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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 561-567, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0019OC

Bacterial Stimulation of Epithelial G-CSF and GM-CSF Expression Promotes PMN Survival in CF Airways

Shahryar Saba, Grace Soong, Steven Greenberg and Alice Prince

College of Physicians and Surgeons, Columbia University, New York, New York

Address correspondence to: Alice Prince, Black Building 416, 650 West 168th Street, New York, NY 10032. E-mail: asp7{at}columbia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cells provide an immediate response to bacterial pathogens by producing chemokines and cytokines that recruit polymorphonuclear leukocytes (PMNs) to the site of infection. This response is excessive in patients with cystic fibrosis (CF) who have bacterial contamination of their airways. We postulated that CF airway pathogens, in activating nuclear factor-{kappa}B–dependent gene transcription in epithelial cells, would promote expression of cytokines that inhibit constitutive apoptosis of recruited PMNs. Epithelial cell culture supernatants from CF (IB-3) and corrected (C-38) epithelial cells stimulated by Staphylococcus aureus or Pseudomonas aeruginosa, increased survival of PMNs by 2- to 5-fold. Enhanced PMN survival was attributed to effects of epithelial granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor expression, which inhibit PMN apoptosis, and was negated by neutralizing antibody to either cytokine. Both CF and normal cells responded to bacteria with increased cytokine production. Granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor expression were activated by ligation of asialoGM1, a receptor for P. aeruginosa and S. aureus, and by S. aureus lipoteichoic acid. Lipopolysaccharide was not a potent stimulus of cytokine expression, and P. aeruginosa algC (lipopolysaccharide) and lasR (quorum sensing) mutants were fully capable of activating epithelial cells. Induced expression of cytokines by airway cells repeatedly exposed to bacteria, as occurs in CF, serves not only to recruit and activate PMNs, but also to enhance their survival.

Abbreviations: cystic fibrosis, CF • transmembrane conductance regulator, CFTR • conditioned media, CM • extracellular signal-related kinase, ERK • fetal calf serum, FCS • granulocyte colony-stimulating factor, G-CSF • granulocyte–macrophage colony-stimulating factor, GM-CSF • mitogen activated protein kinase, MAPK • nuclear factor-kB, NF-kB • polymorphonuclear leukocytes, PMNs • reverse transcriptase-polymerase chain reaction, RT-PCR • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cells respond to inhaled bacteria by producing cytokines and chemokines to recruit and activate polymorphonuclear leukocytes (PMNs). The expression of several proinflammatory cytokines is stimulated by adherent bacteria or shed bacterial components that interact with epithelial receptors. Transcription of interleukin (IL)-8, the CXC chemokine, by airway epithelial cells is activated by Staphylococcus aureus and Pseudomonas aeruginosa, which chronically infect the airways of patients with cystic fibrosis (CF) (1). These bacteria recognize asialylated gycolipid receptors and stimulate the transcription of cytokines and chemokines by activation of nuclear factor (NF)-{kappa}B and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK) (2). IL-6 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are also produced by airway epithelial cells following P. aeruginosa exposure or in response to the proinflammatory cytokines tumor necrosis factor (TNF)-{alpha} and IL-1ß (3). Epithelial signaling is an important component of innate immunity, providing the initial response to inhaled pathogens that elude mechanical and mucociliary clearance. In CF, a disease characterized by PMN-dominated airway inflammation, bacterial contamination of the airways is a major stimulus for the accumulation PMNs. However, even in the absence of detectable infection, substantially elevated amounts of proinflammatory cytokines, chemokines, and PMNs are found in the airways of young infants with CF (4). These clinical findings suggest that the regulation of PMN recruitment and activation is altered in CF. There is evidence of endogenous activation of NF-{kappa}B in cells with CF transmembrane conductance regulator (CFTR) dysfunction, even in the absence of exogenous stimulation of airway epithelial cells (5, 6). Although expression of several relevant chemokines and cytokines have been quantified in CF and control epithelial cells, the effects of these products on PMN function have not been directly assessed.

