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Am J Physiol Lung Cell Mol Physiol 291: L1267-L1276, 2006. First published July 21, 2006; doi:10.1152/ajplung.00097.2006
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Functional and genomic changes induced by alveolar transmigration in human neutrophils

Christopher D. Coldren,1 Jerry A. Nick,1,2 Katie R. Poch,2 Malcolm D. Woolum,1 Brian W. Fouty,1 James M. O’Brien,1 Michael P. Gruber,1 Martin R. Zamora,1 Daiva Svetkauskaite,1 Don A. Richter,1 Qianbin He,1 Jong Sung Park,1 Katherine H. Overdier,1 Edward Abraham,1 and Mark W. Geraci1

1Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and 2Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado

Submitted 17 March 2006 ; accepted in final form 27 June 2006


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: ~15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-{alpha} release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.

acute respiratory distress syndrome; acute lung injury; sepsis; inflammation

ACUTE LUNG INJURY (ALI) is characterized histologically by the accumulation of large numbers of polymorphonuclear leukocytes (neutrophils) in the lungs, as well as inflammatory injury, including elevation in proinflammatory cytokines and loss of endothelial and epithelial integrity (25, 31). In experimental models of ALI, systemic or pulmonary administration of bacteria or bacterial products such as endotoxin (i.e., lipopolysaccharide or LPS) is followed by the rapid entry of neutrophils into pulmonary parenchyma and their subsequent migration into the alveolar space. Neutropenic animals exhibit attenuated lung vascular permeability and other indexes of lung injury relative to control animals in these models, highlighting the importance of neutrophils in the development and severity of ALI (1, 3, 35).

In epidemiologic studies, the most common predisposing etiology for ALI is infection. Gram-negative organisms account for ~50% of the infections predisposing to ALI (25, 34), and endotoxin is an important mediator of organ system dysfunction and death associated with severe gram-negative infections. In animal experiments, endotoxin exposure results in ALI as characterized by neutrophil accumulation in the lungs, increased expression of proinflammatory cytokines, loss of epithelial and endothelial integrity, widening of the alveolar-arterial O2 gradient, and increased interstitial pulmonary edema (2, 3, 28). Human studies demonstrate that administration of endotoxin directly into a pulmonary subsegment results in acute inflammatory changes including increased proinflammatory cytokine levels and activation of procoagulant and antifibrinolytic cascades, followed by the migration of neutrophils into the air spaces of the endotoxin-exposed lungs (29).

Both circulating and pulmonary neutrophils appear to be affected in ALI but often demonstrate different phenotypes in this setting. For example, neutrophils lavaged from the lungs during the course of ALI exhibit decreased apoptosis, whereas apoptosis among peripheral blood neutrophils shows minimal alteration (4, 24, 26, 27). Circulatory neutrophils from patients with ALI demonstrate increased nuclear accumulation of the transcriptional factor NF-{kappa}B, alterations in chemotaxis, and activation of intracellular signaling pathways, including kinases such as Akt (4, 40). In the present study, we used gene expression and functional analyses to examine differences between pulmonary and circulating neutrophils that occur early in endotoxin-induced lung inflammation. In particular, these experiments were aimed at characterizing alterations in gene expression and phenotype that exist between neutrophils that have migrated into the air spaces in response to endotoxin exposure and those present in the peripheral circulation. These changes are contrasted with the alterations induced in circulatory neutrophils by pulmonary endotoxin instillation and by in vitro exposure of circulatory neutrophils to endotoxin. In addition to the direct relevance of these differences in the study of disease states such as ALI that are characterized by tissue-resident neutrophils, circulating neutrophils are the source of much of our understanding of neutrophil biology, and our study points to many ways in which these cells may not be representative of neutrophils in pathogenic states.


   METHODS
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Materials. Endotoxin-free reagents and plastics were used in all experiments. Endotoxin isolated from Escherichia coli was repurified by a second phenol extraction to eliminate contaminating glycoprotein (17). Reference endotoxin isolated from E. coli strain 0111:B3 was obtained from National Institutes of Health (29).

Human model of endotoxin-induced pulmonary inflammation. All subjects were admitted to the General Clinical Research Center (GCRC) at the University of Colorado Hospital. This research protocol was approved by the Colorado Multiple Institution Review Board; written informed consent was provided according to the Declaration of Helsinki. The experimental outline for the study is shown in Fig. 1. A more detailed description of subject eligibility, research protocols, and poststudy follow-up are available in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site. Twenty-four hours before admission to the GCRC, 30 ml of blood was collected for in vitro treatment with endotoxin. After admission, 10 ml of saline was bronchoscopically instilled into a lung subsegment (either the right middle lobe or lingula) followed by instillation of the test dose of reference endotoxin (4 ng/kg) into the contralateral lung. The subjects were randomized to left or right lungs for endotoxin or saline instillation. Following reconstitution of the endotoxin, a quantitative Limulus amebocyte lysate test was done to verify the proper reconstitution and dosage of endotoxin. At the time of the first bronchoscopy, 30 ml of peripheral blood was obtained for isolation of neutrophils.


