with infants without BPD (P < .01, .008, .001 and 0.001). When the 2 groups of infants with BPD were combined, sICAM-1 levels increased postnatally in both groups. When the BPD groups were combined, study of sICAM-1 levels showed an increasing trend that was significantly only on day 28 for infants with BPD compared with infants without BPD (P < .005, RMANCOVA; Fig 4).



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  		Fig 4. sICAM-1 concentrations in non-BPD, BPD d28, BPD 36 weeks, and combined BPD neonates in first 4 weeks of life. Data are means ± SD (unadjusted data shown). sICAM-1 level was significantly increased with postnatal age in all groups of infants (P < .001 each, RMANOVA). sICAM-1 level was significantly increased on day 28 for BPD d28 infants (P < .01) and on days 3, 14, 21, and 28 for BPD 36 weeks infants compared with infants without BPD (P < .01, .008, .001, and .001, Mann-Whitney U test used and Bonferroni corrections made). After adjusting for weight and gestational age, the levels were significantly increased in combined BPD infants only on day 28 compared with infants without BPD (P < .005, RMANCOVA used).

 
Effect of Dexamethasone Treatment on Soluble Adhesion Molecules
Because maternal dexamethasone treatment might affect the infant, we also examined the effect of antenatal betamethasone on day 1 levels of soluble adhesion molecules. We found no effect. Thus, adhesion molecule levels in infants who were exposed to antenatal steroid were comparable to that of infants who were not exposed to antenatal steroid.

To study the effect of dexamethasone on plasma sE-selectin, sICAM-1, and sL-selectin concentrations, we compared mean values of adhesion molecules before initiation, 2 to 3 days after initiation, and 2 to 3 days after discontinuing dexamethasone therapy, as shown in Fig 5. Dexamethasone treatment was associated with increased concentration of sL-selectin (P < .001) and decreased concentration of sE-selectin (P < .004) but did not significantly affect sICAM-1 levels.



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  		Fig 5. sE-selectin, sL-selectin, and sICAM-1 concentrations before (presteroid), 2 to 3 days after (on-steroid), and after discontinuation (off-steroid) of dexamethasone. Data presented as mean ± SD. Dexamethasone decreased sE-selectin (P < .004), increased sL-selectin (P < .001), and did not affect sICAM-1 (RMANOVA used). ICAM indicates intercellular adhesion molecule.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first longitudinal study of sL-selectin, sE-selectin, and sICAM-1 adhesion molecules in a relatively large group of micropremies with RDS at risk for the development of BPD. We found that reduced levels of sL-selectin were associated with subsequent development of BPD. It is interesting that dexamethasone treatment restored the level of sL-selectin. Increasing levels of sE-selectin were observed after birth in infants who developed BPD compared with those who did not. Changes in ICAM-1 levels were not as consistently associated with the development of BPD, although increased levels were significant in the infants with BPD at day 28.

Current studies suggest that sL-selectin, sE-selectin, and sICAM-1 are functionally active and reflect fundamental inflammatory processes including pulmonary disease.33–35 Previous studies14 have shown that sL-selectin was reduced in premature infants compared with term infants and support the findings of our longitudinal study. The data of Ramsey et al,13 who examined cord blood and serum samples from days 1, 3, and 7 in premature infants, are generally supportive of our results. They found higher levels of sE-selectin in serum samples obtained from cord blood and on postnatal days 1 and 3 in infants who developed BPD. When our data were adjusted for weight and gestational age, they showed that increasing levels of sE-selectins were observed only in infants who developed BPD. Increased levels of sICAM were also observed in a study of tracheal aspirates from infants with BPD.36 Our findings are somewhat different with respect to sICAM-1 because we observed that all infants with RDS showed increasing plasma levels of sICAM-1 and the inferences relevant to BPD were limited to day 28.

A fundamental question is whether there are developmentally related changes in adhesion molecules after birth that could suggest that the observed adhesion molecule changes related to BPD are superimposed onto an underlying program. A recent report showed that some soluble adhesion molecules, including sL-selectin, did not change in full-term healthy infants during early postnatal life.37 However, we found that all premature infants with RDS showed a postnatal age-related increase in sL-selectin, which was significantly less in the infants with BPD. In contrast, a postnatal increase in sE-selectin was seen only in those who did develop BPD. Additional postnatal changes in sICAM-1 were observed in all RDS groups, and the increased difference between BPD and non-BPD was significant only on day 28. Thus, the changes that we observed seem to be adhesion molecule specific and likely to reflect biological processes. Other previous reports38,39 support the suggestion that immaturity of granulopoiesis may be critical in the determination of which premature infants with RDS are at risk for development of BPD.