As originally characterized in hematopoietic cells, both granulocyte colony-stimulating factor (G-CSF) and GM-CSF stimulate granulocyte proliferation and maturation, and are important mediators of the host response to infection (7). G-CSF and GM-CSF expression in the lung are involved in the response to foreign antigens (8), allergens, and microbial pathogens (3). The effects of GM-CSF on the maturation and activation of neutrophils (PMNs) and eosinophils have been well-documented (9). Normally, PMNs are programmed to undergo apoptosis, but this process can be substantially delayed by the effects of GM-CSF on Janus kinase/STAT, phosphatidylinositol 3-kinases (10), and ERK-dependent signaling pathways (11). GM-CSF similarly promotes the longevity of eosinophils (9). A major role for GM-CSF in the host response to pathogenic bacteria as well as transiently inhaled commensal organisms was suggested by the initial description of the GM-CSF -/- mouse, which exhibits chiefly pulmonary pathology (12). The importance of GM-CSF in pulmonary infection was further demonstrated in the defective response of the GM-CSF -/- mouse to group B streptococcal lung infection (13). In addition to its effects on granulocytes, GM-CSF is involved in the maturation and activation of dendritic cells (14) and promotes the normal metabolism of surfactant and lipids by alveolar macrophages (15).

Airway epithelial cells produce GM-CSF following exposure to allergens, viral infection, or bacteria. Epithelial expression of GM-CSF requires activation of NF-{kappa}B, protein kinase C, and ERK1/2 MAPKs (16). Less is known about the regulation of G-CSF by airway epithelial cells. Exogenous G-CSF treatment increases the recruitment of PMNs to the lung, in part by upregulating expression of PMN adhesins (17). G-CSF has also been shown to contribute to neutrophil chemotactic activity induced by lipopolysaccharide (LPS) treatment of lung epithelial cells (18).

Although CF is not considered to be primarily an inflammatory disorder, there is accumulating evidence that CFTR dysfunction in airway epithelial cells results in increased activity of NF-{kappa}B and production of some NF-{kappa}B–dependent chemokines and cytokines (5). Because the systemic immune response in CF is apparently normal, effects of mutant CFTR on NF-{kappa}B activity and cytokine/chemokine expression are likely to be limited to cells of the airway. Greater numbers of bacterial receptors on CF cells are also associated with greater cytokine and chemokine production (1). Thus, there are both endogenous and exogenous factors in CF that lead to amplified transcription of at least some NF-{kappa}B–dependent proinflammatory genes. We postulated that expression of G-CSF and GM-CSF by CF cells stimulated by respiratory pathogens would affect PMN recruitment and survival.

In this study we examined the expression of G-CSF and GM-CSF by airway epithelial cells in response to S. aureus or P. aeruginosa. We demonstrate that these cytokines are expressed in response to both Gram-positive and Gram-negative bacterial components, by both normal epithelial cells and cells with CFTR dysfunction, and that they contribute to PMN survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial Strains
S. aureus strain RN6390 was obtained from A. Cheung (Dartmouth Medical School, Hanover, NH) and grown in CYGP media (19). The following P. aeruginosa strains have been described: PAO1 (wild type); PAO/NP, a pilA mutant the construction of which by gene replacement has been previously characterized (1); PAO/NP/fliA, a pilin-deficient, flagellin-deficient double mutant (20); PAO-R1, a lasR mutant lacking a central regulatory gene involved in quorum sensing and expression of several exoproducts including elastase and alkaline protease (21); and PAO1pmm, an algC mutant lacking phosphomannomutase activity resulting in a defective LPS core, lack of O side chains, and defective alginate synthesis (22). P. aeruginosa strains were grown in Luria Broth media overnight at 37°C with aeration. Aliquots were resuspended in PBS and diluted to give a concentration of 1 x 107 cfu/ml to stimulate the epithelial monolayers. Reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.