Figure 1
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  		Fig. 1. Experimental timeline for the endotoxin-induced pulmonary inflammation study.

 
A second bronchoscopy was performed 16 h after the initial bronchoscopy. At the time of the second bronchoscopy, both the endotoxin- and placebo-instilled subsegments were lavaged with 150 ml of normal saline, and 60 ml of peripheral blood was obtained for the isolation of circulating neutrophils (28).

Isolation of neutrophils from peripheral blood and lavage fluid. Human neutrophils were isolated from peripheral blood using the plasma Percoll method (16) and from lavage fluid through a negative selection strategy against human major histocompatibility complex HLA-DR and glycophorin A tetramer (28). Because peripheral blood and lavage fluid differ greatly in their cellular and extracellular composition, different polymorphonuclear leukocyte (PMN) isolation protocols were required, which introduces the possibility that a portion of the differences observed between air space and circulating PMNs was an artifact of these separation methods. To evaluate this possibility, we repurified an aliquot of peripheral PMNs using the lavage fluid method. Comparison of the repurified sample to the original peripheral sample revealed no artifactual changes in gene expression. This is detailed in the online supplement.

Endotoxin stimulation of circulating neutrophils in vitro. Immediately following isolation, circulating neutrophils were resuspended in RPMI 1640 and 5% fetal calf serum medium at a final concentration of 5 x 106 cells/ml and were incubated in the presence of 0 ng/ml (control) or 100 ng/ml endotoxin for 60 min at 37°C with gentle agitation.

Microarray analysis of neutrophil gene expression. RNA stabilization, isolation, and microarray sample labeling were carried out using standard methods for reverse transcription and one round of in vitro transcription (13). HG-U133A microarrays were hybridized with 10 µg cRNA and processed per the manufacturer’s protocol (Affymetrix, Foster City, CA). A MIAME checklist (7) containing extensive experimental details can be found in the online data supplement. Hybridization signals and detection calls were generated in BioConductor, using the gcrma and affy packages (38), and have been deposited in the National Center for Biotechnology Information GEO Database, accession no. GSE2322. Microarray data were analyzed using BRB ArrayTools v3.3b developed by Dr. Richard Simon and Amy Peng Lam. Cluster analysis and class comparison using the paired univariate t-test were performed using the set of 14,131 genes that was reliably detected on two or more arrays. Multivariate permutation tests to determine false discovery rates were based on 1,000 random class label permutations. Gene Ontology (GO) analysis was performed using GenMAPP and MAPPfinder (10).

Neutrophil functional and chemotaxis assays. Standard ELISA assays for the release of cytokines (28) and a cytochrome c reduction assay for superoxide anion release (14) were employed. Neutrophil necrosis was determined by the percent of total lactate dehydrogenase (LDH) released (Cytotoxicity Detection Kit; Roche, Mannheim, Germany). Apoptosis was quantified by immunoassay for cytoplasmic histone-associated DNA fragments (Roche) and reported as an apoptosis index, the ratio of histone-bound DNA in a given sample to the level of histone-bound DNA detected in endotoxin-stimulated circulatory neutrophils. Migration assays were performed as reported previously (28) and reported as a chemotaxis index, which is the ratio between the fluorescence intensity of directed and undirected migration through the membrane for each sample. Data were analyzed using JMP statistical software (SAS Institute, Cary, NC). Studies of neutrophil chemotaxis were expressed as means ± SE. Significance of the difference in neutrophil chemotaxis over time was determined by two-way ANOVA. The significance of the difference in levels of released cytokines (GoGoFig. 4), superoxide anion (Fig. 3C), LDH (Fig. 3B), as well as indexes of apoptosis (Fig. 3A) and priming (Fig. 3D), was assessed with a Student’s t-test for nonpaired samples. For all tests, P < 0.05 was considered significant.


