Published online before print December 16, 2004
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B activation
,1
* Departments of Pathology, Free Radical Research Group, and
Paediatrics, Christchurch School of Medicine and Health Sciences, University of Otago, New Zealand
2 Correspondence: FRRG, Department of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand. E-mail: margret.vissers{at}chmeds.ac.nz
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B, which causes a delay in neutrophil apoptosis, was also investigated. We obtained 68 tracheal aspirate samples from 27 infants with median gestation and birthweight of 26 weeks and 860 g, respectively. Apoptosis was assessed by immunofluorescent detection of the active form of caspase-3, this assay being validated with peripheral blood neutrophils. Activation of NF-B was monitored by the nuclear translocation of the p65 subunit, detected by immunofluorescence. Cleaved caspase-3 was detected in 11 of the 68 samples, and a median of 40% of the neutrophils showed activated caspase-3 (range 3–92%). A majority of the samples did not show evidence of apoptosis. Caspase activation was seen in cells with multilobed nuclear morphology, suggesting that early apoptosis was detectable. There was no significant difference in respiratory outcomes between infants with or without neutrophil apoptosis. Seventeen of the 68 samples (25%) had evidence of activated NF-B, and a median of 20% (range 6–41%) of neutrophils showed activation. In all but one tracheal aspirate sample, there was a mutually exclusive relationship between activated caspase-3 and NF-B activation, which supports in vitro observations that NF-B activation delays neutrophil apoptosis.
Key Words: activated caspase-3 • neutrophil apoptosis • chronic lung disease
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The infiltration of neutrophils into the lungs of infants with hyaline membrane disease (HMD) [7 ] and the persistence of these cells in the airways have been associated with the subsequent development of chronic lung disease (CLD) [8 ]. Evidence of neutrophil apoptosis in the lungs of newborn infants with airway inflammation was first demonstrated by Grigg et al. [9 ], who speculated that this represents a mechanism by which tissue injury is reduced during the resolution of inflammation. Recent studies have suggested that impaired neutrophil apoptosis promotes lung injury in premature neonates [10 , 11 ]. Very low proportions of apoptotic neutrophils were found in tracheal aspirate samples from newborns on mechanical ventilation during the first 4 days of life, and apoptosis was lowest in preterm infants who went on to develop CLD [10 , 11 ]. It has also been suggested that neutrophils from cord blood have a decreased capacity to undergo apoptosis when compared with adult peripheral blood cells [12 , 13 ].
Inflamed lungs are likely to be exposed to proinflammatory cytokines, which are known to delay neutrophil apoptosis [14
]. Indeed, granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-CSF have been shown to prolong the survival of neutrophils in bronchoalveolar lavage of patients with acute respiratory distress syndrome [15
]. Nuclear factor (NF)-B, a transcription factor that regulates the expression of a host of proinflammatory proteins including adhesion molecules and cytokines, also generates antiapoptotic signals [16
17
18
19
]. We have recently shown that NF-B is activated in tracheal aspirate samples from premature infants and that this correlates with the presence of proinflammatory cytokines [20
].
The limitation in obtaining adequate samples from preterm infant lungs has meant that few studies have been carried out on patient samples and that detection of apoptosis must be made by direct observation of the cells themselves. Previous studies have defined apoptotic neutrophils on the basis of their nuclear morphology [9
, 10
, 11
]. These nuclear changes are middle-to-late events in the apoptotic program and are closely associated with phosphatidylserine exposure and clearance by macrophages [9
, 21
, 22
], thereby reducing the chances of detection. We have instead examined an early apoptotic event, caspase-3 activation, and measured this in tracheal aspirate neutrophils from infants with HMD using an immunocytochemical technique. We have also probed for evidence of proinflammatory signals, which may delay apoptosis by comparing NF-B activation with caspase activation in these neutrophils.
