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Originally published online as doi:10.1189/jlb.0605289 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1408-1418.)
© 2005 by Society for Leukocyte Biology

Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor

Scott D. Kobayashi1, Jovanka M. Voyich, Adeline R. Whitney and Frank R. DeLeo2

Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana

2 Corresponding author: Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840. E-mail: fdeleo{at}niaid.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear leukocytes (PMNs or neutrophils) are the most prominent cellular component of the innate immune system in humans and produce an array of potent cytotoxic molecules. It is important that neutrophils undergo constitutive (spontaneous) apoptosis as a mechanism to facilitate normal cell turnover and immune system homeostasis. Conversely, several proinflammatory cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), prolong neutrophil survival. The molecular mechanisms that regulate PMN apoptosis or survival remain incompletely defined. To that end, we compared global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Genes encoding proteins that inhibit apoptosis, such as myeloid cell leukemia sequence 1, caspase 8 and Fas-associated via death domain-like apoptosis regulator (CFLAR), B cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa-interacting protein 2 (BNIP2), and serum/glucocorticoid-regulated kinase (SGK), were down-regulated coincident with neutrophil apoptosis. In contrast, those encoding apoptosis inhibitor 5, BCL2-like 1, BNIP2, CFLAR, SGK, and tumor necrosis factor {alpha}-induced protein 8 were up-regulated in PMNs cultured with GM-CSF. Correspondingly, GM-CSF delayed PMN apoptosis (P<0.03), increased cell viability (P<0.03), and prolonged neutrophil phagocytic capacity (P<0.05). Prolonged functional capacity was paralleled by striking up-regulation of proinflammatory genes and proteins, including CD14, CD24, CD66, and human leukocyte antigen-DR. In addition, expression of SGK protein diminished during PMN apoptosis but was restored by culture with GM-CSF, suggesting SGK is involved in leukocyte survival. These studies provide a global view of the molecular events that regulate neutrophil survival and apoptosis.

Key Words: inflammation • microarray • polymorphonuclear leukocytes • serum/glucocorticoid-regulated kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human polymorphonuclear leukocytes (PMNs; neutrophils or granulocytes) are critical for innate host defense and comprise the single greatest cellular component of the immune system. Approximately 60% of all human leukocytes are granulocytes [1 ]. Neutrophils contain or produce an array of cytotoxic molecules, including numerous proteases and reactive oxygen species (ROS) [2 3 4 5 ], which have potential to cause damage to host tissues during inflammation processes. Therefore, it is critical that neutrophil homeostasis and turnover are highly regulated. As such, ~1011 neutrophils turnover per day in the average human adult [6 ], and this dramatic PMN turnover is mediated by apoptosis [7 8 9 ].

Most inflammation-related processes alter neutrophil apoptosis. For example, phagocytosis of bacteria accelerates apoptosis significantly, presumably to facilitate clearance of effete or "spent" PMNs containing dead bacteria [10 , 11 ]. Conversely, some bacteria-derived products, such as lipopolysaccharide, prolong neutrophil survival [12 13 14 ]. In addition, several proinflammatory cytokines, chemokines, or growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF), delay neutrophil apoptosis [12 , 15 ]. The ability of GM-CSF and other proinflammatory molecules to delay PMN apoptosis is likely important for effective clearance of invading microorganisms. Previous studies demonstrated that the ability of GM-CSF to prolong PMN survival is dependent on new transcription and protein synthesis [15 ]. In addition, Lyn kinase, phosphoinositide-3 kinase (PI-3K), extracellular signal-regulated kinase, Janus kinase/signal transducer and activator of transcription, and CD137 play prominent roles in GM-CSF-mediated neutrophil survival [16 17 18 19 20 ]. PI-3K has been reported to regulate GM-CSF-mediated neutrophil survival through regulation of Mcl-1 and Bad, key modulators of apoptosis [19 , 21 22 23 ].