Figure 2
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  		Fig. 2. Gene expression in air space and circulating neutrophils. Global analysis of gene expression using the 14,131 genes reliably detected for at least 2 of the 58 samples. A: dendrogram illustrating the relatedness of the 58 samples based on centered correlation, displayed with average linkage. The scale on the y-axis is the Pearson correlation; branches of the dendrogram that merge low on the y-axis represent samples with increasingly similar patterns of gene expression. The uniform segregation of air space neutrophil samples (AIR), pre- and postendotoxin instillation circulatory neutrophil samples (PRE and POST), and in vitro incubated neutrophils [both control (CON) and endotoxin (LPS)] reflects consistent differences in gene expression between these cellular populations. B: overabundance plot showing the number of genes with significant differences in expression between air space and circulating neutrophils plotted vs. the indexed significance level for the statistical test (paired t-test). The number of observed differences between air space neutrophils and preendotoxin circulating neutrophils (green line) or postendotoxin circulating neutrophils (red line) exceeds the number of differences found in the comparison of pre- and postendotoxin instillation circulating neutrophils (blue line); each of these exceeds the number of differences expected under the matching null hypothesis (black line) regardless of the significance level chosen for the test. This illustrates the robustness and the magnitude of the difference in gene expression. C: magnification of B, extreme lower left, showing the region where P < 0.05.

 

Figure 3
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  		Fig. 3. Divergent patterns of survival and respiratory burst by air space and circulating neutrophils. Analysis of apoptosis, necrosis, and superoxide anion release in neutrophils isolated from the lung 16 h after endotoxin instillation compared with neutrophils isolated from peripheral blood pre- and postadministration of endotoxin to the airways. A: spontaneous apoptosis. Histone-bound DNA isolated from unstimulated air space and circulatory neutrophils, expressed as an apoptosis index. Air space neutrophils demonstrated significantly less spontaneous apoptosis than either pre- or postendotoxin neutrophils in circulation. B: neutrophil necrosis. The percent of neutrophils that underwent necrosis after 4 h in culture was measured by the release of lactate dehydrogenase (LDH), expressed as the percentage of the total LDH content of an equal number of neutrophils. Air space neutrophils demonstrated a significantly greater percentage of necrosis than unstimulated (open bar) but not LPS-treated (gray bar) circulating neutrophils pre- or postendotoxin. PMN, polymorphonuclear leukocyte. C: superoxide anion release. Superoxide anion release after 30 min from unstimulated (open bar), formyl-Met-Leu-Phe (fMLP)-stimulated (hatched bar), or endotoxin-primed and fMLP-stimulated neutrophils (gray bar) was compared in circulating pre- and postendotoxin administration and air space neutrophils. Significantly greater superoxide anion release was generated by airway neutrophils for each condition studied. D: priming for superoxide anion release. Superoxide release as expressed in a ratio (priming index) between endotoxin-primed and unprimed neutrophils following fMLP stimulation. Air space neutrophils had a significantly lower priming index than circulating neutrophils pre- and postendotoxin administration. Values are shown as means ± SE (n = 14 for circulating samples, n = 7 for air space samples), *P < 0.05 compared with the air space sample as analyzed by Student’s t-test.

 

Figure 4
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  		Fig. 4. Divergent patterns of cytokine release by airway neutrophils compared with circulating neutrophils. Neutrophils isolated from the lung 16 h after endotoxin instillation were compared with circulating neutrophils pre- and postendotoxin administration in the presence or absence of in vitro endotoxin stimulation. The quantity of each cytokine and chemokine (picograms per million cells) released from neutrophils stimulated in vitro with endotoxin (filled bar) or left unstimulated (open bar) is depicted following 2 or 4 h of incubation, as measured by immunoassay. Air space neutrophils produced quantities of IL-8 (2 h) and monocyte chemoattractant protein-1 (MCP-1; 4 h) that were intermediate to values of cytokine released by endotoxin-stimulated and unstimulated circulating neutrophils. Airway neutrophil release of macrophage inflammatory protein-1beta (MIP-1beta) was similar to LPS-treated circulating neutrophils, while the quantity of tumor necrosis factor-{alpha} (TNF-{alpha}) release was equivalent to unstimulated circulating neutrophils. A: IL-8 release. B: MIP-1beta release. C: TNF-{alpha} release. D: MCP-1 release. Cytokines are expressed as means ± SE (n = 14 for circulating samples, n = 7 for air space samples), *P < 0.05 compared with the air space sample as analyzed by Student’s t-test.

 

   RESULTS
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 RESULTS
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No adverse events occurred in the study group as a result of endotoxin instillation. All 19 volunteers completed the experimental protocol; however, not all experimental measurements were collected from every volunteer sample. Table 1 shows the distribution of the measurements collected among the volunteer samples. Instances where measurements were not collected typically reflect experimenter choices regarding the use of the limited number of cells rather than a technical failure of the assay.


