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Originally published online as doi:10.1189/jlb.1004567 on January 26, 2005

Published online before print January 26, 2005
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(Journal of Leukocyte Biology. 2005;77:669-679.)
© 2005 by Society for Leukocyte Biology

Heterogeneity of functional responses in differentiated myeloid cell lines reveals EPRO cells as a valid model of murine neutrophil functional activation

Peter Gaines, Jeffrey Chi and Nancy Berliner1

Section of Hematology, WWW 428, Yale University School of Medicine, New Haven, Connecticut

1 Correspondence: Section of Hematology, WWW 428, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. E-mail: nancy.berliner{at}yale.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mature neutrophils display multiple functional responses upon activation that include chemotaxis, adhesion to and transmigration across endothelial cells, phagocytosis, and pathogen destruction via potent microbicidal enzymes and reactive oxygen species. We are using myeloid cell line models to investigate the signaling pathways that govern neutrophil functional activation. To facilitate these studies, we have performed a direct comparison of functional responses of human and murine myeloid cell line models upon neutrophil differentiation. Our results show that EPRO cells, promyelocytes that undergo complete neutrophil maturation, demonstrate a full spectrum of functional responses, including respiratory burst, chemotaxis toward two murine chemokines, and phagocytosis. We also extend previous studies of granulocyte-colony stimulating factor-induced 32Dcl3 cells, showing they domonstrate chemotaxis and phogocytosis but completely lack a respiratory burst as a result of the absent expression of a critical oxidase subunit, gp91phox. Induced human leukemic NB4 and HL-60 cells display a respiratory burst and phagocytosis but have defective chemotaxis to multiple chemoattractants. We also tested each cell line for the ability to up-regulate cell-surface membrane-activated complex-1 (Mac-1) expression upon activation, a response mediating neutrophil adhesion and a surrogate marker for degranulation. We show that EPRO cells, but not 32Dcl3 or NB4, significantly increase Mac-1 surface expression upon functional activation. Together, these data show that EPRO and MPRO cells demonstrate complete, functional activation upon neutrophil differentiation, suggesting these promyelocytic models accurately reflect the functional capacity of mature murine neutrophils.

Key Words: gp91phox expression • chemotaxis • phagocytosis • degranulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils constitute the first line of host defense mounted against bacterial and certain fungal infections. Upon activation, neutrophils undergo a complex series of functional responses culminating in the destruction of invading microbes. These functions include chemotaxis to sites of infection, transmigration across capillary endothelium, phagocytosis of opsonized microbes, and killing of the engulfed pathogens. During maturation, neutrophils produce multiple enzymes critical to these functional responses, each packaged into primary [azurophil; e.g., myeloperoxidase (MPO), defensins, serine proteases] or secondary (specific) granules (e.g., neutrophil gelatinase, lactoferrin) [1 ]. Neutrophils also produce superoxide anions (O2–), generated by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, which are converted into potent microbicidal reactive oxygen species (ROS), such as hydroxyl radical, hydrogen peroxide (H2O2), and hyperchlorous acid [2 , 3 ]. To date, studies of neutrophil functional responses have largely depended on the use of purified neutrophils from peripheral blood (reviewed in ref. [4 ]). However, as these cells are terminally differentiated and short-lived, their usefulness in studies aimed at dissecting the molecular signaling pathways important to neutrophil function is limited. Myeloid cell lines provide alternative models of neutrophil development that are amenable to genetic manipulation and can be induced to undergo terminal neutrophil maturation. These include two human leukemic models, NB4 and HL-60, which have been well characterized and undergo partial differentiation into morphologically mature neutrophils in response to all-trans retinoic acid (ATRA; NB4) or dimethyl sulfoxide (DMSO; HL-60). NB4 cells carry the t(15;17) translocation and show distinct promyelocytic characteristics [5 ]. HL-60 cells, isolated from a patient with acute myeloblastic leukemia-M2, are described as myeloblasts [6 ]. Functional responses of differentiated NB4 and HL-60 cells described previously include the ability to produce a respiratory burst in response to phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ) [7 8 9 10 ]. Differentiated NB4 and HL-60 cells also demonstrate phagocytosis in response to latex particles [7 , 11 , 12 ]. In addition, HL-60 cells have been reported to migrate in response to certain chemoattractants [13 , 14 ]; however, these data are contradicted by other reports, indicating that differentiated HL-60 cells lack formyl-Met-Leu-Phe (fMLP) or interleukin (IL)-8-induced chemotaxis [14 15 16 ] or show defective responses as compared with normal neutrophils [14 , 17 ]. The failure of NB4 and HL-60 cells to form secondary granules [18 19 20 ] causes a mis-localization of membrane-bound cytochrome b558 to the plasma membrane [9 , 21 ], further altering the functional profile of these leukemic cell models.