Although progress has been made, the molecular basis for the ability of GM-CSF to prolong neutrophil survival is incompletely defined. Inasmuch as the cytokine also primes neutrophils for enhanced responses to subsequent stimuli [24 25 26 27 28 29 ], the mechanisms underlying effects of GM-CSF are likely complex. To that end, we used Affymetrix microarrays coupled with TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunoblotting to generate a comprehensive view of human PMN responses to GM-CSF. Our findings provide new insight into mechanisms that modulate neutrophil function during inflammatory processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Human GM-CSF was purchased from Roche Applied Sciences (Indianapolis, IN). RPMI-1640 medium was obtained from Invitrogen (Carlsbad, CA). Flourescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) specific for human leukocyte antigen (HLA)-DR (clone G46-6), CD14 (clone M5E2), CD24 (clone ML5), CD66 (clone B6.2), CD89 (clone A59), CD119 (clone GIR-94), CD123 (clone 9F5), mouse immunoglobulin G2a (Ig2a; clone G155-178), and mouse IgG1 (clone MPOC-21) were purchased from BD PharMingen (San Diego, CA). Annexin-V conjugated with allophycocyanin (Annexin-V-APC) was also purchased from BD PharMingen. Polyclonal antibody specific for serum/glucocorticoid-regulated kinase 1 (SGK1) was obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Unless specified, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Isolation of human PMNs
Human PMNs were isolated from venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH; Hamilton, MT). Heparinized blood was mixed 1:1 with 0.9% sodium chloride (Irrigation USP, Baxter Healthcare, Deerfield, IL) containing 3.0% Dextran T-500 (Amersham Biosciences Corp., Piscataway, NJ) and incubated for 20 min at room temperature to sediment erythrocytes. The leukocyte-rich supernatant was centrifuged at 670 g for 10 min and resuspended in 35 ml 0.9% sodium chloride. The leukocyte suspension was underlaid with 10 ml Ficoll-PaquePLUS (1.077 g/liter, Amersham Biosciences Corp.) and centrifuged for 25 min to separate PMNs from peripheral blood mononuclear cells (PBMCs). Following aspiration of the PBMC layer and remaining supernatant, sides of the gradient tubes were wiped with sterile cotton swabs to remove any residual cells. Erythrocytes were lysed with water (Irrigation USP, Baxter Healthcare) for 15–30 s followed by immediate mixing with 1.7% sodium chloride. Purified PMNs were centrifuged at 380 g, resuspended in RPMI-1640 medium buffered with 10 mM HEPES (RPMI/H; pH 7.2), and enumerated by microscopy. Isolation of PMNs was performed at room temperature, and purity of neutrophil preparations and cell viability was assessed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). Cell preparations contained ~99% granulocytes (PMNs; neutrophils, eosinophils, and basophils). We have shown previously that eosinophils typically comprise ~5.8% (n=13) of the PMNs in our preparations [30 ]. For simplicity, the terms PMN and neutrophil are used interchangeably. All reagents used contained <25.0 pg/ml endotoxin (Limulus amebocyte lysate assay, Fisher Scientific, Suwanne, GA).

Neutrophil phagocytosis and ROS production
Human PMNs were cultured in RPMI/H ± 100 ng/ml GM-CSF at 37°C and 5% CO2 for 0, 24, and 48 h. For phagocytosis experiments, antibody and complement-coated latex beads (IgG/C3bi LB; 2.0 µm, Polysciences, Inc., Warrington, PA) were prepared as described previously [31 ] but were labeled with FITC [0.75 µg/ml in Dulbecco’s phosphate-buffered saline (DPBS)] for 15 min at room temperature. FITC-labeled IgG/C3bi-LB were washed in DPBS and used immediately for phagocytosis assays. PMNs (107) were combined on ice with or without IgG/C3bi-LB-FITC (8x107) in wells of a 96-well tissue-culture plate precoated with normal human serum (NHS). Plates were centrifuged at 380 g for 8 min at 4°C to synchronize phagocytosis. Following centrifugation, plates were incubated at 37°C in a CO2 incubator for 30 min. Phagocytosis was measured with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA), after addition of 0.2% Trypan blue as described [32 ]. Percent phagocytosis was calculated with the equation (FITC-positive PMNs/total number of PMNs) x 100. Ten thousand events were collected for each sample, and data were analyzed with CellQuest Pro software (BD Biosciences). The assay measures the percentage of PMNs with ingested IgG/C3bi-LB.