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  		Table 1. Measurement distribution among volunteer samples

 
Recovery of neutrophils from lavage fluid and peripheral blood. The accumulation of inflammatory cell populations, primarily neutrophils, was found in lavage fluid recovered from the pulmonary subsegment where endotoxin had been instilled but not in lavage fluid from the contralateral lung subsegment. Bronchoalveolar lavage was performed 16 h following endotoxin instillation and yielded from 35 to 500 million neutrophils from the endotoxin-instilled lung; fewer than 300,000 cells were recovered from the saline-exposed contralateral lung. Peripheral blood yielded between 1 and 5 x 108 neutrophils from 30 ml. Neutrophil purity was determined by counting cells on Diff-Quick-stained cytospin preparations from each air space and circulatory cell sample; these were found to contain at least 98% PMNs. Further support for this high level of PMN comes from the observed absence of expression of genes encoding the lymphocyte markers CD4, CD8A, and the monocyte marker CSF1R, each of which is highly expressed in peripheral blood mononuclear cells (details in online supplement). Endotoxin concentration was measured in cell-free lavage fluid by the Limulus amebocyte lysate test and was found to be similar in samples recovered from saline- and endotoxin-exposed pulmonary subsegments [39 ± 50 and 59 ± 58 pg/ml (means ± SD), respectively].

Gene expression data and analysis. High-quality microarray data were obtained from the 58 samples indicated in Table 1. Each microarray passed a stringent set of quality controls (36). Figure 2A depicts an unsupervised clustering analysis of all 58 samples, based on the expression level of all 14,131 genes reliably detected on two or more arrays. The uniform segregation of the circulatory and pulmonary samples indicates that large and consistent differences in gene expression exist between the two groups. Segregation of the in vitro cultured samples (both endotoxin-treated and control) within the larger group of circulatory samples is also apparent.

Comparisons between sample and treatment groups were performed using the paired univariate t-test, with a nominal P value cutoff of 0.001. In each comparison, samples were grouped by volunteer, and only complete sets were considered (n = 12). There were 1,832 and 1,674 genes that met this nominal criterion in the comparison of air space to pre- and postendotoxin instillation circulating neutrophils (Supplemental Tables S1 and S2), indicating that ~15% of expressed genes are modulated in response to emigration into the lung. False discovery rate analysis (20) suggests with 90% confidence that 10 or fewer false discoveries are listed among either set of genes. In contrast, comparison of pre- and postendotoxin instillation circulating neutrophils yielded only 114 nominally significant differences (Supplemental Table S3). These large changes in gene expression are further illustrated in Fig. 2, B and C, an "overabundance plot" (5). From this plot it can be appreciated that a greater number of gene expression differences are observed between both sets of circulating neutrophils and the air space neutrophils than would be expected by chance, regardless of the P value cutoff chosen. The number of significant differences can be seen to be similar in the comparison of air space to either circulating sample and smaller for the comparison between circulating samples. Exposure of neutrophils to endotoxin in vitro resulted in 166 genes meeting the nominal P value cutoff of 0.001 (Supplemental Table S4), with 17 or fewer false discoveries.

We determined the dominant themes of gene expression changes by GO categories as implemented in MAPPfinder, which allows the identification of functional groups of genes that are differentially expressed more frequently than expected by chance. Analysis of the genes listed in Supplemental Tables S1–S3 identified several Molecular Function and Biological Process categories with significantly elevated prevalence in each of these groups. Table 2 presents GO Biological Process and Molecular Function categories overrepresented in the comparison of postpulmonary endotoxin circulating neutrophils to air space neutrophils. Comparison of preendotoxin circulating neutrophils to air space cells resulted in a very similar pattern, whereas comparison of post- to preendotoxin circulating cells demonstrates a much more limited pattern of increases in immune and stress response genes (GO 6955 and 6950). Exposure to in vitro endotoxin also elicits this general response as well as categories specific for LPS and hyaluronic acid binding (GO 1530 and 5540).


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  		Table 2. Gene Ontology categories with a superabundance of differentially expressed genes in air space neutrophils relative to postpulmonary endotoxin circulating neutrophils

 
Survival of neutrophils isolated from air space and circulation. Changes in the expression of large numbers of apoptosis-related genes occur after neutrophil emigration into the site of pulmonary inflammation (Table 2), and a large part of the changes in gene expression can be interpreted as antiapoptotic. We tested parameters of neutrophil survival at a 4-h time point following the isolation of neutrophils recovered from the air spaces and from the circulation before and after pulmonary endotoxin administration to determine if these gene expression changes were reflected in decreased apoptosis. Neutrophils isolated from the air space did not receive additional stimulation in vitro, whereas circulating neutrophils were examined under both unstimulated and endotoxin-stimulated settings. In the absence of exogenous stimulation, neutrophils undergo apoptosis within hours (15, 30), but exposure to endotoxin typically delays this process (9, 21). Air space neutrophils demonstrated significantly less spontaneous apoptosis than both pre- and postpulmonary endotoxin circulating neutrophils in the absence of in vitro endotoxin (Fig. 3A). Neutrophils that do not undergo apoptosis die by necrosis, characterized by the release of the cytosolic contents. A significantly greater percentage of air space neutrophils underwent necrosis than unstimulated circulating neutrophils (Fig. 3B).