Epithelial Cell Lines
IB-3 cells ({Delta}F508/W1282X), a human bronchial epithelial cell line, and C-38 cells, "corrected" cells with normal physiology which express an episomal, truncated form of CFTR (23), were obtained from P. Zeitlin (Johns Hopkins University, Baltimore, MD) and grown in LHC-8 media (Biofluids, Rockville, MD) and 10% fetal calf serum (FCS). The IB-3 cells used in these studies contain the empty vector. The cells were weaned from serum for 24 h before cytokine enzyme-linked immunosorbent assay experiments, but were maintained in LHC-8 media and 10% FCS for PMN viability assays and for reverse transcriptase-polymerase chain reaction (RT-PCR) studies.

ELISA for GM-CSF and G-CSF Secretion
Confluent monolayers of epithelial cells were grown in 10 cm dishes. Cells were treated for 60 min with washed P. aeruginosa PAO1 (1 x 107 cfu/ml) resuspended in LHC-basal. The monolayers were washed three times in media containing 100 µg/ml gentamicin and re-incubated for 18–24 h. The culture supernatant was then removed, stored at -70°C, and used for G-CSF and GM-CSF assays. GM-CSF and G-CSF concentrations in the supernatant were detected using human GM-CSF and human G-CSF Quantikine kits from R&D Systems (Minneapolis, MN). ELISAs were performed as per manufacturer's instructions.

Statistical Analysis
Each G-CSF and GM-CSF enzyme-linked immunosorbent assay data point was determined in quadruplicate or quintuplicate. Means and standard deviations were calculated, and statistical significance was evaluated with GraphPad InStat version 3.0 (GraphPad, San Diego, CA) using a one-way analysis of variance. Bonferroni's post test was used to test the null hypothesis that there was no difference in the amount of the outcome variable (G-CSF or GM-CSF production) as compared with the untreated control.

Expression of GM-CSF and G-CSF by RT-PCR
Confluent monolayers of epithelial cells were grown in 10-cm dishes. Cells were treated for 60 min with either washed bacteria (1 x 107 cfu/ml) resuspended in LHC-basal, or isolated bacterial components; S. aureus lipoteichoic acid, P. aeruginosa LPS, antibody to asialoGM1 (Wako Chemical Co., Richmond, VA), or TNF-{alpha}. The monolayers were washed three times and lysed with RLT Buffer as per RNeasy Mini Kit protocol (Qiagen, Valencia, CA). Lysate was homogenized using Qiashredder and total RNA isolated using RNeasy Mini Kit. Reverse transcription was performed using Omniscript Reverse Transcriptase (RT) from Qiagen and Oligod(T) from Perkin Elmer (Boston, MA). PCR (35 cycles) was done using Taq from Roche Biochemicals (Indianapolis, IN). PCR primers were 5'-GCTTAGAGCAAGTGAGGAAG and 5'-AGGTGGCGTAGAACGCGGTA for human G-CSF, 5'-GGAGCATGTGAATGC CATC and 5'-ATCTGGGTTG CACAGGAAG for GM-CSF, 5'-TACTCCAAACCTTTCCAA CCC and 5'-AACTTCTCCACAACCCTCTG for IL-8, and 5'-GTGGGCCGCTCTAGGCACCA and 5'-GGTTGGCCTTAG GGTTCAGGGGGG for ß-actin.

Isolation of Neutrophils
Under sterile conditions, blood from medication-free donors was drawn using a heparin-coated syringe and immediately mixed with 0.9% NaCl. The blood was layered on a discontinuous Histopaque gradient and centrifuged at 2,000 RPM for 20 min. The neutrophil band was collected and centrifuged at 1,500 RPM for 15 min at 4°C. The resulting pellet was resuspended with ice-cold 0.2% NaCl, then mixed with 1.6% NaCl to lyse the residual erythrocytes. This mixture was centrifuged at 6,000 RPM for 8 s and the pellet was resuspended in neutrophil buffer (1x PBS containing 25% human serum albumin and 0.1% glucose). Cells were counted using a hemacytometer.