PMN ROS production was measured using a published method [31 , 33 ]. Briefly, neutrophils were incubated with 25 µM 2',7'-dihydrodiclorofluroscein diacetate (Molecular Probes, Eugene, OR) for 30 min at room temperature in RPMI/H. PMNs (106) and IgG/C3bi-LB (8x108) were combined in wells of a 96-well microtiter plate at 4°C, and samples were centrifuged for 5 min at 380 g. Following centrifugation, plates were transferred to a microplate fluorometer (Spectramax Gemini, Molecular Devices, Sunnyvale, CA), and ROS production was measured for up to 90 min at 37°C with excitation and emission wavelengths of 485 and 538 nm, respectively. Maximum velocity (Vmax) was calculated as the maximum rate of ROS production within a 10-min time period using Softmax Pro Version 3.1.2 (Molecular Devices).

Neutrophil RNA preparation/gene expression analysis
Human PMNs were cultured in RPMI/H ± 100 ng/ml GM-CSF at 37°C and 5% CO2 for up to 24 h as indicated. At the indicated time-points, tissue culture medium was aspirated from each well, and PMNs were lysed with RLT buffer (Qiagen, Valencia, CA). RNA was isolated as described [11 , 31 , 34 ] and was subsequently used to prepare labeled cRNA target (12 µg) for analysis on Hu95Av2 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). Labeling of samples, GeneChip hybridization, and scanning were performed according to standard Affymetrix protocols (see http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Total PMN RNA was visualized with an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Wilmington, DE) to detect potential degradation of RNA species, and GeneChips had 5' to 3' RNA ratios of >3 and a scaled noise factor of >5 (GeneChip SuiteTM, Affymetrix). Each experiment was performed with three separate blood donors, except for the Time 0 controls. Nine blood donors were used to generate the Time 0 control samples.

Gene expression data were analyzed as described previously with GeneSpring expression analysis software Version 6.1 (Silicon Genetics, Redwood City, CA) [11 ]. Briefly, genes were defined as differentially transcribed if the average expression level changed at least twofold compared with unstimulated cells (0 h, those not cultured at 37°C) over the three experiments. Further, up-regulated genes must have been called "Present" in at least two individuals by Microarray SuiteTM (Affymetrix), and alternatively, down-regulated genes had to be Present in six of nine individuals at Time 0. All of the genes included as differentially transcribed were up- or down-regulated at least twofold in one of the treatments. Microarray data are posted on the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, accession number GSE2803).

Assays for PMN apoptosis
PMNs (2x106) were plated directly into 24-well plates precoated with NHS and incubated in the presence or absence of human GM-CSF (100 ng/ml final concentration) for up to 48 h. Four separate assays were used to determine PMN apoptosis. DNA fragmentation was determined with a modified terminal deoxyuridine triphosphate nick-end labeling (TUNEL) assay as described by the manufacturer (Apo-BRDUTM apoptosis detection kit, BD Biosciences). DNA content in apoptotic PMNs was detected by flow cytometry as described [31 , 35 ]. Briefly, cells (1x106/0.1ml) were resuspended in 2x hypotonic fluorochrome solution [propidium iodide (PI), 100 µg/ml, in 0.2% sodium citrate plus, 0.2%, Triton X-100] and incubated overnight at 4°C. Surface exposure of phosphatidylserine, a well-known indicator of early apoptosis, was determined with Annexin-V-APC as described by the manufacturer (BD PharMingen). Samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson). Ten thousand events were collected for each sample, and data were analyzed with CellQuest Pro software (BD Biosciences). Alternatively, morphological assessment of neutrophil apoptosis (condensed nuclei) was determined by microscopy [11 , 36 ]. For each sample, ~250 cells from five separate fields of view were analyzed.

TaqMan real-time RT-PCR analysis (TaqMan analysis)
PMNs were cultured for 18 h or 24 h ± GM-CSF, and RNA preparation for TaqMan analysis was done with conditions similar to those used for the microarray analysis. Contaminating DNA was subsequently removed from RNA samples by treatment with DNA-Free (Ambion, Austin, TX) [31 ]. Primers and probe sets were designed with Primer ExpressTM software Version 1.5a (Applied Biosystems, Foster City, CA) and were manufactured by Applied Biosystems as follows: forward primer, 5'-AGAACATTGAACACAACAGCACAACT-3'; reverse primer, 5'-AAGC-ACCTCAGGTGCGAGAT-3'; and probe for SGK, 5'-CACCTTCTGTGGCACGCCGGAG-3'. TaqMan analysis of triplicate samples from three blood donors was performed with an ABI 7500 thermocycler (Applied Biosystems) as described previously [31 ].

