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BPD is a cause of significant morbidity and mortality in the premature infant. Multiple insults thought to contribute to the onset of this fibrotic disease include hyperoxia, barotrauma, and infection. The unmitigated fibroproliferation that characterizes this disease may involve dysregulation of cytokines including IL and TNF-α, possibly in response to inflammation(1, 2). Chronic pulmonary inflammation, as described by the cytology of TA, is correlated with developing fibrosis(3–7). However, in one study, neither cell counts nor differentials were associated with the development of BPD(8). Importantly, these and other studies analyzed airway cells and secretions as part of routine endotracheal suctioning or using suction catheters inserted just beyond the tip of the endotracheal tube. Additionally, the repertoire of cytokine expression may not be reflected by changes in cellularity as cells may become activated without concomitant recruitment of inflammatory cells. Furthermore, evidence of proximal airway inflammation (that may be evident in TA samples) may not accurately reflect the pathogenic processes in the distal airways, sites more likely involved in the development of BPD.

In this preliminary study we collected TA and DPL from premature infants on 1, 7, and 28 d of age. We hypothesized that cells isolated from distal lung compartments display unique cytokine expression patterns. These specific patterns of cytokine expression (mRNA phenotype)(9) could then be tested to predict the ultimate development of BPD. Described in this preliminary report is our approach to testing this hypothesis using the sensitive method of RT-PCR.

METHODS

Collection of pulmonary cells by TA and DPL. Informed consent under the University of Rochester Human Subjects Review Committee's guidelines was obtained to have DPL, preceded by tracheal aspiration, performed on intubated premature babies in our Neonatal Intensive Care Unit. Samples were to be obtained from babies at 1, 7, and 28 d of age.

After pulmonary toilet, a Bodai Neo2-Safe neonatal suction valve (Baxter Healthcare Corp., Valencia, CA) was installed in the ventilator circuit. For the TA collection, sterile, preservative-free 0.9% NaCl at 1 mL/kg body weight(maximum of 1 mL) was instilled in two aliquots into the endotracheal tube after disconnecting the ventilator. After administration of the first aliquot, the ventilator was reconnected for three breaths before collecting secretions by suction through a 5 French feeding catheter (Bard, Inc., Cranston, RI) into a sterile suction trap (Busse, Hauppauge, NY). The catheter was premeasured so that its tip did not go past the end of the endotracheal tube. The procedure was repeated with the remaining aliquot of saline. The catheter was then rinsed with 0.5 mL of saline and collected into the trap.

The DPL was next performed. A 5 French Umbili-Cath (Gesco International Inc., San Antonio, TX) was aseptically passed through the Bodai suction port and down the endotracheal tube. The catheter was gently threaded until resistance was met, at which time the catheter was withdrawn 0.5 cm. One mL/kg normal saline was flushed into the lung and quickly aspirated using a syringe attached to the end of catheter. At least 30 s were allowed to elapse before repeating the procedure two more times. The catheter was then rinsed with 0.5 mL of saline and collected into the trap. Figure 1 shows radiographically the position of the DPL catheter relative to the endotracheal tube within the chest of a baby.

Figure 1
figure 1

Chest x-ray showing the placement of the DPL catheter(arrow) through the endotracheal tube (arrowhead) into the lower right segmental bronchus. The TA sample was collected via a catheter whose tip did not exceed the end of the endotracheal tube.

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Both DPL and TA samples were placed on ice and transported for immediate processing (within 15 min of collection). Fluid volumes were determined with a polystyrene serologic pipette, transferred to 50-mL polypropylene tubes, and centrifuged at 300 × g at 4°C for 6 min in a swinging bucket rotor without braking. Cell pellets were resuspended in 30 mL of ice-cold Hank's buffered saline solution (Life Technologies Inc., Grand Island, NY) by gentle swirling and inversion of the tube. Cells were repelleted with braking and resuspended in 1 mL of Hank's buffered saline solution. A 10-μL aliquot was then counted by use of an improved Neubauer hemacytometer. Cells were cytocentrifuged (Cytospin 2; Shandon, Pittsburgh, PA) onto slides for determination of cell differentials by Diff-Quik staining(Baxter, McGraw Park, IL). Remaining cells (2-500 × 104) were lysed in guanidine isothiocyanate and stored at -80°C until acidic phenol-chloroform extraction of RNA was performed(10). Data were expressed as mean ± SD.

RT-PCR conditions. Total RNA was reverse transcribed by avian myeloblastosis virus RT in the presence of oligo(dT)15 using reagents supplied by the manufacturer (Promega, Madison, WI). The reaction was allowed to proceed for 20 min at 42°C, then for 10 min at 50°C. Each reaction preparation then was heated for 4 min at 94°C, usually after dilution with water. Primer pairs (Amplimers) for the IL, TNF-α, and for β-actin were used under conditions recommended by the manufacturer (Clontech, Palo Alto, CA) using the thermostable DNA polymerase AmpliTaq (Hoffmann La Roche, Nutley, NJ) and the PCR enhancement reagent Taq Extender(Stratagene, La Jolla, CA). A master mix was prepared and aliquoted to PCR tubes that contained Amplimers and 2.5-10% of each cDNA reaction preparation. PCR was performed for 30 cycles: 45 s at 94°C, 45 s at 60°C, and 2 min at 72°C. The reaction was continued for 7 min at 72°C and held at 4°C until analysis by ethidium bromide agarose gel electrophoresis.