Neutrophil Viability Assay
Neutrophil viability was monitored in 96-well cell culture plates with each well containing 4 x 105 neutrophils. Cell culture supernatants (conditioned media) were harvested from confluent IB-3 or C-38 cell monolayers that were exposed to control conditions (media alone) or incubated with 1 x 107 cfu/ml of P. aeruginosa for 60 min, washed three times in media containing 100 µg/ml gentamicin, and re-incubated for 18–24 h. The conditioned media was mixed with neutralizing antibody to either GM-CSF (10 µg/ml), G-CSF (5 µg/ml), an equivalent amount of matched IgG isotype, or with both anti–GM-CSF and G-CSF (R&D Systems). The conditioned media (in doses ranging from 10–100%/well volume) was then added to the neutrophils and incubated for 18 h. Control wells were incubated with cell culture media (LHC-8 with 10% FCS) with or without G-CSF (1 ng/ml) or GM-CSF (500 U/ml). Following 18-h incubation at 37°C with CO2, neutrophil viability was determined using a colorimetric Procheck cell viability assay (Intergen, Purchase, NY) and monitored spectrophotometrically. This assay is based on the ability of viable neutrophils to reduce the tetrazolium salts present in the assay dye during a 1- to 2-h incubation. Each assay was performed in triplicate, a mean and standard error determined, and repeated at least three times using different PMN donors. Statistical significance was evaluated using analysis of variance as described above.

Quantitation of Neutrophil Apoptosis Using Flow Cytometry
The number of neutrophils undergoing apoptosis or necrosis following exposure to epithelial conditioned media was determined using Vybrant Apoptosis Assay Kit #2 from Molecular Probes, Inc. (Eugene, OR), which is composed of Alexa Fluor 488 annexin V and propidium iodide. In brief, 5 x 105 neutrophils were incubated with conditioned media harvested from C-38 or IB-3 cells that had been exposed to 1 x 107 cfu/ml of P. aeruginosa PAO1 for 1 h, human recombinant GM-CSF (5,000 U/ml), or media alone. After 42 h, cells were stained with Alexa Fluor 488 annexin V and propidium iodide and analyzed using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) and CellQuest software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMN Viability Is Increased by Epithelial Cell Culture Supernatants
To determine how epithelial cytokine expression affects PMN viability, conditioned supernatants from CF (IB-3) and control (C-38) cells were tested for effects on PMN survival following an 18-h incubation (Figure 1). Conditioned media harvested from uninfected C-38 or IB-3 cells had no effect in delaying the death of PMNs as compared with control media (not exposed to epithelial cells). In contrast, conditioned media from P. aeruginosa-exposed CF or corrected C-38 cell cultures prolonged PMN survival by 2- to 3-fold (P < 0.001) (Figure 1). A dose–response effect was noted as PMN viability increased as a function of the amount of conditioned media added up to 80%, after which cytotoxicity was observed (Figure 1A). To determine if the effects of conditioned media in prolonging PMN survival were due to G-CSF and/or GM-CSF secreted by the epithelial cells, we first tested how exogenous G-CSF and GM-CSF, as well as neutralizing antibody to each, influences PMN survival (Figure 1B). The addition of either G-CSF or GM-CSF to the media enhanced PMN viability by 2.5-fold (P < 0.001) and this effect was inhibited by the addition of neutralizing antibody to either of the cytokines. To determine if epithelial G-CSF and/or GM-CSF expression induced by P. aeruginosa enhances PMN survival, anti–G-CSF, anti–GM-CSF, or both antibodies were added to conditioned media harvested from CF (IB-3) or corrected (C-38) cells (Figure 1C). Conditioned media from P. aeruginosa stimulated IB-3 cells caused a 4.3-fold increase in the number of viable PMNs as compared with the conditioned media from unstimulated cells. Addition of anti–GM-CSF (10 µg/ml) decreased PMN viability by 23% (P < 0.001) in contrast to the isotype control IgG1 that had no effect. Antibody to G-CSF (5 µg/ml) diminished the effect of conditioned media on PMN survival by 35% (P < 0.001).