Several factor-dependent, nontransformed mouse models of neutrophil differentiation have been developed, which can be induced to undergo more complete maturation with morphologic changes accompanied by expression of the full spectrum of neutrophil-specific genes. 32Dcl3 cells are an IL-3-dependent myeloblastic cell line, which undergoes neutrophil differentiation in response to granulocyte-colony stimulating factor (G-CSF) in the absence of IL-3 [22 ]. Upon G-CSF induction, 32Dcl3 cells exhibit a marked up-regulation of several important secondary granule genes, including lactoferrin and neutrophil gelatinase [23 , 24 ]. Recent studies have demonstrated the ability of differentiated 32Dcl3 to perform several functional responses, including shape changes in response to the mouse keratinocyte-derived chemokine (KC), phagocytosis of bacterial and latex particles, and the release of the primary granule enzyme MPO [25 , 26 ]. However, we recently demonstrated that fully differentiated 32Dcl3 cells completely lack a respiratory burst in response to PMA [27 ], a result confirmed by Guchhait et al. [26 ]. The biochemical cause of this defect was not described previously but may reflect defects in the assembly of the NADPH oxidase complex, as is commonly found in patients with chronic granulomatous disease [28 ]. Two murine ATRA-responsive myeloid models have also been established and characterized, EPRO and MPRO cells. These cell lines are granulocyte-macrophage-CSF-dependent promyelocytes derived from bone marrow cells transduced with a dominant-negative retinoic acid receptor {alpha}. MPRO cells were generated directly from transduced bone marrow cells, whereas EPRO cells were derived from multipotent EML cells [29 , 30 ]. Both lines undergo rapid maturation into morphologically mature neutrophils in response to superphysiologic levels of ATRA and express multiple secondary granule genes characteristic of normal, mature neutrophils [31 ]. Whether these cells also exhibit normal functional responses, however, has not been investigated.

To facilitate studies using cell line models to elucidate the molecular pathways important to neutrophil maturation, we have performed a comprehensive comparison of the functional responses of differentiated human and mouse myeloblastic and promyelocytic cell line models. Here, we describe the functional responses of ATRA-induced EPRO and MPRO cells and compare these responses to those in normal neutrophils, 32Dcl3 cells, and NB4 and HL-60 cells. Our results show that differentiated EPRO and MPRO cells demonstrate high levels of respiratory burst activity, chemotaxis, and phagocytosis. G-CSF-induced 32Dcl3 cells also demonstrate chemotaxis and phagocytosis but completely lack a respiratory burst as a result of absent expression of gp91phox, the large subunit of cytochrome b558, which functions as the redox center of the NADPH oxidase complex. By comparison, differentiated NB4 and HL-60 cells show respiratory burst activity and phagocytosis but fail to migrate in response to fMLP or two human chemokines. Finally, we show that differentiated MPRO and EPRO cells, but not 32Dcl3 or NB4 cells, exhibit increased cell-surface expression of CD11b/CD18 [membrane-activated complex-1 (Mac-1)] upon PMA stimulation, a surrogate marker for granule release [32 ]. Together, these studies provide a comprehensive characterization of the functional responses of multiple cell line models of neutrophil maturation and suggest that EPRO and MPRO cells constitute the most valid models for investigations aimed at identifying signaling pathways important to neutrophil functional activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
EPRO cells were derived from EML cells (provided by Dr. Schickwann Tsai, University of Utah Hematology Division, Salt Lake City) and were maintained in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 20% horse serum (Invitrogen, Carlsbad, CA) and 10% baby hamster kidney (BHK)/HM5-conditioned medium as described previously [30 ]. MPRO cells were maintained in AIM V (Gemini Bio-products, Woodland, CA), supplemented with 5% fetal bovine serum (FBS; Gemini Bio-products) plus 10% BHK/HM5-conditioned medium. 32Dcl3 cells were maintained in IMDM, supplemented with 10% FBS and 10% WEHI-3B-conditioned medium. NB4 and HL-60 cell lines were maintained in RPMI supplemented with 10% FBS. All growth medium was supplemented with 5 U/mL penicillin, 5 µg/mL streptomycin sulfate, and 2 mM L-glutamine.