Superoxide anion release by neutrophils isolated from air space and circulation. Superoxide anion (O2–) release is a major component of respiratory burst by neutrophils in response to a variety of proinflammatory stimuli. In the absence of stimulation, circulatory neutrophils release negligible amounts of O2–. Neutrophils in the primed state also release little O2– but demonstrate significantly enhanced O2– release when subsequently stimulated with the G protein-coupled receptor agonist formyl-Met-Leu-Phe (fMLP). Air space neutrophils were found to release significantly more O2– than circulating neutrophils (before or after pulmonary endotoxin) both in response to stimulation by fMLP and in response to fMLP stimulation after endotoxin priming (Fig. 3C). The ability of substimulatory concentrations of endotoxin to induce priming (i.e., priming index) is determined by the ratio of O2– release in response to fMLP stimulation with and without endotoxin priming. Air space neutrophils demonstrated a significantly lower priming index compared with the circulating neutrophils (Fig. 3D), a reflection of prior priming in the process of endotoxin-induced emigration of neutrophils into the air space.

Divergent patterns of cytokine release by air space neutrophils. Neutrophils are capable of releasing a limited number of cytokines and chemokines, some of which are synthesized de novo in response to proinflammatory stimuli (8, 39). Cytokine gene expression is induced by pulmonary endotoxin and by in vitro endotoxin treatment (Table 3). Within the set of genes differentially expressed between air space and postendotoxin instillation circulating neutrophils are 20 members of the GO Molecular Function "cytokine activity" category [Gene Ontology ID (GOID)] 5125; this category is inclusive of GOID 8009, "chemokine activity". Three chemokines [monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1beta (MIP-1beta)] and one cytokine [tumor necrosis factor-{alpha} (TNF-{alpha})] were selected for further analysis because of their established roles in the pathogenesis of ALI. Spontaneous release of TNF-{alpha}, MCP-1, IL-8, or MIP-1beta by air space neutrophils was compared with spontaneous and LPS-induced release of these cytokines by the circulating neutrophils pre- and postendotoxin administration to the airways. After 2 h, the release of IL-8 by air space neutrophils was intermediate to unstimulated and LPS-stimulated circulatory neutrophils (Fig. 4A), whereas the release of MIP-1beta by air space neutrophils was significantly greater than the unstimulated circulating neutrophils. Conversely, air space neutrophils failed to release TNF-{alpha} by 4 h (Fig. 4C), whereas release of MCP-1 by air space neutrophils was intermediate to unstimulated and LPS-stimulated circulatory neutrophils (Fig. 4D). Together, these data demonstrate a pattern of cytokine release by air space neutrophils that is distinct from both unstimulated and circulating neutrophils stimulated with LPS either in vivo or ex vivo.


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  		Table 3. Cytokine gene expression changes

 
Air space and circulatory neutrophil migration. Chemotaxis represents the coordination of complex processes linking chemokine receptors with cytoskeleton assembly (and disassembly) as well as cell-cell and cell-substrate adhesion via intracellar signaling events. A number of GO categories for which differential expression is induced by pulmonary endotoxin (Table 2) and by in vitro endotoxin treatment relate to chemotaxis, but based on patterns of gene expression it is difficult to predict if neutrophil migration will be increased or decreased under the conditions studied. Airway neutrophils demonstrate a superabundance of increased expression for members of the GO Molecular Function "C-C chemokine receptor activity" category (GOID 16493) as well as GO Biological Process "heterophilic cell adhesion" category (GOID 7157) and "cell surface receptor-linked signal transduction" category (GOID 7166). However, there is also an increase in the GO Molecular Function "MAP kinase phosphatase activity" category (GOID 17017), which could result in suppression of p38 MAP kinase signal transduction, required for neutrophil migration.

IL-8, a potent stimulus of neutrophil migration, was used to test the relative capacity of airway neutrophil to undergo additional chemotaxis. IL-8-induced chemotaxis was significantly decreased relative to nondirectional migration in air space neutrophils compared with both circulatory neutrophils pre- and postendotoxin administration to the airways when analyzed ex vivo (Fig. 5).


Figure 5
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  		Fig. 5. IL-8-induced chemotaxis in air space neutrophils is diminished compared with circulating neutrophils. Neutrophils isolated 16 h after endotoxin instillation from bronchoalveolar lavage fluid (filled circle), preendotoxin instillation peripheral blood (open triangle), or postendotoxin instillation peripheral blood (open square). Air space neutrophils demonstrated a significant decrease in IL-8-induced migration ex vivo over a 1-h period compared with both groups of neutrophils recovered from the circulation. Neutrophil isolated from circulation postendotoxin administration demonstrated a significant decrease in chemotaxis index compared with cells isolated preendotoxin administration. Values are mean indexes ± SE (n = 4 for each group), P < 0.001 by 2-way ANOVA at all time points from 10 through 60 min.