Analysis of surface-expressed proteins by flow cytometry
Human PMNs were cultured as described above and subsequently stained with the indicated mAb (5 µg/ml for 30 min at 4°C) as described by the manufacturer (BD PharMingen). PI (0.5 µg/ml final concentration) was used to identify dead cells. Ten thousand events for each sample were collected on a FACSCalibur flow cytometer (Becton Dickinson), and data were analyzed with CellQuest Pro software (BD Biosciences). Percent positive neutrophils were determined with a marker defined by the boundary of the isotype-matched control antibody. Alternatively, cells were stained simultaneously with antibodies specific for CD24 or CD66, Annexin-V-APC, and PI. Quadrants were set based on isotope control antibodies (mouse IgG2a and IgG1) and fresh PMNs (Annexin-V-APC and PI). Dead cells were excluded with a single gate. Dot plots were retraced using CorelTrace12, Version 12 (Corel Corp., Ottawa, Ontario, Canada).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
Neutrophils (106) were cultured ± GM-CSF (100 ng/ml) in 96-well plates for up to 24 h as indicated. At the desired time, cells were boiled in standard Laemmli SDS-PAGE sample buffer (Bio-Rad, Hercules, CA) containing 2-mercaptoethanol for 5 min. Proteins were resolved by SDS-PAGE (10–20% Tris-HCL gels, Bio-Rad) and transferred to nitrocellulose. Immunoblots were probed with rabbit antibody specific for human SGK1 (5 µg/ml, Upstate Cell Signaling Solutions) for 1 h and detected with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:1000, Amersham Biosciences). Images were cropped and assembled in CorelDraw graphics suite Version 12 (Corel Corp.) An open lane between 3 h and 9 h was cropped out of each immunoblot.

Statistics
Statistics were performed with a paired Student’s t-test (Microsoft Excel 2002, Microsoft Corp., Bellevue, WA), one-way ANOVA, and Bonferroni’s or Dunnett’s post-test for multiple comparisons (GraphPad Prism Version 4.0 for Windows, GraphPad Software, Inc., San Diego, CA) unless indicated otherwise.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neutrophil apoptosis and the effects of GM-CSF on viability and function
As a first step toward gaining a better understanding of PMN apoptosis, we characterized constitutive or spontaneous neutrophil apoptosis and the ability of GM-CSF to prolong neutrophil survival using our assay conditions (Fig. 1A 1B 1C 1D ). After 24 h in culture, spontaneous neutrophil apoptosis was 66.3 ± 4.8% by morphological assessment, 79.5 ± 2.2% using TUNEL, and 78.7 ± 2.8% with PI staining (Fig. 1B 1C 1D) . Apoptosis was inhibited significantly by 100 ng/ml GM-CSF after 24 h of culture (78.6±3.1% for untreated cells vs. 41.3±6.9% for those cultured with GM-CSF, P<0.01, n=5; Fig. 1A ). Prolonged survival was temporal, as apoptosis in PMNs cultured with GM-CSF continued to increase by 48 h, albeit to a reduced level compared with untreated cells (Fig. 1B) . The ability of GM-CSF to delay apoptosis under our assay conditions was confirmed with three separate assays (Fig. 1A 1B 1C 1D) . During the onset of apoptosis, PMN viability decreased slightly over time (to 80.7±4.5% at 24 h) but was rescued completely by GM-CSF (94.7±1.1% at 0 h vs. 95.1±0.8% at 24 h with GM-CSF; Fig. 1E ). These findings are consistent with previous studies by Colotta et al. [12 ] and Brach et al. [15 ], who first described the ability of proinflammatory cytokines to prolong neutrophil survival.



