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Neutrophil Activation in Preterm Infants Who Have Respiratory Distress Syndrome

Irmeli Nupponen, MD^*,, Eero Pesonen, MD, PhD, Sture Andersson, MD, PhD^,||, Aila Mäkelä, MD^*,, Riikka Turunen, BM^*,, Hannu Kautiainen, BA^¶ and Heikki Repo, MD, PhD^*,#

^* Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
Department of Anesthesiology, University of Helsinki, Helsinki, Finland
Hospital for Children and Adolescents, Helsinki, Finland
^|| Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland
^¶ Rheumatism Foundation Hospital, Heinola, Finland
^# Department of Medicine, Division of Infectious Diseases, University of Helsinki, Helsinki, Finland

	ABSTRACT

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Objective. To study neutrophil activation in circulation as a sign of systemic inflammation in preterm infants with respiratory distress syndrome.

Methods. The study comprised very low birth weight preterm infants who had respiratory distress syndrome and required intubation and mechanical ventilation (n = 51), 1-day-old preterm infants who had no need for mechanical ventilation (n = 12), term infants (n = 47), and adult volunteers (n = 25). Neutrophil surface expression of CD11b was quantified with flow cytometry.

Results. In preterm infants with respiratory distress syndrome, neutrophil CD11b expression during the first day of life was higher than in cord blood (mean: 165 relative fluorescence units [RFU] [standard deviation [SD]: 53], n = 29 vs 83 RFU [SD: 21], n = 11; 95% confidence interval [CI] for difference: 59–106) or in preterm infants without mechanical ventilation (106 RFU [SD: 33], n = 12; 95% CI for difference: 17–90). CD11b expression decreased by age of 10 days. CD11b expression was lower in preterm cord than in term cord blood (95% CI for difference: 5–53). However, in preterm infants with respiratory distress syndrome aged 2 to 5 days, it was higher than in term infants of that age.

Conclusions. The observations demonstrate an early transient postnatal neutrophil activation indicative of systemic inflammation that may contribute to the tissue injury in preterm infants with respiratory distress syndrome.

Key Words: CD11b/CD18 • C-reactive protein • neutrophil activation • preterm infant • respiratory distress syndrome • systemic inflammation

Abbreviations: ARDS, adult respiratory distress syndrome • FMLP, formyl-methionyl-leucyl-phenylalanine • RDS, respiratory distress syndrome • BPD, bronchopulmonary dysplasia • FIO₂, fraction of inspired oxygen • SD, standard deviation • FACS, fluorescence-activated cell sorter • RFU, relative fluorescence unit • ANOVA, analysis of variance • CI, confidence interval

	INTRODUCTION

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A host’s first reaction to inflammatory stimuli such as infection, trauma, and immune-mediated tissue injury may be a systemic inflammatory response,¹ which is frequently complicated by development of organ failure such as adult respiratory distress syndrome (ARDS). The major pathogenetic mechanisms of ARDS are considered to involve systemic release of proinflammatory cytokines, which stimulate vascular endothelium, and activation and subsequent pulmonary sequestration of circulating phagocytes, ie, neutrophils and monocytes.^2,3 Activated neutrophils generate toxic oxygen species and release proteolytic enzymes, which may result in microvascular injury with subsequent increase in pulmonary vascular permeability, a pathognomonic feature of ARDS.

The activated endothelium is considered to play a crucial role in neutrophil pulmonary sequestration. The initial neutrophil endothelial adhesion, a reversible event, is mediated by the selectin family of cell adhesion molecules⁴ and is considered to result in activation of neutrophils by locally available agonists such as platelet activating factor.⁵ Neutrophil activation is characterized by emergence of neo-epitopes on CD11b/CD18 (Mac-1, _Mß₂, CR3),⁶ a ß₂-integrin constitutively expressed at low levels on resting neutrophils, which renders neutrophil-endothelial adhesion irreversible, and, furthermore, by an increase in CD11b/CD18 complex density on neutrophils.^7,8 The latter derives from translocation of CD11b/CD18 molecules from the intracellular storage granules, ie, secretory vesicles and specific granules including gelatinase granules, to the cell surface.⁸ In unstimulated neutrophils, only 5% of the total cell content of CD11b/CD18 complexes is expressed on the cell surface, whereas 75% is as membrane components of specific granules and 20% as membrane components of secretory vesicles.⁹