 

   DISCUSSION
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 ABSTRACT
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Characterization of various features of neutrophil response in the human lung is a central theme in studies relating to inflammatory lung diseases. However, the difficulties inherent in collecting pulmonary neutrophils have led to the widespread use of in vitro systems of neutrophil activation, which typically utilize a single stimulus to evoke responses relevant to the pathogenesis of lung injury. Whereas studies of single stimulus response are essential to define individual intracellular signaling pathways, it is apparent that no single stimulus can recreate the complex series of events that result in neutrophil accumulation in the airway. Specifically, the neutrophil responds to a gradient of multiple chemokines, cytokines, and endotoxin, as well as the physical contact with adhesion molecules and transmigration from the circulation to the airway. Each of these stimuli and events can evoke both common and unique features of neutrophil activation.

Data presented in this report represent the summation of multiple stimuli that contribute to human neutrophil recruitment to a site of endotoxin-induced pulmonary inflammation and demonstrate that a broad pattern of altered gene expression and functional changes are induced. Significant overlap, as well as significant differences, exist between the gene expression changes observed in our system with those observed in previous studies of neutrophils isolated from circulation and challenged in vitro with endotoxin (11). Likewise, the results support and extend previous studies demonstrating reduced neutrophil apoptosis upon transmigration across an in vitro lung epithelial monolayer (18). Although the present experiments examined neutrophils from a single time point in the progression of pulmonary inflammation, the study provides an unprecedented level of detail regarding changes in gene expression in alveolar neutrophils; furthermore, these changes are, in part, consistent with the functional alterations observed ex vivo. Of importance is the fact that alveolar neutrophils in the setting of the acute respiratory distress syndrome (ARDS) exhibit similar functional alterations in apoptosis to those induced experimentally in the present study (27). Differences between in vivo functional changes and those observed in vitro provide potentially valuable insight into the complexity of neutrophil activation in vivo and the strengths and weaknesses of existing model systems.

Expression changes in cytokine and chemokine genes. The ability of massive quantities of airway neutrophils to release cytokines is a potential mechanism by which excessive airway inflammation could be prolonged. Cytokine gene expression induced by pulmonary endotoxin and subsequent transmigration and by in vitro endotoxin treatment (Table 3) was compared with mean fold change in cytokine release for IL-8, MIP-1beta, TNF-{alpha}, and MCP-1 (Fig. 4). In vitro endotoxin treatment leads to correlated increased expression of a given cytokine gene and the increased release of the corresponding protein (Fig. 6). The same correlation is found with pulmonary endotoxin stimulation, with the exception of TNF-{alpha}, where gene expression levels were observed to increase without a simultaneous increase in protein release. Overall, these observations support the concept that modulation of neutrophil gene expression at the transcriptional stage plays an active role in the production of cytokines and chemokines in the inflammatory milieu. However, under the conditions tested, certain gene products are not translated despite robust changes in gene expression. It is notable that the degree of correlation between gene expression and protein expression varies widely within this narrow functional group.


Figure 6
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  		Fig. 6. Correlation between changes in cytokine gene expression and protein release by airway and circulating neutrophils. The mean fold change in gene expression for air space neutrophils (compared to unstimulated circulating neutrophils) and LPS-stimulated neutrophils (compared to unstimulated neutrophils; Table 3) were compared with the mean fold change in cytokine release for IL-8, MIP-1beta, TNF-{alpha}, and MCP-1 (Fig. 4). Changes in gene expression for both air space (triangle) and LPS-stimulated neutrophils (circle) correlate strongly with protein release for the chemokines IL-8, MIP-1beta, and MCP-1 (R2 = 0.84, P = 0.0097). However, release of TNF-{alpha} correlated poorly with changes in gene expression, and when TNF-{alpha} is included in the analysis of correlation between gene expression and protein release the correlation no longer reaches significance (R2 = 0.36, P = 0.12).

 
Whereas a similar pattern of cytokine gene expression increases is observed whether pre- or postendotoxin instillation circulating neutrophils are compared with air space neutrophils, we observed a different pattern with in vitro endotoxin exposure (Table 3). In many cases, the level of cytokine gene induction was greater in vitro (i.e., TNF-{alpha}, IL-1{alpha}, and CCL3), whereas for CCL2, in vivo stimulation elicited a stronger induction. This pattern is similar to the pattern that was detected in cytokine release (Fig. 4). In addition to the differences in the inflammatory environment between the in vitro and in vivo models, another cause for the difference in these patterns may be the length of time elapsed in each case: air space neutrophils were collected 16 h after endotoxin instillation, whereas the in vitro samples were exposed to endotoxin for 60 min. Comparison of pre- and postendotoxin instillation circulating neutrophils revealed no significant expression changes in cytokine genes (Supplemental Table S3).