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  		Figure 1. Effects of CF (IB-3) and corrected (C-38) cell culture supernatants (conditioned media) on PMN viability. (A) Dose response–PMN survival was assessed after an 18-h incubation with dilutions of conditioned media harvested from unstimulated IB-3 cells or IB-3 cells stimulated with PAO1 for 60 min, and compared with the viability of PMNs exposed to LHC-8 and 10% FCS (0% control). The number of viable PMNs following an 18-h incubation with media alone was considered to be 100% (control). PMN viability was assessed spectrophotometrically using the Procheck Cell Viability Assay. The experiment was done twice with similar results and a representative experiment is presented. (B) Effects of neutralizing antibody to G-CSF and/or GM-CSF on PMN viability. The effects of exogenous GM-CSF (500 U/ml) or exogenous G-CSF (1 ng/ml) +/- respective neutralizing antibodies (10 µg/ml anti–GM-CSF and 5 µg/ml anti–G-CSF) on PMN viability are shown as compared with cells exposed to media alone (control). These experiments were done three times and representative data is presented. (C). Effects of neutralizing antibody on conditioned media harvested from CF (IB-3) and corrected (C-38) cells. PMN viability was compared following 18 h incubation with conditioned media (40%) harvested from P. aeruginosa stimulated CF (IB-3) cells (black bars) or corrected C-38 cells (hatched bars) in the presence of neutralizing antibody to GM-CSF (10 µg/ml), an IgG1 isotype control antibody (10 µg/ml), anti–G-CSF (5 µg/ml) or both antibodies. Each experimental condition was tested in triplicate and a mean and standard error of the mean were calculated. The error bars, once the data is converted to percent control, are less than 1% and thus, are contained within the data bars.

 
Similar but smaller effects were obtained for the C-38 cells. There was a 2.8-fold increase in PMN viability in the presence of P. aeruginosa stimulated C-38 conditioned media that was decreased by 38% (P < 0.01) with the addition of anti–GM-CSF (10 µg/ml) and decreased by 12% with 5 µg/ml of anti–G-CSF. An additive effect was seen with both antibodies (10 µg/ml GM-CSF and 5.0 µg/ml of anti-G-CSF), which together decreased PMN viability by 50% (P < 0.01).

Expression of G-CSF and GM-CSF in Matched CF and Control Cell Lines
Bacterial induction of G-CSF and GM-CSF in the CF and corrected cell lines were compared. Induction of IL-8 expression, which is increased in cells with CFTR dysfunction (24), was included as a control. G-CSF mRNA was induced by either P. aeruginosa or S. aureus in both C-38 (control) and IB-3 (CF) cell lines (Figure 2). mRNA for G-CSF, GM-CSF, and IL-8 was detected in unstimulated normal cells (C-38), and increased expression was detected 24 h following stimulation with either P. aeruginosa or S. aureus. The corresponding IB-3 (CF) cells appeared to have slightly more G-CSF and GM-CSF expression than the normal cells under unstimulated conditions; there was significantly more IL-8 expression by the CF cells.



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  		Figure 2. Expression of cytokines and chemokines in cell lines with CFTR dysfunction. Matched CF (IB-3) and corrected (C-38) cell lines under unstimulated conditions or following exposure to P. aeruginosa PAO1 or S. aureus RN6390 for 1 hour were washed with PBS, sterilized with 100 µg/ml of gentamicin for 24 h, and analyzed by RT-PCR for G-CSF, GM-CSF, IL-8, and ß-actin expression. Shown is a representative experiment.

 
To verify that the RT-PCR results, which are not quantitative, correlate with actual cytokine secretion, the G-CSF and GM-CSF concentrations in epithelial cell culture supernatants were measured by ELISA in three separate experiments (Figure 3). In response to P. aeruginosa stimulation, there were 6.6- and 7.5- fold increases in G-CSF production in the C-38 and IB3 (CF) cells, respectively (not significantly different; P > 0.05 for all of the data analyzed together) (Figure 3A). GM-CSF production in C-38 and IB3 cells increased by 5.9- and 16.6-fold, respectively; following exposure to P.aeruginosa, a significantly greater increase was associated with the CF cells as compared with the corrected C-38 cells (Figure 3B) (P < 0.001).