RT-PCR sensitivity of detection. In an attempt to estimate the sensitivity of our overall RT-PCR, we simultaneously isolated total RNA from TA cell pellets of 500 000, 50 000, and 5 000 cells. Each RNA was then dissolved in a 20-μL volume of water, and 9.8 μL of each RNA preparation were reversed transcribed as described above. Each reaction was performed under identical conditions except for the initial RNA concentration. Reaction preparations were diluted to a final volume of 80 μL and heated to 94°C to quench enzyme activity. PCR was performed as described above on 2 μL of each dilute cDNA for 30 cycles in the presence of β-actin Amplimers as described above. Accounting for the dilutions, the equivalent of 6000, 600, and 60 cells, respectively, were subjected to PCR. One fifth of each PCR(1200-, 120-, and 12-cell equivalents) was visualized by electrophoresis through a 1.5% agarose gel containing ethidium bromide.

RESULTS

Six babies were enrolled in this study, ranging in weight from 650 to 1050 g and in gestational age from 25.5 to 30.5 wk. Neonates tolerated both TA and DPL procedures and exhibited no untoward effects. A brief elevation in the fraction of inspired O2 (Fio2) was occasionally needed during these procedures because of transient oxygen desaturation. Percent recovered DPL volumes ranged from 44.3 to 70.3% (59.7 ± 9.2%). TA percent recovered volumes ranged from 16.7 to 62.3% (38.7 ± 17.0%).

DPL cell recovery ranged from 0.333 to 57.4 × 105 cells (10.6× 105 ± 19.5 × 105), and the TA cell recovery ranged from 0.927 to 3.38 × 105 cells (16.9 × 105± 12.4 × 105). Cell differentials also exhibited a wide variability. Macrophages comprised 6.0-90% of the cells. This variability was reflected in the abundance of polymorphonuclear leukocytes which ranged from 6.25 to 90.7%. Lymphocytes ranged from 0 to 6.25%. Other cells included epithelial cells and those which could not be assigned to the other groups. Differentials from five TA/DPL pairs were compared, and only patient F showed similar TA and DPL differentials (Table 1). Cell preparations from patients D and E yielded insufficient numbers of intact cells to analyze on the cytospins.

Table 1 DPL and TA cells
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The sensitivity of RT-PCR reactions is illustrated byFigure 2. We show that we can extract and reverse-transcribe sufficient RNA for amplification of cDNA from 50 000 TA cells under the conditions examined. Lane 2 represents one fifth of a PCR reaction that was performed on cDNA equivalent to that derived from 600 cells. No attempt was made to assess the differential efficiencies of RNA extraction, avian myeloblastosis virus RT, or Taq Polymerase that may have been caused by differences in cell number. Our intent was not to examine critically each parameter that may alter stepwise yields but to examine the summed results of these variables in toto.

Figure 2
figure 2

Sensitivity of RT-PCR. RNA was extracted from a 10-fold dilution series of cells as described in “Methods.” RT-PCR ofβ-actin was performed and loaded onto a ethidium bromide agarose gel.M, 100-bp ladder. Lanes 1, 2, and 3: 20% of PCR performed on 6000, 600, and 60 cells, respectively.

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Seven paired DPL and TA samples were analyzed by RT-PCR, the results of which are summarized in Table 2. RNA was extracted from 0.11-46 × 105 cells. PCR was performed on cDNA derived from 540 to 58 000 cells and one fifth of each reaction was analyzed by agarose gel electrophoresis. Two DPL samples did not have readily detectable levels ofβ-actin mRNA (patient D, and patient E at d 28). These samples likewise did not express any cytokine mRNA, nor were any intact cells seen after cytocentrifugation of patient D DPL. IL-8 was detected in all β-actin positive samples (n = 12) and thus served as an unintended second control, indicating that our reaction conditions were sensitive enough for RT-PCR detection of labile cytokine mRNAs. Interestingly, no intact cells were seen on TA cytospins from patients D and E but each of the cytokine mRNAs were expressed. No cytokine was found exclusively in one pulmonary compartmentversus the other. Further, cells from both sources could express each cytokine. In four of seven TA samples, all five cytokines were detected, whereas no DPL sample expressed all five cytokines. The cytokine profiles of cells from the two pulmonary compartments differed in four of five eligible TA/DPL pairs tested. Figure 3 is a photograph of a representative gel of the RT-PCR results for a pair of TA and DPL samples(patient C).