Secretory vesicles are readily mobilized in response to a weak neutrophil stimulus that barely mobilizes the granules,¹⁰ whereas a strong neutrophil agonist, such as formyl-methionyl-leucyl-phenylalanine (FMLP), promotes exocytosis of secretory vesicles and, in part, also of specific granules.⁹ Although enhanced expression of CD11b/CD18 is insufficient for neutrophil adhesion,¹¹ it serves as an activation marker of neutrophils.^12–14 Indeed, increased neutrophil CD11b/CD18 expression is well documented in adult patients with systemic inflammation triggered by sepsis,^15,16 trauma,¹⁷ and burn injury¹⁸ and may even predict the development of organ failure in patients with cirrhosis of the liver¹⁹ and septic shock.²⁰

Surfactant deficiency in the lungs of preterm infants leads to respiratory distress syndrome (RDS). Similar to the patients with early ARDS, premature infants with RDS evidently also have an early inflammatory reaction in their lungs.^21,22 Pulmonary inflammation is probably a key factor also in the pathogenesis of bronchopulmonary dysplasia (BPD),^23,24 the development of which begins during the first weeks of life.²⁵

Recent data show that the plasma level of elastase/ ₁-proteinase inhibitor complex per neutrophil correlates directly and platelet-activating factor-inhibiting capacity of plasma correlates indirectly with the severity of RDS, providing indirect evidence of systemic neutrophil activation.^26,27 The present study was undertaken to evaluate neutrophil activation as a sign of systemic inflammation in preterm infants with RDS during the first postnatal week in relation to pulmonary morbidity.

	METHODS

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Patients
This study was conducted between March 1998 and January 2001 at the Hospital for Children and Adolescents, University of Helsinki (Helsinki, Finland). The study protocol was approved by the institutional review board, and informed consent was obtained from the parents. The present series consists of 4 separate groups: preterm infants requiring intubation and mechanical ventilation (n = 51; Table 1), 1-day-old preterm infants with no need for mechanical ventilation (n = 12; Table 1), healthy term infants (n = 47), and adult volunteers (n = 25). For the preterm infants with RDS, surfactant treatment (Curosurf, 100 mg/kg; Chiesi Farmaceutici SPA, Parma, Italy) was started when the arterial alveolar oxygenation ratio was below 0.22. The first dose of surfactant was given at the age of 1 to 24 hours. As standard treatment, every patient received ampicillin 200 mg/kg and netilmicin 6 mg/kg from the first day of life for at least 7 days. Concerning complications of pregnancy, the criteria for chorioamnionitis were 1) clinical signs of the mother (fever, tenderness of the uterus), 2) leukocytosis (blood leukocyte count >14 x 10⁹/L), and 3) C-reactive protein concentration in plasma >50 mg/L. The criteria of preeclampsia were 1) elevated blood pressure and 2) proteinuria. All mothers received glucocorticoid treatment ante partum (1–5 courses of betamethasone given as 2 doses of 12 mg at a 12-hour interval) to reduce the frequency of RDS. Four preterm infants died by the age of 10 days.

Blood Samples
From preterm infants with RDS delivered by cesarean section, cord blood samples were taken from the umbilical vein with a pyrogen-free syringe within 2 to 4 minutes after the placenta was detached. From 4 of these and from the other 40 preterm infants with RDS, blood samples were taken, whenever possible, from the arterial cannula on the first day of life between 6 and 24 hours after birth and on the second, third, fifth, seventh, and tenth days. The first sample was always taken after the first dose of surfactant. From each of the 12 preterm infants without RDS, 1 peripheral blood sample was drawn concurrently with the clinical samples requested by the clinician.