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  		Figure 3. P. aeruginosa induction of G-CSF and GM-CSF expression by CF (IB-3) and corrected (C-38) cells. (A) G-CSF and (B) GM-CSF in C-38 and IB-3 epithelial cell culture supernatants were measured by ELISA under control conditions or following a 60 min incubation with P. aeruginosa PAO1. Each experiment was performed on 3 separate days in quintuplicate and all of the data assayed by ELISA and analyzed for statistical significance. The data shown are the averages of three experiments. G-CSF production increased in response to P. aeruginosa stimulation by 6.6-fold in C-38 cells and 7.5-fold in IB-3 cells (P > 0.05). GM-CSF production increased by P. aeruginosa stimulation by 5.9-fold in C-38 cells and 16.6-fold in IB-3 cells (P < 0.001).

 
Bacterial Induction of G-CSF and GM-CSF Expression
To identify which bacterial components activate the expression of these cytokines, IB-3 (CF) and C-38 (control) cell lines were stimulated with individual bacterial components or TNF-{alpha}, and cytokine transcription was estimated by RT-PCR. Antibody to the P. aeruginosa pilin receptor asialoGM1, a stimulus for NF-{kappa}B activation and IL-8 expression that is differentially expressed in CF and normal cells, was included as a control (24). Constitutive expression of G-CSF was not detected in either the CF or corrected cell lines under control conditions by this assay (Figure 4). Antibody to asialoGM1, S. aureus lipoteichoic acid, and TNF-{alpha}, but not LPS, activated G-CSF expression in both the C-38 and IB3 (CF) cell lines. GM-CSF expression was induced by all of the stimuli and was detected in unstimulated CF cells as well.



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  		Figure 4. Comparison of G-CSF and GM-CSF expression in CF and corrected cells induced by bacterial components. Expression of G-CSF, GM-CSF and an actin control are compared in CF (IB-3) and corrected cells (C-38) in response to specific bacterial ligands. Cell cultures were treated with media or the following reagents for 2 h prior to RT-PCR analysis: Control cells, unstimulated; antibody to asialoGM1 (10 µg/ml); P. aeruginosa LPS (0.1 mg/ml); S. aureus lipoteichoic acid (LTA) (10 µg/ml); TNF-{alpha} (50 ng/ml), positive control.

 
To determine what bacterial components activate G-CSF and GM-CSF expression, P. aeruginosa mutants with defects in specific adhesins or virulence factors were tested for their ability to stimulate epithelial cytokine expression (Figure 5). The P. aeruginosa adhesins pili and flagella appear to be involved in stimulating G-CSF expression (Figure 5A), whereas lack of mature LPS (PAOpmm) did not affect G-CSF stimulation, nor did the lack of coordinated exoproduct expression (PAOR1). P. aeruginosa mutants lacking pili stimulated approximately half as much GM-CSF expression as the parental strain (Figure 5B), whereas mutants with defective LPS structure or lacking exoproducts (PAOR1) were not impaired in GM-CSF stimulation.



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  		Figure 5. Induction of G-CSF and GM-CSF expression by P. aeruginosa mutants—the expression of (A) G-CSF and (B) GM-CSF in IB-3 cell culture supernatants were measured by ELISA following a 60 min incubation with a 1 x 107 cfu/ml inoculum of the following strains: Control, no bacteria; PAO1 (wild type); PAO/NP (pilA lacks pilin); PAOpmm (algC) (phosphomannomutase mutant with defective LPS); PAO-R1 (lasR lacks quorum sensing and some exoproduct expression); PAO/NP/fliA (lacks both pili and flagella). Experiments were performed on 3 separate days and each experimental condition was performed in quintuplicate. A representative experiment is shown.

 
GM-CSF- and P. aeruginosa–Stimulated Conditioned Media Inhibit PMN Apoptosis
Having found that P. aeruginosa–stimulated conditioned media prolongs PMN viability, and that this effect is attributable to induction of G-CSF and GM-CSF expression, we expected to find that this effect is due to inhibiting PMN apoptosis. To determine whether this is the case, PMNs exposed to either GM-CSF alone, or conditioned media from P. aeruginosa–stimulated IB3 and C-38 cells were screened for apoptosis using Alexa Fluor 488 annexin V and for necrosis using propidium iodide (Figure 6). Under control conditions, > 60% of PMNs exposed to media alone are apoptotic in 42 h, as compared with ~ 30% apoptotic PMNs exposed to media containing GM-CSF or conditioned media. Fewer than 5% of the untreated PMNs were viable at 42 h, as compared with > 40% viability of PMNs incubated with conditioned media from IB3 or C-38 cells, or cells treated with GM-CSF.