Table 2 RT-PCR mRNA phenotyping of DPL and TA cells
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Figure 3
figure 3

RT-PCR products. Representative gel from patient C demonstrating heterogeneity of cytokine mRNA expression by TA and DPL cells.

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DISCUSSION

We determined the cell differentials from five paired TA/DPL samples at 1, 7, and 28 d of age. Cell differentials varied widely, with similarity in only one case, suggesting that different cell populations were being sampled by each technique. This is to our knowledge the first analysis of freshly isolated TA and DPL from premature infants. Grigg et al.(11) using a technique similar to our DPL found that the percentage of neutrophils decreased in the second BAL compared with the first, whereas cell numbers remained unchanged. Our first and second DPL samples were pooled and thus not available for comparison. The significance of these findings from our small sample is unclear at present. However, the cell type distribution found in the endotracheal tube probably does not reflect the relative distribution of distal airway cells.

By RT-PCR we easily detected IL-1α, IL-1β, IL-6, IL-8, and TNF-α mRNAs in cells isolated from these pulmonary fluids. These cytokine mRNAs previously have been detected in BAL from adults and children(12–14). We believe this is the first report to demonstrate that pulmonary cells isolated from airways of premature infants can contain mRNAs for these inflammatory mediators. Differences in cytokine profiles cannot be explained by cell differentials. Patient A displayed markedly different cytology between TA and DPL cells yet had identical cytokine profiles. In contrast, patient F had similar differentials and dissimilar mRNA phenotypes.

One could postulate that upper airway secretions may display phenotypes of both upper airway cells and distal cells translocated by mucociliary transport. It may also be argued that our TA technique allows some TA cells to be aspirated into the lung so that subsequent DPL could contain TA cells. However, we found no compelling cytologic evidence to indicate either of these scenarios. Further, of the five paired TA/DPL samples examined, we found cytokine profile identity once, suggesting that mixing of upper and lower respiratory secretions is either not common or inefficient. Grigg et al.(11) found evidence suggesting that sequential BAL samples cells from increasingly distal segments. We could not test whether this was manifested at the level of mRNA diversity because we pooled our sequential DPL. Four TA and no DPL samples expressed all five cytokines, perhaps indicating a more heterogenous TA cell population. The greater DPL intrasample phenotypic diversity may benefit our attempts to exploit these cells in predicting the transformation of respiratory distress syndrome to BPD.

One criticism of our RT-PCR procedure is that we did not normalize our cDNA or PCR reactions to include equal amounts of input mRNA and cDNA, respectively. It was previously reported that differences in cytokine profiles of adult human BAL could be demonstrated by RT-PCR in a similarly nonquantitative assessment(12–14). We likewise made no attempt to be quantitative in this initial study. Our intent was to develop a safe means of retrieving distal airway cells that could be assessed both cytologically and with molecular biologic techniques. However, simple differences in numbers of cells subjected to RT-PCR cannot account for all cytokine expression patterns. For example, fewer TA cells from patient A than patient C were subjected to RT-PCR, but patient A displayed three cytokines versus one cytokine detected from patient C. Our samples contained relatively equal amounts of amplified β-actin. In cases whereβ-actin was not readily detectable, no cytokine was amplified. Interestingly, IL-8 was present in all β-actin positive samples and served as an unintended qualitative control.

Increased levels of IL-6 bioactivity in lung fluid has been correlated with the onset of BPD in neonates(8). Interestingly, these higher levels of bioactivity were not reflected by increased immunoreactivity as measured by ELISA. In the same study, TNF-α was implicated in the development of BPD but statistical significance was not demonstrated. The authors suggested that IL-6 and TNF-α may be involved with acute and chronic injury processes in the ontogeny of BPD. We show that, by using the relatively simple technique of RT-PCR, these two cytokines are detectable in cells from TA and DPL from 1- to 28-d-old premature babies, and that they display both interand intracompartmental variability in expression. We show that TA and DPL can independently express IL-6 and TNF-α, suggesting that upper and lower airways are alternate sources of these cytokines.

In summary, we demonstrated that we can detect a panel of cytokines including IL-1α, IL-1β, IL-6, IL-8, and TNF-α from both TA and DPL cells. This pilot study provides a basis for collecting more samples to perform a careful analysis of potential differences in these cell populations. We further show that, by the technique of RT-PCR, relatively small numbers of cells may be exploited to define a cytokine mRNA expression pattern. Clear differences in mRNA phenotypes both between patients and between distal and tracheal airway cells were revealed by RT-PCR, suggesting that studying DPL cells may be an alternate means to evaluate pulmonary inflammation and parenchymal injury. Experiments in progress include standardizing reaction conditions to allow for semiquantitative analysis,in situ mRNA hybridization and immunocytochemistry to identify cell-specific cytokine synthesis, and testing new primer pairs to expand the cytokine mRNA profiles of lavageable pulmonary cells from intubated babies.