Recent studies suggest that L-selectin shedding has a regulatory effect on leukocyte recruitment such that experimental inhibition of shedding increased transmigration by affecting velocity over time and increasing firm adhesion mediated by CD11b/CD18.40 Other studies have shown that transit time of leukocytes rolling through venules affects leukocyte recruitment such that increased rolling caused by some types of inflammation may decrease recruitment.41 Although neither study is directly related to this investigation, both support a critical role for soluble selectin in regulation of neutrophil recruitment. More relevant are the studies of Koenig,42 which suggest that diminished sL-selectin in cord blood is associated with impaired shedding from activated neutrophils. We observed that lower levels of plasma sL-selectin were associated with development of BPD. In related studies, we have found reduced surface expression of L-selectin on neutrophils31 in infants with RDS who developed BPD compared with those who did not. Therefore, it seems that low sL-selectin in BPD neonates is probably related to decreased shedding from neutrophils associated with decreased surface expression of these molecules and presumably would promote greater neutrophil transmigration.

Soluble adhesion molecules have been studied in several clinical conditions in children.8,15–21,43,44 Plasma levels of these adhesion molecules are generally increased in a number of infectious16,17,21 as well as in inflammatory,17–20 endocrine,43 and cardiovascular conditions,44 a finding that possibly reflects enhanced cellular expression or shedding. The cellular expression of E-selectin and sICAM-1 has been shown to be upregulated by endotoxins,45 by proinflammatory cytokines including tumor necrosis factor-{alpha} and interleukin-1, and by soluble adhesion molecules that are released from activated endothelial cells in vitro.46

Plasma sL-selectin levels are increased in systemic inflammatory conditions such as sepsis and trauma as a result of increased shedding of L-selectin from leukocyte surface.47,48It has also been found to be elevated in adult patients of diffuse panbronchiolitis.49 However, patients with multiple trauma, pancreatitis, and perforated bowel who progressed to adult respiratory distress syndrome had significantly decreased initial plasma levels of sL-selectin compared with those who did not and also compared with healthy adults.35

In this investigation, we found that dexamethasone treatment increased low sL-selectin levels and decreased sE-selectin levels in neonates with BPD. In addition, these levels dropped to the baseline after discontinuation of dexamethasone. We do not know the relevance of these findings with respect to chronic lung disease and the long-term outcome of these infants. Studies done in healthy adult volunteers have also shown that sE-selectin and sICAM-1 concentrations were reduced by 22% and 15%, respectively, after dexamethasone treatment at a dose of 1 mg/kg twice a day for 2 days.25 In contrast, in the present study, the level of sICAM-1 was not significantly affected by dexamethasone. This finding may be supported by a report that steroid treatment did not affect sICAM-1 in patients with multiple sclerosis.50 These differences may also reflect that sICAM-1 is a product of many cells and that steroid would suppress different activated cells in different clinical conditions. This study is the first to show that dexamethasone treatment led to increased levels of sL-selectin in infants with BPD. Related work has shown that that L-selectin (CD62L) was decreased on circulating neutrophils and monocytes isolated from premature infants with BPD who had received dexamethasone.51 Therefore, we speculate that dexamethasone may increase L-selectin shedding, leading to increased levels in the plasma and decreased levels on the surface of neutrophils, which would be expected to reduce neutrophil-endothelial interaction through CD18/11b and decreased delivery of neutrophils to the inflamed lung of the infant with BPD. It has been reported that lower cortisol levels during days 2 to 7 of life correlate with development of BPD.52 Banks et al53 also recently reported that there is a borderline significant association between low cortisol and increased risk of BPD (P = .08). We have observed that dexamethasone decreased sE-selectin and increased sL-selectin in plasma. Therefore, it is possible that low cortisol levels may be an underlying cause of high sE-selectin and low sL-selectin in neonates with BPD. In this study, we did not find any significant effect of antenatal betamethasone on day 1 level of adhesion molecules. This is probably because of low concentrations of betamethasone achieved in blood of the newborns as a result of short half-life of betamethasone in mothers (approximately 6 hours), consistent with lower cord blood levels and maternal ratio of betamethasone (0.37–0.4). In addition, mothers receive variable doses of betamethasone at different times, anywhere from a few weeks to 1 hour before the delivery of the neonate.54

These studies suggest that additional investigation of adhesion molecules in premature infants who are at risk for BPD may reveal fundamental biological relationships that could lead to new understanding of this critical disorder.


   ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health grant RR 06020 and NCI 129502.


   FOOTNOTES
 
Received for publication Feb 26, 2002; Accepted Aug 12, 2002.

Reprint requests to (S.C.-R.) New York Presbyterian Hospital-Weill Medical College of Cornell University, 525 E. 68th St, New York, NY 10021. E-mail: scrundle{at}med.cornell.edu

Dr Ballabh’s current affiliation is Division of Newborn Medicine, Westchester Medical Center, Valhalla, New York.

Presented at a meeting of the Society of Pediatric Research; April 29, 2001; Baltimore, MD.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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