Expression changes in apoptosis-related genes. We observed that changes in the expression of large numbers of apoptosis-related genes occur after neutrophil emigration into the site of pulmonary inflammation. Air space neutrophils had a decreased rate of spontaneous apoptosis relative to circulating neutrophils (Fig. 3A), and a large part of the changes in gene expression can be interpreted as antiapoptotic. In particular, the GO categories "antiapoptosis" and "negative regulation of apoptosis" each have a significant number of genes with increased expression in air space neutrophils, whereas the converse categories "induction of apoptosis" and "positive regulation of apoptosis" have approximately the same number of induced genes that would be expected by chance. A number of the cytokines that are upregulated in air space neutrophils have antiapoptotic properties (i.e., SPP1, TNF-{alpha}, and IL-1{alpha}). In addition, increases in expression of genes coding for inhibitors of both TNF- and NF-{kappa}B-mediated apoptosis are present in the air space neutrophils. For example, bacloviral IAP repeat-containing (BIRC) family members diminish TNF-mediated apoptosis by binding TNF receptor-associated factors (TRAFs) and inhibiting caspase-3, caspase-7, and caspase-9 (22). The genes encoding BIRC2 (c-IAP1), BIRC3 (c-IAP2), and BIRC4 (x-IAP) each show increased expression, from two- to sevenfold in air space neutrophils. Additional effects in the NF-{kappa}B pathway are expected from increases in NFKBIE (24.7-fold), NFKBIA (3-fold), TNFAIP3 (15-fold), IER3 (14-fold), NFKB1 (4.8-fold), and RELA (2.1-fold) expression, and are represented as a group by the GO category 7253, "cytoplasmic sequestering of NF-{kappa}B." Statistically significant changes were also observed in genes with direct roles in mitochondrial membrane processes associated with apoptosis: BCL family members BCL2A1 (up 2.7-fold), BCL2L1 (up 6.3-fold), and MCL1 (down 2-fold). Although it is reasonable to interpret these changes as antiapoptotic, the data provide incomplete information regarding the expression of alternatively spliced variants (particularly important in BCL2A1 or BCLXL/XS) and no information about the translational and posttranslational modulations of apoptosis-related molecules.

Differentiation-related gene expression changes. One of the most striking increases in gene expression among air space neutrophils is that of the pan-cyclin-dependent kinase inhibitor CDKN1A (p21cip1), which is induced by an average of 140-fold in endotoxin-elicited alveolar neutrophils. A prior study has established that mature circulating neutrophils and their precursors have no detectable CDKN1A transcript or protein (19). In fact, expression of this gene was not detected in circulating neutrophils. However, robust expression of CDKN1A was found in every sample of air space neutrophils, as well as neutrophils cultured in vitro, and transcription is induced by incubation with endotoxin (1.7-fold, P = 0.01). CDKN1A expression is induced by all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cell lines harboring the PML-RAR{alpha} fusion protein (6). In addition, more modest but significant induction of cyclin D2 (8-fold), one of the binding partners of CDKN1A, was observed in air space neutrophils. No significant change in the expression of CDK4 was observed. The broad pattern of gene expression changes found in air space compared with peripheral neutrophils (~15% of neutrophil-expressed genes are modulated), together with the marked induction of CDKN1A upon pulmonary transmigration but not by in vitro exposure to endotoxin, suggests that the air space neutrophil may be viewed as being in a more advanced developmental state rather than simply an altered state of activation.

Modulation of cytotoxic gene expression. Constitutive production of perforin and granzyme B, the active and functional agents of antibody-dependant cellular cytotoxicity, has been described in neutrophils (37) and may provide a molecular basis for the damage that occurs through the induction of apoptosis in tissues infiltrated by neutrophils. We observed that the expression of both cytotoxic mediators is strongly induced upon recruitment to the air space: perforin gene expression by 13.2-fold and granzyme B by 26.2-fold. Additionally, we found a 9.6- and 8.7-fold increase in granzymes A and K, respectively. In contrast to other reports (32), we did not detect expression of the gene encoding FAS ligand (FASLG, TNFSF6) in air space, or circulating or cultured neutrophils, suggesting that extrinsic routes to induced epithelial apoptosis may not be mediated by neutrophils under these circumstances.