Induction of differentiation
Proliferating EPRO, MPRO, and NB4 cells were differentiated by the addition of ATRA (10 µM for EPRO and MPRO; 5 µM for NB4 cells; Sigma Chemical Co., St. Louis, MO) to the growth medium. 32Dcl3 cells (5x105/mL) were washed twice in 1x phosphate-buffered saline (PBS) and then grown in IMDM, supplemented with G-CSF (100 ng/mL; Amgen, Thousand Oaks, CA). Medium was replaced every 3 days during the induction procedure, and cell densities were maintained at or below 1.0 x 106 cells/mL. HL-60 cells were induced in the presence of 1.25% DMSO plus 1 µM ATRA as described previously [9 ].

Primary granulocytes
Human neutrophils were isolated from venous blood using Polymorphprep (Axis-Shield, Oslo, Norway) according to the manufacturer’s protocol. Mouse granulocytes were isolated from peripheral blood collected from cardiac puncture using Ficoll-Hypaque (ICN Biomedicals, Aurora, Ohio) and dextrose sedimentation using standard procedures [33 ]. Serum isolation and OZ were performed following standard protocols [33 ].

Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA was extracted from cell lines using the TRIzol reagent (Life Technologies, Rockville, MD), according to the manufacturer’s protocol. For Northern analysis, 10 µg total RNA was electrophoresed, blotted, and probed as described previously [34 ]. For RT-PCR, cDNA was synthesized from total RNA using SuperScript II (Invitrogen), according to the manufacturer’s specifications. Primers for PCR amplification of murine NADPH oxidase genes were as follows: p22, 5'-AGGGGTCCACCATGGAGCGA-3' and 5'-GCTCAATGGGAGTCCACTGC-3'; p47phox, 5'-AGAACAGAGTCATCCCACACCT-3' and 5'-TCTCTGTTCCCGAACTCTTCTC-3'; p67phox, 5'GCATCAACAGAGACAAGCACTC-3' and 5'CTCAGTTTAGTGTGTTCTGGCG-3'; and gp91phox, 5'-CTGGAAACCCTCCTATGACTTG-3' and 5'-GGTCTTGAACTCGTTATCCCAG-3'. The mouse ß-actin gene primers were 5'-GTGGGCCGCTCTAGGCACCA-3' and 5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'. PCR reactions were carried out in a PTC-100 programmable thermal controller (MJ Research, Watertown, MA) for the indicated number of cycles using the following conditions: 94°C x 30 s, 60°C x 30 s, and 72°C x 1 min. Reaction products were then analyzed on ethidium bromide-stained 1% agarose gels.