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  		Figure 1. GM-CSF delays neutrophil apoptosis and prolongs cell function. (A and B) Apoptosis was measured by morphological assessment of neutrophil nuclei at the indicated concentrations of human GM-CSF. (A) Apoptosis was measured after 24 h of culture. (B) Cells were cultured ± 100 ng/ml GM-CSF. *, ANOVA with Dunnett’s post-test. (C) PMN apoptosis was measured by staining of subdiploid DNA with PI. (D) Neutrophil apoptosis was measured with a modified TUNEL assay. (E) Percent intact PMNs was determined by exclusion of PI as described in Materials and Methods. (F) Neutrophils were cultured for the indicated times ± 100 ng/ml GM-CSF, and phagocytosis assays were performed as described in Materials and Methods. Phagocytosis is the percentage of neutrophils containing ingested IgG/C3bi LB. Statistics were performed with repeated-measures ANOVA and Bonferroni’s post-test. (B–E) Statistical analyses were made with a one-way ANOVA and Bonferroni’s post-test. Results are displayed as the mean ± SEM of the indicated number of experiments.

 
Inasmuch as neutrophil survival was prolonged significantly by culture with GM-CSF, we next tested whether there was concomitant retention of neutrophil function. Neutrophil phagocytosis of antibody- and complement-opsonized latex beads (IgG/C3bi-LB) was used as a measure of cell function (Fig. 1F) . The ability of human neutrophils to ingest IgG/C3bi-LB was reduced significantly after 24 h in culture (from 34.2±1.6% with freshly isolated cells to 8.2±0.8% at 24 h, P<0.001; Fig. 1F ). These findings are in accordance with studies by Whyte and coworkers [36 ], who demonstrated reduced chemotaxis, phagocytosis, superoxide production, and degranulation in aged neutrophils. Conversely, there was marked retention of phagocytic capacity after 24 h of culture with GM-CSF (8.2±0.8% in untreated cells at 24 h vs. 19.7±0.4% in those cultured with GM-CSF, P<0.05). Similar results were obtained by measuring production of ROS after phagocytosis (not shown).

Global changes in neutrophil gene expression during spontaneous apoptosis and culture with GM-CSF
To gain new insight into the molecular processes that regulate PMN apoptosis and cytokine-enhanced survival, we screened ~12,500 human genes for changes in gene expression over a 24-h period of time in the presence and absence of GM-CSF (Figs. 2 and 3 and Supplemental Table 1, which is published as online supplemental material). To facilitate analysis, differentially expressed genes were categorized by reported or putative function (Figs. 2 and 3) . We focused mainly on genes whose expression patterns differed between control (untreated) and GM-CSF-treated culture conditions.



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  		Figure 2. Global changes in PMN gene expression during spontaneous neutrophil apoptosis and culture with GM-CSF. Human neutrophils were cultured in a humidified CO2 incubator at 37°C for the indicated times ± 100 ng/ml GM-CSF. Differential gene expression was measured with AffymetrixTM Hu95Av2 human oligonucleotide microarrays as described in Materials and Methods. Results are presented as the mean fold-increase or -decrease of three separate experiments using PMNs from three separate individuals (comparison is with freshly isolated PMNs at the start of culture, i.e., at 0 h). *, Genes encoding proteins known to inhibit apoptosis.

 


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  		Figure 3. GM-CSF modulates global changes in PMN genes encoding regulators of metabolism, protein synthesis, and unknown function. Neutrophil gene expression was measured as described in the legend of Figure 2 .

 
Transcriptional regulation of proinflammatory modulators
Expression of dozens of genes encoding PMN proinflammatory molecules diminished over 24 h in culture (Fig. 2) . For example, there was significant down-regulation of genes encoding interleukin-1 receptor 1 (IL1R1), IL1R2, IL-1R antagonist, Ig superfamily member 6, interferon-{alpha}, -ß, and -{omega} receptor 2 (IFNAR2), IFN-{gamma} receptor 1 (IFNGR1 or CD119), suppressor of cytokine signaling 1 (SOCS1), SOCS3, proviral integration site 1 (PIM1), CD14, CD32, complement receptor 1 (CD35), CD58, L-selectin (CD62L), CD89, CD97, GM-CSF receptor-{alpha} (CD116), GM-CSF receptor-ß (CD131), sialomucin (CD164), Toll-like receptor 1 (TLR1), TLR2, IL-13R {alpha}1 (IL13RA1), and matrix metalloproteinase 9 (or gelatinase) at or before 24 h of culture (Fig. 2 and Supplemental Table 1). Notably, decreases in a large number of proinflammatory genes corresponded well with induction of neutrophil apoptosis (compare Figs. 1 and 2 ). Previous studies by Dransfield et al. [37 ] demonstrated that surface expression of CD35, CD58, and CD62L diminishes coincident with apoptosis, and we recently reported that phagocytosis-induced apoptosis triggers down-regulation of proinflammatory capacity [30 ].