Cell surface receptor gene expression changes. One of the dominant themes in gene expression change in the air space neutrophil is an increase in the expression of genes encoding proteins with receptor activity. The largest induction was observed for the orphan chemokine receptor CCRL2, which increased 168-fold. CCRL2 increases 12.6-fold upon in vitro endotoxin stimulation as has been previously described (12) and is one of a cluster of chemokine receptor genes on 3p21. Three other members of this cluster, CCR1, CCR5, and CXCR6, also exhibited increased expression (4.7-, 5.5-, and 3.6-fold, respectively) in air space neutrophils; however, other members of the cluster (CCR2 and CCR3) are unaffected. Robust induction of both C3AR1 (18.1-fold) and its ligand C3 (15.4-fold) were observed as well as downregulation of C5R1 expression (down 6.7-fold). Expression levels increased for a number of less well-characterized G protein-coupled receptors were GPR171 (20.7-fold), GPR41 (26.95-fold), GPR43 (5.1-fold), GPR65 (3.5-fold), and GPR109B (4.1-fold). If translated, these represent opportunities for the integration of extracellular signals that are available to the air space but not the circulating neutrophil. This difference may have important implications for in vitro studies using circulating neutrophils. Moreover, induction of a number of cell adhesion surface molecules, including ICAM-1, CD2, and CD44 (9.5-, 8.0-, and 27.9-fold, respectively) was observed, whereas ICAM-3 expression decreases (5.3-fold). Expression of CD44 by neutrophils has been shown to be critical for the resolution of lung inflammation (33), whereas a specific role for ICAM-1 expression by these cells is unknown. In contrast, exposure to endotoxin in vitro does not produce broad expression changes in cell surface receptors.

Functional properties of air space neutrophils. A functional analysis of neutrophil survival, respiratory burst, cytokine synthesis, and chemotaxis demonstrates that neutrophils recruited to the air space in response to endobronchial endotoxin are distinct from both unstimulated and endotoxin-stimulated circulating neutrophils. Compared with circulating neutrophils from the same subject, air space neutrophils died through cell necrosis rather than apoptosis. Further evidence of the activated state of the air space neutrophil comes from an enhanced release of O2– and decreased ability to respond to endotoxin priming. However, the air space neutrophil, recruited in response to endotoxin, is distinct from the circulating neutrophil stimulated in vitro with endotoxin, as evidenced by a failure to release TNF-{alpha} (Fig. 4C). The air space neutrophil is also much less capable of migration towards IL-8 than the circulating neutrophil (Fig. 5). The unique state of air space neutrophil activation may result from the series of priming and activation steps neutrophils are subjected to in the course of migration into the lungs. Although endobronchial endotoxin was the initial inflammatory stimulus, it is likely that neutrophils recovered from the lung 16 h later encountered only trace amounts of endotoxin. Neutrophils rapidly internalize and degrade endotoxin (23), and lavage fluid recovered with the endotoxin-elicited air space neutrophils contained no more endotoxin than fluid recovered from the contralateral, saline-exposed lung. Instead, neutrophil migration into the alveoli and bronchioles presumably occurred in response to chemokines (especially IL-8) released primarily by residential lung macrophages, with subsequent activation through adhesion molecule binding. It is also possible that the population of neutrophils recovered from the air spaces is older than the circulating neutrophil population; this supposition is supported by a significantly greater number of cells undergoing necrosis ex vivo (Fig. 3B).

Overall, the present experiments demonstrate that neutrophils undergo profound alterations in gene expression and functional properties when they migrate from the peripheral circulation into the lung in response to an endotoxin challenge. The characteristics of the air space neutrophils collected in this study are not likely to have been acquired through direct exposure to endotoxin, but rather as a result of contact with pulmonary cell populations, such as endothelial and epithelial cells, encountered during migration from the blood to the lung and through exposure to the proinflammatory milieu induced in the alveoli by endotoxin-stimulated macrophages and epithelial cells. Nevertheless, the contrasting functional and gene expression profiles of air space and circulatory neutrophils underline the profound alterations in neutrophil phenotypes produced by migration into the lung. This study represents the first well-controlled examination of these differences in a human model of pulmonary inflammation and as such provides unprecedented detail about a process central to ALI.


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 ABSTRACT
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This study was supported by National Institutes of Health Grants HL-68743, HL-72340, CA-046934, and M01-RR-00051, and a Cystic Fibrosis Genome Analysis Program Grant from the Cystic Fibrosis Foundation.


   DISCLOSURES
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 ABSTRACT
 METHODS
 RESULTS
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REFERENCES
 
This study was supported in part by Eli Lilly and Company. E. Abraham has served as a paid consultant for Eli Lilly and Company.


   ACKNOWLEDGMENTS
 
Present addresses: B. W. Fouty, Center for Lung Biology, Division of Pulmonary Medicine, University of South Alabama School of Medicine, Mobile, AL 36688; J. M. O’Brien, Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. D. Coldren, Division of Pulmonary Sciences and Critical Care Medicine, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., C272, Denver, CO 80262 (e-mail: chris.coldren{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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