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  		Figure 6. Effects of conditioned media and GM-CSF alone on PMN viability and apoptosis. PMNs were incubated with media, GM-CSF (5,000 U/ml), or P. aeruginosa-stimulated CM from C-38 (C-38/PA CM) and IB-3 (IB-3/PA CM) cells for 42 h and analyzed by flow cytometry using Alexa Fluor 488 annexin V as a marker for apoptosis and permeability to propidium iodide as an indication of necrosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Airway epithelial cells play a major role in the immune response to inhaled bacteria. In these studies, we demonstrate that both P. aeruginosa and S. aureus, common airway pathogens, stimulate sufficient epithelial expression of G-CSF and GM-CSF to affect PMN viability through their independent and combined effects in inhibiting PMN apoptosis. Epithelial responses to airway pathogens are particularly important in cystic fibrosis, a disease characterized by impaired clearance of inhaled bacteria, and by what appears to be inappropriate inflammation in the airway (4). In vitro studies indicate that some NF-{kappa}B–dependent gene expression is increased in cells with CFTR dysfunction even in the absence of bacterial stimulation (6). Increased bacterial attachment to cells with CFTR dysfunction has been demonstrated and is directly associated with increased IL-8 expression (1, 25, 26). P. aeruginosa pulmonary infections in cftr -/- mice are associated with significantly greater amounts of inflammation than in wild-type mice consistent with these in vitro observations (27). However, not all epithelial proinflammatory responses are upregulated in cells that express CFTR mutations. Neither TNF-{alpha} (28), nor regulated upon activation, normal T cell expressed and secreted (29) signaling has been found to be increased, nor was neutrophil migration increased through CF epithelial monolayers (30). Our cytokine data similarly indicate more GM-CSF expression associated with bacterial activation of the CF cells, but no differences in G-CSF expression in normal and CF cells.

Although comparable clinical studies are difficult to perform, Noah and coworkers (31) suggested that patients with CF demonstrate more airway inflammation than do normal patients with comparable bacterial infection. In prospective studies of infants with CF, excessive numbers of inflammatory cells, PMN products, proinflammatory cytokines, and chemokines have been found in the airways even in the absence of detectable infection (4). These clinical studies suggest that once an inflammatory response is triggered in the CF airway, whether it is quantitatively greater than normal or not, it may not be regulated appropriately. The kinetics of GM-CSF secretion are altered in CF cells, and cytokine production persists for much longer than in control cells following exposure to P. aeruginosa (3). As presented here, it appears that G-CSF and GM-CSF, along with IL-8, contribute to the accumulation of PMNs in the airways and have a substantial effect in delaying the normal progression of PMN apoptosis, presumably through activation of PI-3 kinase and ERK kinase pathways (10, 11). Thus, epithelial expression of these NF-{kappa}B–dependent chemokines also contributes to the persistent PMN-dominated inflammation typical of CF airway disease.

The contribution of GM-CSF to the general homeostasis of the lung has been well established (12, 15), whereas its participation in mucosal immunity is less well characterized. Epithelial expression of GM-CSF is part of the acute response to foreign antigens, such as bacteria, viruses (32), or allergens (8). In the experiments presented here, diverse bacterial components are shown to stimulate G-CSF and GM-CSF expression. GM-CSF expression is induced by Gram-positive bacteria such as S. aureus or isolated staphylococcal lipoteichoic acid, which does not activate IL-8 expression in these cells (A. Prince, unpublished observations). G-CSF and GM-CSF expression are stimulated by ligands that recognize asialylated glycolipid receptors. As has been shown for bacterial activation of IL-8 expression (1), recognition of asialylated glycolipid receptors by intact bacteria, P. aeruginosa pili, flagella, or antibody, stimulate both G-CSF and GM-CSF production by airway epithelial cells. Piliated P. aeruginosa and S. aureus stimulate epithelial signaling through an asialoGM1 pathway that involves Ca2+-dependent activation of ERK MAPK's and NF-{kappa}B translocation (2), a pathway that is also likely to activate G-CSF and GM-CSF expression. Although it is unlikely that more proximal components of this signaling pathway are shared, it appears that the induction of GM-CSF and G-CSF expression by both bacteria and viruses involves the activation of ERK MAPKs (16, 33).