Western analysis
Cells were washed once with 1x PBS and lysed at 1 x 106 cells/100 µL in 1x radioimmunoprecipitation assay buffer (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions. Total protein lysates (20 µg/lane) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–12% Bis-Tris precast gels (Invitrogen) and probed as described previously [34 ]. Probes used were anti-mouse gp91phox and anti-human p67phox antibodies (BD Transduction Laboratories, San Diego, CA) and anti-ß-actin antibody (Santa Cruz Biotechnology, CA), each at a dilution of 1:1000. Following overnight exposure with primary antibodies at 4°C, blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin G (IgG) antibodies (BD Transduction Laboratories) or HRP-conjugated donkey anti-goat IgG antibodies (Santa Cruz Biotechnology), each diluted 1:2000. Chemiluminescence detection was performed according to the manufacturer’s recommendations (Western Lightning Chemiluminescence, PerkinElmer Life Sciences, Boston, MA).

Respiratory burst
For respiratory burst assays using luminol-enhanced chemiluminescence (ECL), cells (1x106) were centrifuged, washed once in 1x PBS, resuspended in 400 µL Hank’s balanced salt solution (HBSS) containing Mg2+ and Ca2+ (137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4·7H2O, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 4.2 mM NaHCO3, 1 mM MgCl2, 1.3 mM CaCl2, 0.6 mM MgSO4·7H2O, 5.6 mM D-glucose), supplemented with an additional 0.1% D-glucose, and added to 100 µl Diogenes reagent (National Diagnostics, Atlanta, GA). Following incubation at 37°C for 15 min, cells were stimulated with 3.2 µM PMA, and chemiluminescence was read for 10 s immediately after PMA addition and then at 1-min intervals over a period of 5 min. Respiratory burst assays using the fluorescent probe dihydrorhodamine 123 (DHR) were performed as described previously [35 ], where 5 x 105 cells were washed twice in HBSS (without Ca2+ or Mg2+) with 0.1% albumin (human fraction V) and 1 mM EDTA (pH 8.0) and then incubated with 5.2 µL 29 mM DHR and 5 µL catalase (1400 U/µL) at 37°C for 5 min. After 5 min, 1 µL 2 mg/mL PMA or 1 µL DMSO was added to each reaction mix. After an incubation of 15 min at 37°C, each reaction mix was analyzed immediately using a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA) with CellQuest software. For OZ-induced respiratory burst, Zymosan A (Sigma Chemical Co.) particles were suspended in HBSS at 1 mg/mL, sonicated twice, and opsonized with fresh mouse or human serum for 1 h at 37°C. Particles were then washed and resuspended at 20 mg/mL in HBSS. Cells (1x106) were centrifuged, resuspended in 300 µl HBSS buffer plus 100 µl Diogenes, and incubated at 37°C for 15 min. Cells then were stimulated with 100 µl OZ, and luminescence was measured for 10 s at 2-min intervals for 40 min.

Phagocytosis
Fluorescein-conjugated zymosan bioparticles (Molecular Probes, Eugene, OR) were first opsonized with fresh mouse or human serum for 1 h at 37°C and were then washed twice in HBSS. Cells (1x106) were washed in HBSS, resuspended in 100 µl HBSS, and added to 800 µl HBSS containing 100 µl fresh mouse or human serum. To each mixture, 107 fluorescein OZ particles were added, and samples were incubated in 5 ml Falcon polystyrene round-bottom tubes (Becton Dickinson Labware, Franklin Lakes, NJ) at 37°C for 30 min with constant rocking or incubated on ice for negative controls. Cells were then collected, centrifuged at 250 g for 10 min, and then resuspended in HBSS containing 10% FBS plus 2 mM NaF to stop phagocytosis. Cells were vortexed to dissociate adherent particles and then were visualized under a fluorescent microscope, and the number of particles engulfed per cell was counted manually. Each counted cell was carefully examined while rolling past the field of view, which helped to distinguish between adherent and internalized particles.