Cord blood samples were collected from 30 of the 47 healthy term infants after uncomplicated pregnancy and delivery as described above, and postnatal samples were collected from the other 17 at the age of 2 to 5 days. The latter infants were healthy with the exception of physiologic hyperbilirubinemia. The samples of peripheral venous blood were obtained concurrently with the clinical samples taken for serum bilirubin measurement. Adult blood samples were obtained by venipuncture.

Immediately after being drawn, each blood sample was divided into 2 portions. For studying basal neutrophil CD11b expression, an aliquot was added to a pyrogen-free tube containing citrate phosphate dextrose (Baxter Health Care Ltd, Norfolk, England; 0.14 mL/mL blood) and was immediately cooled to 0°C in an ice-water bath to minimize neutrophil activation ex vivo.^15,28 For studying FMLP-stimulated CD11b expression, another portion put in an identical tube was immediately placed in a 37°C water bath.

Stimulation of Neutrophils With FMLP
Aliquots of a stock solution of FMLP (Sigma Chemical Co, St. Louis, MO) dissolved in dimethylsulfoxide at concentration of 2 x 10^{– 4} M were stored at –20°C. The working solution was 10^–6 M FMLP in phosphate-buffered saline at 37°C. An aliquot of working solution (2.5 µL) was added to a 25-µL aliquot of blood sample kept at 37°C and further incubated at 37°C for 10 minutes. After incubation, the sample was kept at 0°C until stained for flow cytometry.

Determination of CD11b Expression by Flow Cytometry
Neutrophil CD11b expression was assessed as described previously.^15,28 FMLP-stimulated and unstimulated samples, both at 0°C, were processed for flow cytometry within 1 hour.

Neutrophils in 25-µL aliquots of whole blood were labeled with saturating concentrations of the R-phyco-erythrin conjugate of mouse anti-CD11b immunoglobulin G1 antibody, clone 2LPM19c, and its control antibody Aspergillus niger glucose oxidase immunoglobulin G1-R-phyco-erythrin, clone DAK-GO1, purchased from Dako (Glostrup, Denmark). After labeling, contaminating erythrocytes were lysed by addition of 2 mL of a 1/10 diluted ice-cold fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson, San Jose, CA). After a 3-minute incubation on ice, the leukocytes were centrifuged for 5 minutes at 4°C at 1400 x g, and a second incubation with 2 mL of FACS lysing solution was performed for 5 minutes at room temperature. After centrifugation, leukocytes were resuspended in 1% formalin at 0°C. A FACScan flow cytometer (Becton Dickinson) and CellQuest analysis software (Becton Dickinson) were used for the acquisition and analysis of the data. Neutrophils were identified on the basis of their light-scattering properties. For each sample, 5000 events were recorded. CD11b expression is reported in relative fluorescence units (RFU), ie, as the mean channel of the positive fluorescing cell population.

Data Analysis
CD11b expression levels in preterm infants with RDS were evaluated at 3 time points: on the first day of life, at days 2 to 5, and at days 7 to 10. Arithmetic means were used when 2 or 3 samples had been obtained at days 2 to 5 and 7 to 10, respectively. Statistical comparisons between groups were made by analysis of variance (ANOVA), t test, or Welch’s test. Post hoc multiple comparisons were made by the Tukey or Tamhane’s T2 method. Significance of repeated measures was tested by repeated-measures ANOVA. The normality of variables was evaluated by Shapiro-Wilk statistics. The most relevant descriptive values were expressed with a 95% confidence interval (CI). Correlation was estimated by Spearman’s coefficient method. The level was set at 0.05 for all tests.