LPS was not a potent stimulus for GM-CSF or G-CSF expression, as has been shown previously for IL-8 in the same cell lines (1). The PAO1algC mutant with defective LPS core and O-side chain glycosylation was fully capable of activating expression of either cytokine. Similarly, the PAOlasR mutant was unimpaired in its stimulation of cytokine production. This mutant has multiple defects, including limited protease expression. Airway epithelial cells have been shown to express GM-CSF following stimulation of the proteinase-activated receptor-2 (9); however, P. aeruginosa proteases are apparently not necessary to evoke a GM-CSF response in this fashion. Epithelial GM-CSF expression is also inducible by IL-1ß, or TNF-{alpha} (16) cytokines that are elaborated by epithelial cells (12); thus, it is possible that there are also autocrine effects regulating GM-CSF expression.

Having demonstrated that both S. aureus and P. aeruginosa stimulate G-CSF and GM-CSF expression by airway epithelial cells, a biologically important effect of these cytokines on airway inflammation may be prolongation of PMN survival. Our results demonstrate that G-CSF and GM-CSF secreted by airway cells exposed to bacteria enhance PMN survival by delaying constitutive apoptosis, presumably through activation of PI-3 kinase and ERK kinase pathways (10, 11). This effect may be especially significant in the CF lung, considering the potential for exogenous stimulation of cytokine expression. Organisms persisting in the airways or trapped in mucin can shed components to activate airway cytokine responses. Although the epithelial cells did not constitutively express sufficient cytokine to affect PMN survival in the absence of infection, once either normal or CF epithelial cells are exposed to bacteria, sufficient amounts of G-CSF and GM-CSF are expressed to prolong PMN survival.

The cytokine expression associated with stimulated CF cells and subsequent effects on PMN viability may help to explain the clinical observations that bronchoalveolar lavage fluids from patients with CF often contain PMNs, even in the absence of detectable infection. Transient contamination of the airways and delayed clearance of bacteria, as is common in CF, stimulate cytokine release by CF cells. The anti-apoptotic effects of both G-CSF and/or GM-CSF would contribute to persistence of the PMNs in the airways. Lysed bacteria, or even shed bacterial components, would be sufficient to stimulate epithelial cytokine responses even if the organisms were killed by mucosal defenses. As there is little evidence for CFTR-associated defects in professional immune cells, it seems likely that the exaggerated inflammatory responses typical of CF airways are a manifestation of abnormal mucosal immunity as exhibited by airway epithelial cells, which are substantially affected by CFTR dysfunction.

Epithelial cytokine secretion is clearly relevant to the pathogenesis of airway inflammation in CF. The data here presented do not reflect major differences in the basal level of cytokine expression in these CF and corrected cell lines. Neither of the cell lines produced sufficient G-CSF or GM-CSF under basal conditions to affect PMN apoptosis. However, once stimulated by bacteria, both normal and CF cells produce cytokines that effect PMN survival. The combined effects of both cytokines and chemokines, and prolonged epithelial exposure to bacterial stimuli in the CF lung, could account for much of the PMN-dominated airway inflammatory response characteristic of this disease process (4, 27, 31). Although this is not a primary cause of the excessive inflammatory response in CF, these observations help to explain why inflammation, once initiated in CF, is so persistent and progressive.


   Acknowledgments
 
This work was supported by National Institutes of Health grant RO1 HL56194 (A.P.).

Received in original form February 8, 2002

Received in final form May 3, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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