Chemotaxis
Chemotaxis assays were performed using 6.5 mm-diameter Corning Transwell 24-well plates with a 3.0-µm pore polycarbonate membrane (Corning Life Sciences, Acton, MA) essentially as described previously [36 ]. Briefly, 1 x 106 cells were collected by centrifugation at 250 g for 5 min, washed in 1x PBS, and resuspended in 100 µL IMDM with 0.5% bovine serum albumin (mouse cell lines) or RPMI plus 2% FBS (NB4 and HL-60 cells). Prior to each assay, wells were pre-equilibrated with cell resuspension medium for 1 h at 37°C/5% CO2. Equilibration medium was then removed from each chamber, and 600 µL medium alone or medium with chemoattractant was added to the lower chamber. Chemoattractants used were as follows: for human cells, fMLP (1 µg/mL, Sigma Chemical Co.), human IL-8, and human growth-related oncogene-1 (GRO-1; 0.3 µg/mL, R & D Systems, Minneapolis, MN); for mouse cells, KC and macrophage-inflammatory protein-1 (MIP-2; 0.3 µg/mL, R & D Systems). The washed cells were then added to the upper chamber, and the plates were incubated for 2 h at 37°C/5% CO2. Cells from the bottom well of each sample were collected, and migrated cells were manually counted by trypan blue exclusion with a hemocytometer.

Flow cytometry for Mac-1 expression
Cell-surface Mac-1 expression was analyzed by staining unstimulated or PMA-stimulated cells with phycoerythrin (PE)-labeled anti-CD11b/CD18 (PharMingen, San Diego, CA). For PMA stimulation, 1 x 106 cells were collected, washed, and resuspended in 500 µL HBSS with 0.5% human serum albumin (Sigma Chemical Co.). Cells were then stimulated with 5 µL PMA at 100 ng/mL or DMSO for a control and incubated for 15 min at 37°C. Cells were then washed twice with 1x PBS, 2% FBS, and 0.1% sodium azide (PFN buffer) and resuspended in 100 µl PFN buffer. Mouse cells were preincubated with 1 µl Fc block (PharMingen) for 5 min at 4°C. Cells were labeled by adding 1 µl PE-conjugated rat anti-mouse CD11b (anti-Mac-1) antibody (PharMingen) or 1 µL PE-conjugated rat IgG2b isotype control (BD Biosciences, San Diego, CA) for 30 min at 4°C. Cells were then centrifuged, washed twice in PFN buffer, and resuspended in 300 µl PFN buffer. Mac-1 expression was then analyzed using a FACSVantage flow cytometer with CellQuest software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Respiratory burst produced by multiple cell line models
One of the best-studied, functional responses of neutrophils is the capacity to produce a respiratory burst caused by a rapid increase in molecular oxygen consumption. This process is mediated by the NADPH oxidase complex, which catalyzes the reduction of molecular oxygen to O2– and H2O2 (reviewed in ref. [37 ]). To investigate the functional responses of mouse and human cell line models of neutrophil maturation, differentiated cells were assessed for O2– production. Cells used in each assay were from early passages or in the case of EPRO cells, derived directly from EML cells. Cells were first induced toward terminally differentiated neutrophils using previously established induction regimens (see Materials and Methods). During each induction, the cells were maintained at a concentration that ranged from 5 to 10 x 105 cells/mL, and proliferation rates were observed to diminish as cells reached terminal stages of differentiation (data not shown). Following inductions, Wright-Giemsa-stained cytospin smears of each induced cell line were assessed for morphologic maturation and compared with the morphology of mature neutrophils isolated from mouse or human peripheral blood (Fig. 1A ). To confirm complete neutrophil maturation of murine cell lines, we also tested for expression of the secondary granule gene lactoferrin. As demonstrated in Figure 1B , each murine cell line demonstrated up-regulated lactoferrin expression following induction. The lactoferrin expressed in uninduced EPRO cells is a result of the effects of maintaining the cells in 20% horse serum, which we have previously shown to induce spontaneous differentiation in EPRO or MPRO cells (personal observations and ref. [31 ]). Undifferentiated and differentiated cells were then tested for O2– production using a luminol-ECL assay [38 ]. As shown in Figure 2A , undifferentiated EPRO cells showed no significant respiratory burst upon PMA stimulation. Differentiated EPRO cells stimulated with DMSO alone also showed background levels of chemiluminescence (less than 5000 light units per 10-s readings; data not shown). In contrast, differentiated EPRO cells stimulated with PMA showed a rapid increase in chemiluminescence, which peaked within the first 1–2 min of stimulation. Response profiles from differentiated EPRO cells were similar to those observed with murine peripheral blood neutrophils (Fig. 2A) , although levels of photon emissions were variable between experiments with peaks as high as 700,000 light units. To account for this variability, experiments to compare levels produced by multiple cell lines were performed in parallel (Fig. 2B) . Differentiated MPRO and human NB4 cells also demonstrated rapid respiratory burst activity in response to PMA, whereas 32Dcl3 cells completely lacked O2– production (Fig. 2B) .