	RESULTS

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Basal CD11b Expression
The results in Fig 1 show that mean basal CD11b expression on neutrophils in cord blood samples of preterm infants with RDS was significantly lower than that of term infants (83 RFU at SD 21 vs 112 RFU at SD 38; 95% CI for difference: 5–53). On the first postnatal day, CD11b expression level on neutrophils of the preterm infants with RDS (165 RFU at SD 53) was significantly higher than the respective levels on neutrophils from cord blood of the preterm infants (difference: 82 RFU; 95% CI: 59–106) and on neutrophils from 1-day-old preterm infants without RDS (difference: 59 RFU; 95% CI: 17–90). During follow-up, the CD11b expression levels in preterm infants with RDS decreased significantly (P value for linear trend: <.001). On postnatal days 2 to 5, neutrophil CD11b expression was significantly higher in preterm infants with RDS than in term infants (P < .001). Basal CD11b expression levels of neutrophils in cord blood samples from term infants, in postnatal samples from term infants at days 2 to 5, and in samples from healthy adults were comparable (P = .95).

View larger version (19K): [in this window] [in a new window]   Fig 1. Basal CD11b expression on neutrophils in cord blood samples from healthy term infants (, n = 30) and preterm infants (•, n = 11); in postnatal samples from preterm infants with RDS ( and whiskers; n = 29 at each time point), preterm infants without RDS on day 1 (, n = 12), and healthy term infants on days 2 to 5 (, n = 17); and in adult healthy volunteers (shaded box and whiskers; n = 25). All blood samples were cooled on ice immediately after sampling and processed for flow cytometry. The box indicates 75th and 25th percentiles with the central line the median. Whiskers represent the range without outliers indicated by asterisks. From preterm infants with RDS, at least 1 postnatal sample was obtained at each of the 3 time points; when additional samples were obtained, arithmetic mean of CD11b density was calculated at days 2 to 5 and 7 to 10, and, finally, the course of CD11b expression was evaluated using linear trend test. Neutrophils in aliquots of some postnatal samples, some cord blood samples, and some adult blood samples were stimulated with FMLP.

	DISCUSSION

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The results show that preterm infants who need mechanical ventilation as a result of RDS develop an early systemic inflammatory reaction as defined by increased neutrophil CD11b density. This reaction gradually vanishes by the age of 7 to 10 days. This reaction failed to occur in 1-day-old preterm infants with no need for mechanical ventilation or in term infants aged 2 to 5 days, indicating that it does not represent a normal physiologic adaptation of birth. However, the possibility cannot be excluded that this reaction observed may occur in age- and weight-matched mechanically ventilated preterm infants without RDS, because such infants are rare and were not studied. While the present article was in preparation, Drossou-Agakidou et al²⁹ reported that, in agreement with our results, neutrophil CD11b expression is enhanced in preterm infants studied during the first day of life.

Several explanations exist for our findings. Preterm infants are extremely susceptible to infection, a typical cause of systemic inflammation and neutrophil activation in adults.^1,30 However, infection may not explain our finding because enhanced CD11b expression occurred in all preterm infants with RDS and was related neither to the presence of risk factors nor to laboratory markers of neonatal infection.

At birth, preterm infants are susceptible to periods of hypoxia and reoxygenation, which may cause neutrophil activation.³¹ However, CD11b expression in asphyxiated and nonasphyxiated infants was similar. Previous studies have shown that the mode of delivery exerts virtually no effect on neutrophil CD11b expression level.^32,33 In the present study, CD11b expression did not correlate with maternal preeclampsia, which is associated with high CD11b expression.³⁴

The enhanced CD11b expression observed most probably derives from exocytosis of the highly mobile secretory vesicles. Secretory vesicles are formed at a late stage of neutrophil maturation.⁸ Their exocytosis occurs in vivo and is controlled by multiple intracellular signaling mechanisms.³⁵ It is not known whether the mechanisms responsible for enhanced CD11b expression are functionally normal in preterm infants. Taken together, these findings suggest that neutrophil activation is related to the development of RDS, treatment with surfactant, or both or to prematurity itself. However, on the basis of our results, it is not possible to conclude whether the transient increase in CD11b expression in preterm neonates derives from RDS itself, surfactant therapy, mechanical ventilation, or a combination of them.