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Tracheal aspirate samples
The NICU policy is to undertake endotracheal suctioning when clinically indicated, and all samples are collected for study. The procedure for suctioning of the endotracheal tube and the collection of tracheal aspirates was according to the method described by Darlow et al. [23
]. Briefly, the tip of the suction catheter was advanced just past the tip of the endotracheal tube and then withdrawn slightly before a set suction pressure was applied. "Dry" suctioning or suction following instillation of 0.5 mL saline was used. Secretions were trapped within the length as well as the hub at the proximal end of each suction catheter. These catheters were stored at 4°C in the NICU and processed within 6 h of collection. Samples that had heavy growth of gram-positive or gram-negative bacteria or yeast, as determined subsequently by the diagnostic laboratory, were excluded from analysis.
Removal of the secretions involved repetitive and gentle flushing of the suction catheters using 1 mL phosphate-buffered saline (PBS). The cells in this suspension were separated by centrifugation at 3000 rpm for 5 min. The cell pellet was resuspended in 500 µL PBS and filtered through a 48-µm nylon mesh to remove mucous from the samples. Cell number and viability were determined using a hemocytometer and the trypan blue exclusion test. The cells were >95% viable and were suspended in PBS at a final concentration of 5–10 x 105 cells/mL.
Assay for apoptosis
Peripheral blood neutrophils isolated from healthy adult volunteers [24
] were used to validate the caspase-3 assay before applying it to tracheal aspirate neutrophils. To generate apoptotic cells, neutrophils were "aged" in vitro in the presence and absence of G-CSF (600 ng/mL). The cells were suspended at 106/mL in endotoxin-free RPMI 1640 with 10% autologous serum and incubated at 37°C in 5% CO2. At intervals up to 48 h, the cells were harvested, and pellets or cytospins were prepared. The morphology of the cells was assessed after Giemsa staining, and apoptotic cells with pyknotic nuclei and condensed chromatin were counted. Caspase activity was measured by immunofluorescence and by monitoring the cleavage of the fluorescent substrate Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) in a lysed cell pellet [25
]. Microscopic examination was carried out by at least two investigators, who were blinded to the sample identification.
Proteolytic activation of caspase-3 occurs when initiator caspases cleave the 32-kDa procaspase-3 into p20/p17 and p12 subunits. We used a monoclonal rabbit anti-human-cleaved caspase-3 antibody (Cell Signaling Technology, Inc., Beverly, MA), which recognizes the p20/p17 subunit in the cytoplasm of apoptotic cells. A volume of 100 µL suspended cells was cytospun at 250 rpm over 5 min onto high-binding microscope glass slides (SuperFrostPlus, Menzel-Glaser GmbH, Braunscheig, Germany). Two slide specimens were prepared from each sample, one for the assessment of apoptosis and the other for NF-B. Cells were fixed with 4% (w/v)-buffered paraformaldehyde (pH 7.4) for 30–60 min, rinsed three times with PBS, and further fixed with chilled (–20°C) 100% methanol for 5–10 min. The cells were blocked and permeabilized for 1 h in PBS with 1% (w/v) bovine serum albumin (PBS/BSA) and 0.1% (w/v) saponin at room temperature and incubated overnight with 100 µl anticaspase-3 antibody (1:250 dilution). After three washes with PBS/BSA, the samples were further blocked with 10% goat serum for 30 min before staining with fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragment goat anti-rabbit immunoglobulin G for 1–2 h in the dark at room temperature. All blocking and washing steps as well as incubation with antibodies required the inclusion of 0.1% saponin. The cells were counter-stained with Hoechst 33342 (10 µg/mL) before they were air-dried and mounted. The assessment of apoptotic neutrophils was performed on a fluorescence microscope. Neutrophils were identified by their multilobed nuclear morphology. An average of 100 neutrophils was assessed in each case.
Slides were processed in batches as tracheal aspirates became available. Positive and negative samples were present within each batch, suggesting that the positive results were not artifactual. Two investigators, who were blinded to the sample identification, independently assessed each slide, and caspase-3 and NF-B were analyzed independently.
Assay for NF-B activation
An immunofluorescence assay using a similar staining protocol to that described for caspase-3 was used to measure translocation of NF-B into the nucleus [20
]. An antibody targeted against the p65 subunit of the NF-B dimer was used [26
, 27
]. Cells were counter-stained with Hoechst 33342 to enable visualization of their nuclei.