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  		Figure 1. Neutrophil differentiation of myeloid cell line models. Differentiation of cell line models was assessed by Wright-Giemsa-stained cytospins and up-regulated expression of the secondary granule gene lactoferrin (mouse cell lines only). (A) Morphologic maturation of induced cell lines was monitored by Wright-Giesma staining of cytospins and analysis by light microscopy. Shown are representative, uninduced cells and cells induced for 3 days with 10 µM ATRA (EPRO and MPRO cells), 5 µM ATRA (NB4 cells), 1 µM ATRA plus 1.3% DMSO (HL-60 cells), or for 7 days with G-CSF in the absence of IL-3 (32Dcl3 cells). Also shown are polymorphonuclear neutrophils (PMN) isolated from mouse (m PMN) or human (h PMN) peripheral blood. Original magnification, x100. (B) Total RNA from uninduced and induced mouse cell lines was isolated and subjected to Northern blot analysis of lactoferrin expression. Cells were induced for the number of days indicated, and 10 µg extracted total RNA was loaded for each blot shown. Shown below is the agarose gel stained with ethidium bromide (EtBr) prior to transfer.

 


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  		Figure 2. Respiratory burst produced by differentiated cell lines in response to PMA and OZ stimulation. Induced cells were analyzed for O2– production by enhanced luminol-ECL upon stimulation with PMA (3.2 µM) or OZ (4 mg/mL). (A) EPRO cells induced with ATRA (EPRO-I; 10 µM) for 3 days or purified peripheral blood neutrophils from three independent mice were stimulated with PMA. Photon emission over a period of 10 s was monitored by a luminometer at 1-min intervals for a total of 5 min. Data for EPRO cells are given as averages ± SD of triplicate samples from ATRA-induced cells. EPRO-U, Uninduced. (B) A comparison of respiratory burst activities produced by differentiated EPRO, MPRO, NB4, and 32Dcl3 cells upon PMA stimulation is shown. Data represent the peak light units emitted over 10 s during a 5 min-period of analysis. (C) A direct comparison of chemiluminescence stimulated by PMA vs. OZ is shown between induced EPRO, MPRO, and 32Dcl3 cells. Shown are peak light units emitted over 10 s during a 5-min, (PMA) or 40-min (OZ) period of analysis. (D) Respiratory burst activity produced by differentiated NB4 and HL-60 cell lines upon PMA or OZ stimulation were assessed and compared to levels produced by human PMN. Data sets are given as averages ± SD from triplicate samples analyzed from each induced cell line and are representative of 3 independent experiments.

 
PMA stimulates neutrophils by directly activating protein kinase C (PKC) [39 ], mimicking the effects of the natural PKC activator, diacylglycerol. To determine whether pathways stimulated by opsonins found in serum, e.g., IgG and complement C3b, also stimulate a respiratory burst in these cell lines, assays using OZ as the stimulus were also performed (Fig. 2C and 2D) . EPRO and MPRO cells responded to OZ within 30 min of stimulation, whereas 32Dcl3 cells again showed no response. Respiratory bursts produced by PMA or OZ in differentiated NB4 and HL-60 cells were also compared. As shown in Figure 2D , both human leukemic models showed high levels of respiratory burst activity in response to PMA or OZ, and peak levels were approximately half that observed in normal human neutrophils.