In contrast, there was dramatic increase in dozens of transcripts encoding proteins that facilitate host defense or play a key role in the inflammatory response after culture with GM-CSF (Fig. 2 and Supplemental Table 1). For example, CD14, CD32, CD24, CD44, CD54, CD66A, CD69, CD74, CD89, CD119, CD74, chemokine (C-C motif) receptor 1 (CCR1), IL1B, IL3RA, IL1R2, gp91phox (CYBB), and genes encoding major histocompatibility complex (MHC) class II molecules were up-regulated during the course of culture with GM-CSF (Fig. 3) . Several of these molecules, including IL1B, IL3RA, CCR1, CYBB, HLA-DR, CD54, and CD69, have been reported to be induced in neutrophils by GM-CSF [38 39 40 41 42 43 44 ]. This remarkable increase in proinflammatory molecules likely accounts, in part, for enhanced neutrophil function and/or the retention of function following exposure to GM-CSF.

Modulation of apoptosis regulators in human neutrophils
Inasmuch as there were significant levels of spontaneous PMN apoptosis after 24 h of culture (Fig. 1A 1B 1C 1D) , we examined carefully genes likely to regulate cell fate or apoptosis (Fig. 2) . Of note, genes encoding the antiapoptosis proteins myeloid cell leukemia sequence 1 (MCL1), caspase 8 and Fas-associated via death domain-like apoptosis regulator (CFLAR), B cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa-interacting protein 2 (BNIP2), and SGK were down-regulated coincident with spontaneous apoptosis (compare Figs. 1 and 2 ). Derout et al. [22 ] and Moulding et al. [23 , 45 ] demonstrated that MCL1 expression and stability are important for neutrophil survival, and decreases in MCL1 transcript and protein levels typically accompany neutrophil apoptosis. In contrast, several transcripts involved in cell survival and inhibition of apoptosis were significantly up-regulated following culture for 24 h with GM-CSF (Fig. 2) . For example, genes encoding BNIP2, tumor necrosis factor {alpha}-induced protein 8 (TNFAIP8), CFLAR, apoptosis inhibitor 5, BCL2-like 1, and SGK were each induced by GM-CSF (Fig. 2) . Of the genes involved in apoptosis and cell fate, SGK was one of the most highly induced transcripts after exposure to GM-CSF (range=seven- to 13.2-fold increase vs. 0 h in culture; Fig. 2 ). As SGK plays a prominent role in promoting cell survival in other cell types [46 47 48 ], it is possible that it participates in the GM-CSF-mediated delay in apoptosis.

Global changes in transcription regulators and genes mediating protein synthesis
Forty-eight genes encoding ribosomal proteins and at least 20 genes involved directly in translation were up-regulated in human neutrophils only after culture with GM-CSF (Fig. 3 , middle panel). Eighteen genes encoding proteasome subunits and ubiquitin-modifying enzymes were similarly up-regulated following cytokine treatment (Fig. 3 , middle panel). These observations provide strong support to the idea that neutrophil protein biosynthesis is increased significantly by prolonged (>3 h) exposure to GM-CSF. There were also many genes encoding proteins with uncharacterized function, such as adaptin ear-binding coat-associated protein 1, ankyrin repeat domain 12, centaurin delta 1, c10orf22, GA17, Ku86 autoantigen-related protein-1-binding protein, and L-type amino acid transporter 1 (LAT1)-3TM, which were strongly up-regulated by extended culture with GM-CSF (Fig. 3) . In contrast, several genes with unknown function were induced during spontaneous neutrophil apoptosis (12–24 h in culture) but remained unchanged after culture with GM-CSF. The importance of these molecules in neutrophil function has yet to be determined.