Statistical analysis
The Mann-Whitney U-rank sum test or Wilcoxon signed rank test was used to compare nonparametric data. The 
2 test was used to examine differences between groups of ordinal data, and the Fisher exact test was used if an expected frequency was less than five. Results are expressed as median with the range from minimum to maximum values. The differences are statistically significant when the P value is <0.05. Analysis of data was performed using a statistical software package, SigmaStat version 1 (Jandel Corporation, San Rafael, CA).
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Figure 1. The correlation of caspase activation with morphological changes associated with apoptosis in aged peripheral blood neutrophils. (A) The activation of caspase-3 as measured by cleavage of the fluorescent substrate DEVD-AMC. a.u., Arbitrary units of fluorescence. (B) The incidence of apoptosis in neutrophils as determined by morphology. The criteria shown in the inset were used to judge morphological appearance of apoptotic (arrows) and preapoptotic cells (arrowhead). Means ± SD of individual estimates of morphology are shown. (•) Control neutrophils; ( ) G-CSF-treated neutrophils. The results are from a single, representative experiment. Similar results were obtained on at least three other occasions.
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Figure 2. The detection of cleaved caspase-3 in adult peripheral blood neutrophils as detected by immunofluorescence. Neutrophils were rendered apoptotic by aging them over 24 h in media. Cleaved caspase-3 antibody (FITC-conjugated) stains the cytoplasm of apoptotic neutrophils green in the presence of activated caspase-3. Nuclear morphology could be identified by staining with Hoechst 33342 (blue fluorescence). (A–D) Control neutrophils after 0 h, 8 h, 16 h, and 24 h, respectively. (E–H) G-CSF-treated neutrophils at 0 h, 8 h, 16 h, and 24 h. Original bars, 5 µm.
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Table 1. Characteristics of the Study of Infants and Their Tracheal Aspirate Samples
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Figure 3. (A) Tracheal aspirate neutrophils stained for cleaved caspase-3 with apoptotic neutrophils exhibiting green fluorescence in their cytoplasm. (B) Tracheal aspirate neutrophils stained with an antibody to the p65 subunit of NF-B and detected by FITC-conjugated secondary antibody. The presence of green fluorescence in the nucleus indicates translocation of NF-B from the cytoplasm. (C) The nuclear translocation is confirmed by the overlay of the blue nuclear fluorescence. Bars, 5 µm.
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Figure 4. Apoptotic activity as measured by cleaved caspase-3 in tracheal aspirate neutrophils from seven premature infants who showed at least one positive sample. Each symbol represents an individual infant; sequential samples from the same infant are joined by lines.
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Most of the mothers of the infants (21/27) had received at least one dose of antenatal corticosteroids. Seven of these 21 infants had samples with caspase activation, compared with none of the six infants whose mothers received no steroids. With this number of patients, the difference was not significant (P=0.15).
Relationship between apoptosis and NF-B activation in tracheal aspirate neutrophils
NF-B activation was assessed by indirect immunofluorescence, and translocation of the transcription factor into the neutrophil nucleus provided evidence of activation (Fig. 3B
and 3C)
. Seventeen of the 68 samples (25%) had evidence of activated NF-B, and a median of 20% (range 6–41%) of neutrophils showed activation. These 17 samples were from 10 babies. Of these 17 samples, all but one was negative for cleaved caspase-3. Eleven aspirates were positive for cleaved caspase-3. A scatter-plot shows that with the exception of one sample, the two activities were mutually exclusive (Fig. 5
).
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Figure 5. Relationship between the percentages of tracheal aspirate neutrophils with cleaved caspase-3 and NF–B activation (n=68). Forty-one samples registered zero for both assays.