As levels of respiratory burst, as measured by luminol-ECL, were variable and as the luminol excitation reaction can depend on the differentiation state of the cell line and levels of MPO expressed, we also examined respiratory burst responses using the fluorescent probe DHR 123 and flow cytometry. DHR is freely permeable, and directly measures the levels of H2O2 and O2– produced by the stimulated cell [35 ]. As shown in Figure 3 , differentiated EPRO and NB4 cells showed significant increased oxidation of DHR to rhodamine 123 upon PMA stimulation. Similar results were obtained from MPRO and HL-60 cells, and as previously observed with the luminol-ECL assay, 32Dcl3 cells showed no significant oxidation of DHR (data not shown). We also found the levels of rhodamine fluorescence in each cell line upon PMA stimulation were variable between experiments (data not shown), similar to our results observed using the luminol-ECL assay. Thus, although these assays may be useful in studies that directly compare the respiratory burst activities between similar cell line models, direct comparisons between different cell line models (e.g., mouse vs. human cell lines) must be interpreted with caution.



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  		Figure 3. Oxidation of DHR by differentiated cells upon PMA stimulation. Differentiated EPRO and NB4 cells were assessed for PMA-stimulated oxidative burst activity by flow cytometric analysis of the oxidation of DHR 123 to rhodamine 123. (A) A representative flow cytometric histogram of EPRO cells induced with ATRA (10 µM) for 3 days and assayed for DHR oxidation before and after stimulation with 2 µg PMA. Shown are the responses of cells analyzed after 5, 10, and 15 min of PMA stimulation. (B) A flow cytometric histogram of NB4 cells induced with 5 µM ATRA for 4 days and stimulated with 400 ng or 2 µg PMA, with assays performed after 5 or 15 min of stimulation. Also shown in each histogram are rhodamine levels exhibited by unstained and unstimulated cells. Data shown are representative of at least three independent experiments.

 
32Dcl3 cells lack gp91phox expression
The enzyme that produces oxygen radicals during the respiratory burst, NADPH oxidase, is a complex of multiple subunits, two of which, gp91phox and p22phox, reside in the membranes of specific granules and secretory vesicles and comprise the cytochrome b558. Upon activation, cytosolic subunits, including p47phox and p67phox, translocate to the membrane, where their association with cytochrome b558 renders the complex enzymatically active. To test whether the lack of respiratory burst activity in differentiated 32Dcl3 cells is a result of defective expression of one or more NADPH oxidase components, expression levels of multiple subunits were assessed in undifferentiated versus G-CSF-induced 32Dcl3 cells. For comparison, levels of expression in uninduced versus differentiated EPRO cells were also assessed. As shown in Figure 4A , gp91phox gene expression was easily detected in differentiated EPRO and MPRO cells, whereas expression was undetectable in differentiated 32Dcl3 cells as assessed by Northern analyses. By comparison, expression of p47phox, p22phox, and p67phox was readily detected in induced 32Dcl3 cells by RT-PCR, and levels were comparable with that expressed in differentiated EPRO cells (Fig. 4B) . As we were able to detect a faint gp91phox gene product amplified by RT-PCR for 30 cycles in differentiated 32Dcl3 cells (data not shown), we also examined protein expression of gp91phox. Western analyses showed high-level gp91phox protein expression in differentiated EPRO, MPRO, NB4, and HL-60 cells (the murine gp91phox protein migrates at 58 kDa compared with the human 91-kDa protein [40 ]) and protein expression of p67phox in these cells and in differentiated 32Dcl3 cells but a complete lack of gp91phox protein in differentiated 32Dcl3 cells (Fig. 4C) . These data therefore indicate the defective respiratory burst observed in 32Dcl3 cells is most likely a result of a complete absence of gp91phox protein expression. Southern blot analysis of the gp91phox locus in 32Dcl3 cells revealed no gross rearrangement or disruption of the gene (data not shown). Furthermore, sequence analysis of the gp91phox RT-PCR product generated from differentiated 32Dcl3 cells, which terminates 147 base pairs upstream from the 3' end of the open reading frame, revealed only two silent mutations, suggesting full-length gene transcription (data not shown).