GM-CSF triggers up-regulation of key surface molecules at the level of gene expression
As we have demonstrated in numerous studies that confirmation of Affymetrix microarray data by TaqMan real-time RT-PCR in human neutrophils is typically 81–100% (89.3±5.8%, n=175 TaqMan assays) [11 , 30 , 31 , 34 , 49 , 50 ], we chose to measure expression of selected surface receptors as functional confirmation of the microarray data (Fig. 4 ). CD14, CD24, CD66, and CD119 were constitutively expressed on freshly isolated neutrophils from each of the individuals tested (Fig. 4A 4B 4C) . Only three of six subjects tested expressed neutrophil CD123 (IL3RA), and none expressed HLA-DR (Fig. 4C) . During the course of culture for 24 h, there were decreases in surface expression of each of molecules tested, except HLA-DR, which was not expressed initially (Fig. 4C) . These findings support the notion that proinflammatory capacity diminishes during neutrophil apoptosis. Culture with GM-CSF increased significantly the percentage of neutrophils with surface-expressed CD14 and HLA-DR (Fig. 5 5A ). Moreover, there were significant increases in CD24 and CD66 at 6 h and/or 24 h of culture with GM-CSF (Fig. 5B and 5C) . There was heterogeneity with regard to increases in expression of CD123, and GM-CSF failed to rescue surface expression of CD119 using our culture conditions (Fig. 5A) . It is possible that the protein is made but not exported to the cell surface. Taken together, there was generally good correlation between gene expression and surface expression of the proteins tested.



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  		Figure 4. Surface expression of PMN receptors during spontaneous apoptosis. (A and B) Representative analyses of cells stained with CD24 or CD66 and Annexin-V conjugated to APC. Results shown are representative of those from four individuals. (C) Neutrophil surface proteins were detected by flow cytometry as described in A and B, except that labeling with Annexin-V-APC was omitted. Dead cells were excluded in the analysis with a single gate (PI-positive). Results are expressed as percent positive PMNs or mean fluorescence as indicated. Each symbol represents a separate individual. IgG1' and IgG2b', samples stained sequentially with IgG1 or IgG2b and FITC-conjugated secondary Ab.

 


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  		Figure 5. Modulation of neutrophil surface molecules after culture with GM-CSF. (A–C) Neutrophil surface proteins were detected by flow cytometry after culture ± 100 ng/ml GM-CSF as indicated. (A and B) Each symbol-line-symbol pair represents a separate individual. (C) Representative flow cytometry histograms of neutrophil CD24 and CD66 during culture ± GM-CSF as indicated.

 
Induction of SGK by GM-CSF correlates with the delay in apoptosis
Given the prominent increase in neutrophil SGK transcript following GM-CSF exposure, and as SGK has been implicated in cell survival mechanisms in tumor cell lines [46 47 48 ], we investigated a possible role for SGK in the delay of apoptosis mediated by GM-CSF. The gene encoding SGK was previously shown by Cowling and Birnboim [51 ] to be induced in neutrophils by GM-CSF, albeit a possible role for the protein was not proposed. First, we used TaqMan real-time RT-PCR to confirm the increases in neutrophil SGK transcript, which we observed in the microarray experiments after culture with GM-CSF (Fig. 6 6A ). Next, we evaluated SGK protein levels during spontaneous neutrophil apoptosis (Fig. 6B , upper panel). Freshly isolated PMNs expressed SGK, and protein levels diminished significantly during the onset of apoptosis (at 24 h, the level was 21.6±6.3% of that at 0 h; Fig. 6C ). Consistent with the transcript data, expression of SGK protein was rescued by culture with GM-CSF (Fig. 6B and 6C) . Notably, the GM-CSF-mediated increase in SGK paralleled prolonged neutrophil survival (compare Figs. 1 and 6 ).



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  		Figure 6. (A) Expression of SGK was measured by TaqMan real-time PCR using conditions identical to those of the microarray experiments. (B) Human neutrophils were cultured with GM-CSF for the indicated times, and SGK was detected by immunoblot analysis. Results are representative of four separate experiments. (C) Quantitation of SGK immunoblots. Results are the mean ± SEM of four experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Constitutive neutrophil apoptosis is important for maintaining immune system homeostasis. During inflammatory states, e.g., infection, neutrophils are exposed to multiple cytokines, chemokines, immunomodulatory agents, and microbe-derived molecules, which have dramatic effects on neutrophil function and turnover. GM-CSF is a potent PMN-priming agent and chemotactic factor [25 , 29 , 52 , 53 ] and thus, promotes enhanced clearance of invading microorganisms [54 55 56 ]. In addition, the cytokine prolongs neutrophil survival and function, a phenomenon that likely further facilitates host defense [12 , 15 , 57 ]. A recent report by Martinelli et al. [58 ] used a microarray-based approach to dissect neutrophil differentiation and IFN-mediated antibacterial defense mechanisms after stimulation with GM-CSF for up to 7 h. However, a comprehensive analysis of neutrophil gene expression during spontaneous apoptosis and after extended culture (12–24 h) with GM-CSF has not been conducted. Inasmuch as constitutive PMN apoptosis and GM-CSF-enhanced neutrophil survival are important for innate immune system function, we used microarrays coupled with functional assays to generate comprehensive views of these processes.