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The use of this method in tracheal aspirates may explain why, in some samples, we measured much higher proportions of cells undergoing apoptosis than have been reported previously. By counting myeloperoxidase-stained neutrophils inside alveolar macrophages, Grigg et al. [9 ] reported 0.6–9.8% of the neutrophil population as apoptotic. Oei et al. [10 ] reported negligible neutrophil apoptosis (median of 0.26%) in first-week tracheal aspirate samples from 31 preterm infants, whereas Kotecha et al. [11 ] estimated 8.6% of lung neutrophils to be apoptotic in infants with resolved HMD. In the latter studies, apoptotic neutrophils were determined morphologically by their pyknotic nuclei. Such assessment of apoptosis in situ by monitoring late events such as nuclear changes may result in an underestimation of the number of apoptotic cells because of their rapid uptake by macrophages or because of the development of secondary necrosis. Our own preliminary analysis of a series of tracheal aspirate samples also suggests that cells with pyknotic nuclei, which could be considered as apoptotic neutrophils, are rarely seen in Giemsa-stained or Hoechst-labeled cytospins (unpublished results). Our current study provides a different measure of apoptosis, which allows detection of early apoptosis in a population of cells that may appear transiently.
Despite the detection of early apoptosis using the cleaved caspase-3 assay, a large proportion of the samples in our study had negligible apoptosis. This suggests that the presence of apoptotic neutrophils is a transient phenomenon, which is detected only occasionally, or it reflects the influence of antiapoptotic factors present in some tracheal aspirates. Neutrophil apoptosis is delayed during active inflammation by the presence of antiapoptotic cytokines such as G-CSF and tumor necrosis factor 
(TNF-) [14
, 15
], and this may be mediated by NF-B [28
]. Indeed, cytokines are elevated in tracheal aspirates obtained from patients with acute respiratory distress syndrome [15
] and infants with HMD who developed CLD [29
, 30
]. We have also recently shown that NF-B activation in neutrophils and macrophages from premature infant lung aspirates is associated positively with elevated levels of TNF- [20
]. Therefore, neutrophils in the neonatal lung are likely to be exposed to proinflammatory mediators, which influence the recruitment, activation, and apoptosis of these cells. Our finding in the present study, which showed that NF-B activation and caspase-3 activation were mutually exclusive events, would support this view.
In our study, several infants with severe respiratory difficulties had some of the highest proportions of apoptotic lung neutrophils. Indeed, two infants had sequential samples that showed persistently high proportions of apoptotic neutrophils, although their clinical outcomes were divergent: One died from severe respiratory failure, and the other had resolved lung disease. This finding runs counter to the suggestions of others [10 , 11 ] that the development of chronic lung disease of early infancy is associated with a failure of the apoptosis program in lung neutrophils. We also found no difference in the proportions of infants with caspase-3 activation between the group that developed CLD and the group that had resolved lung disease, although the lack of statistical significance may be attributed to a small sample size.
In summary, we detected neutrophils with early apoptosis or with NF-B activation in tracheal aspirates from premature infants. That these events did not occur together suggests that the proinflammatory conditions that led to NF-B activation also prevented neutrophil apoptosis. However, the presence of high numbers of apoptotic neutrophils in tracheal aspirates from these infants did not preclude progression to CLD. Hence, it is still unclear whether neutrophil apoptosis alone is important for limiting damage in the immature lung. Further studies will be required to determine the impact of apoptosis and of various antiapoptotic factors such as NF-B in influencing the respiratory outcomes of lung inflammation in these infants.
Received September 16, 2004; revised November 23, 2004; accepted November 24, 2004.
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B and rel proteins in innate immunity Adv. Immunol. 58,1-27[Medline]B in the immune system Annu. Rev. Immunol. 12,141-179[Medline] B in cytokine gene regulation Am. J. Respir. Cell Mol. Biol. 17,3-9B activation is a critical regulator of human granulocyte apoptosis in vitro J. Biol. Chem. 274,4309-4318B activation in pulmonary leukocytes from infants with hyaline membrane disease: associations with chorioamnionitis and Ureaplasma urealyticum colonization Pediatr. Res. in press.B activity in response to oxidants and antioxidants Methods Enzymol. 300,363-374[Medline]B activation in cultured human keratinocytes upon wounding and the effect of low energy HeNe irradiation Free Radic. Biol. Med. 25,998-1005[CrossRef][Medline] B activation Eur. J. Pharmacol. 433,17-27[CrossRef][Medline] and IL-6 in tracheobronchial aspirate fluid indicator of subsequent chronic lung disease in preterm infants Arch. Dis. Child. Fetal Neonatal Ed. 77,F198-F201This article has been cited by other articles:
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