The crucial question is whether in preterm infants increased neutrophil CD11b serves as a marker of systemic inflammation. The results of the present study and of several others show that, when stimulated with FMLP, neutrophils from cord blood of term infants^36–40 and of preterm infants^41–43 and neutrophils of infants aged 10 to 20 days^40,41 all show a reduced ability to enhance CD11b expression. This can be explained by the findings of lower total cell content of CD11b/CD18 complexes in term neonates than in adults^43,44 and correlates with gestational age.⁴³ Despite this defect, there is a 2- to 4-fold increase in neutrophil CD11b expression on the first day of life in infants (mean weight: 2700 g; gestational age: 36 weeks) with blood culture–-positive sepsis.⁴⁵ Our previous studies of neonates with infection⁴⁶ and of adults with septic shock²⁰ showed similar results. Taken together, these findings suggest that enhanced CD11b expression serves as a systemic inflammation marker also in preterm infants.

The level of neutrophil CD11b expression correlated modestly with the severity of RDS, ie, number of doses of surfactant and maximum FIO₂ on the first day of life. This may simply reflect an indirect association of 2 independent factors, both of which are present in preterm infants with RDS. Because natural porcine surfactant has both anti-inflammatory and proinflammatory actions, the possible role of surfactant in neutrophil activation remains unclear.^47–50 Another possibility is an actual pathophysiologic relationship between neutrophil activation and pulmonary morbidity or mechanical ventilation as such. In an experimental setting, hyperoxic lung injury begins in the pulmonary capillary endothelium,⁵¹ indicating that high FIO₂ may result in endothelial damage in the region of those alveoli with sufficient ventilation. Subsequently, endothelial injury may promote neutrophil activation, which may ultimately amplify hyperoxic lung injury. Indeed, in experimental hyperoxia, neutrophil depletion ameliorates but does not totally prevent lung injury.⁵² However, local neutrophil activation in the lung may not fully explain the observation of activated neutrophils in the circulation. It is possible that systemic neutrophil activation itself contributes, at least partially, to the pulmonary injury. In both clinical and experimental settings, systemic inflammatory response and neutrophil activation as a part of it may result in ARDS.⁵³ In any case, whether neutrophil activation is the inducer or the result of lung injury, our observation of correlation of systemic neutrophil activation with pulmonary morbidity is in line with previous findings indicating that in infants who have RDS, neutrophils accumulate in the lung tissue during the first postnatal days.^21,54–56

	CONCLUSION

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We demonstrate a systemic inflammatory reaction, indicated by increased CD11b expression on circulating neutrophils, in preterm infants with RDS. This reaction is transient; it occurs on the first day of life and gradually diminishes during the first 10 postnatal days, and it seems not to be caused by neonatal infection or to be any part of normal postnatal adaptation. The reaction seems to be related to the severity of RDS correlating weakly to number of doses of surfactant and maximum FIO₂. Additional studies are needed to determine whether the reaction is related to RDS itself, surfactant therapy, mechanical ventilation, or a combination of them. Although the mechanism of the transient increase in CD11b expression and its clinical significance, if any, remain unclear, systemic inflammation and neutrophil activation may contribute to the tissue injury in preterm infants with RDS.

	ACKNOWLEDGMENTS

 
This study was supported by grants from the Foundation for Pediatric Research (Helsinki, Finland), the Helsinki University Central Hospital Research Funds (Helsinki, Finland), and the Paulo Foundation (Helsinki, Finland).

	FOOTNOTES

 
Received for publication Jun 26, 2001; Accepted Dec 13, 2001.

Address correspondence to Irmeli Nupponen, MD, Department of Bacteriology and Immunology, Haartman Institute, Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. E-mail: irmeli.nupponen{at}kolumbus.fi

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