Neutrophil apoptosis was accompanied by significant loss of phagocytic capacity (Fig. 1F) , a finding consistent with down-regulation of the receptors typically involved in phagocytosis. Whyte et al. [36 ] previously reported decreased phagocytic capacity in apoptotic neutrophils, although the molecular basis for the reduced function was not elucidated. Our data indicate that decreased uptake of antibody- and complement-coated latex beads was possibly a reflection of diminished expression of transcripts encoding CD11b, CD16, CD18, CD32, CD35, and CD64 (Fig. 2 and Supplemental Table 1). Attenuation of neutrophil phagocytic capacity was inhibited significantly by GM-CSF, and correspondingly, transcript levels for the receptors likely involved (CD11b, CD18, CD32, and/or CD35) failed to decrease (Fig. 2 and Supplemental Table 1). Spontaneous PMN apoptosis was also accompanied by dramatic decreases in transcript levels for genes encoding prominent surface receptors and associated signal transduction molecules (Fig. 2) . Expression of many of these genes was rescued by culture with GM-CSF, and transcript increases were generally accompanied by increases in cell surface expression of the proteins selected for confirmation by flow cytometry (Fig. 5) . There were GM-CSF-mediated increases in at least six genes encoding MHC class II molecules, which were paralleled by significant increases in neutrophil HLA-DR surface expression after 24 h of culture with the cytokine (Fig. 5) . Previous studies have demonstrated that granulocytes cocultured with IFN-{gamma} and GM-CSF or those plus IL-4 express MHC class II proteins and can acquire dendritic cell characteristics [59 , 60 ]. Further study of an extended role of neutrophils in the immune response, i.e., antigen presentation, is clearly warranted.

Genes encoding several key survival molecules, including BNIP2, CFLAR, and SGK, were down-regulated during neutrophil apoptosis. These genes, along with TNFAIP8, were up-regulated by culture with GM-CSF (Fig. 2) . We recently demonstrated that BNIP2, CFLAR, and TNFAIP8 were induced in human neutrophils following uptake of Anaplasma phagocytophilum [50 ], a pathogen that delays PMN apoptosis, not unlike GM-CSF. Therefore, these genes may play a general role in the modulation of neutrophil apoptosis. Although previous work demonstrated that the gene encoding SGK is induced by GM-CSF [51 ], the function of this protein in neutrophils is unclear. In our studies, transcript and protein levels decreased coincident with apoptosis, and these levels increased after treatment with GM-CSF. Wu et al. [48 ] identified SGK1 as a survival gene associated with apoptosis in mammary epithelial cells, and at least two other reports indicate that SGK is associated with glucocorticoid-mediated protection from apoptosis in similar cell lines [46 , 47 ]. Taken together, these data suggest that SGK may play a role in delaying neutrophil apoptosis.

In summary, our data provide the first genome-wide analysis of spontaneous neutrophil apoptosis and importantly, a global analysis of GM-CSF-mediated priming and survival responses. These studies provide novel insights into the mechanisms underlying constitutive PMN turnover and those that facilitate neutrophil survival and function during inflammatory states.


   ACKNOWLEDGEMENTS
 
This research was supported by the Intramural Research Program of the NIH, NIAID. The authors are grateful to M. Quinn (Montana State University, Bozeman) for critical review of the manuscript. We thank R. Lempicki and J. Yang (NIH, Frederick, MD) for performing the RNA hybridizations and scanning of the Affymetrix GeneChips.


   FOOTNOTES
 
1 Current address: University of Idaho, Department of MMBB, Life Science 142, Moscow, ID 83844-3052. Back

Received June 1, 2005; revised July 27, 2005; accepted August 26, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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