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  		Figure 4. NADPH oxidase expression in myeloid cell lines following neutrophil differentiation. Human and mouse myeloid cell lines were analyzed for expression of multiple subunits of the NADPH oxidase complex during differentiation toward mature neutrophils. (A) Total RNA from uninduced and differentiated EPRO, MPRO, and 32Dcl3 cells was blotted and hybridized sequentially with 32P-labeled probes for gp91phox and ß-actin. (B) RNA samples from uninduced and differentiated EPRO and 32Dcl3 cells were reverse-transcribed to generate cDNAs, which were then analyzed by PCR for gene expression of the cytochrome b558 subunits gp91phox and p22phox and the NADPH oxidase cytoplasmic subunits p47phoxand p67phox. Amplifications were performed for the indicated number of cycles. Control reactions contained H2O substituted for cDNA, and amplifications were performed for the maximum number of cycles shown. Also shown is ß-actin, amplified from the same cDNAs for each condition. (C) Total cell lysates (1x106 cells/100 µl lysis buffer) were harvested from uninduced and differentiated cells. Protein expression of gp91phox and p67phox was then assessed by Western blot analysis. Blots were sequentially probed with antibodies against gp91phox and p67phox and then probed with an antibody against ß-actin as a control for the total amount of protein loaded in each lane.

 
Murine but not human myeloid models show significant chemotaxis
Chemotaxis of neutrophils to sites of infection is a process critical for the effective control of invading microbes. Chemotaxis is mediated by a variety of chemoattractants, including those produced by the invading organisms themselves, such as the formylated bacterial peptide fMLP, and host-produced factors such as the human CXC chemokines IL-8 and GRO-{alpha} or the murine orthologs KC and MIP-2. Early studies of HL-60 cells demonstrated chemotaxis of differentiated cells in response to fMLP or IL-8 [13 , 17 ]; however, these data are complicated by more recent studies showing that levels of migration were not significantly different between medium alone or medium supplemented with fMLP or IL-8 [14 , 16 , 41 ]. Whether the murine cell lines used here display chemotaxis in response to murine chemokines has not yet been examined, but 32Dcl3 cells have previously been shown to express the murine homologue of the CXC chemokine receptor 2 (CXCR2), which binds with high affinity to the murine chemokine MIP-2 [42 ]. We therefore tested whether differentiated EPRO cells migrate in response to the murine chemokines KC or MIP-2 using a standard migration assay and how responses compare with that of differentiated 32Dcl3 cells. Assays comparing different cell lines directly were performed in parallel. As shown in Figure 5A and 5B , differentiated EPRO cells exhibited significant, increased migration in response to KC or MIP-2 above background levels in medium alone. Differentiated MPRO cells also exhibited an increased response to both chemokines (three- to fourfold for either chemokine; data not shown). G-CSF-induced 32Dcl3 cells also exhibited increased migration, although overall levels of migration were consistently less than those of EPRO cells. In contrast, differentiated NB-4 cells showed little migration above background levels (medium alone) in response to multiple chemotactic agents, which included fMLP, human IL-8, and human GRO-{alpha} (Fig. 5C ; the P value of greater than 0.05 is considered not statistically significant). Similar results were observed for differentiated HL-60 cells (data not shown).



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  		Figure 5. Chemotaxis of differentiated cell lines to recombinant chemokines. Chemotaxis was analyzed by determining the total number of differentiated cells that migrate in response to medium alone or to medium containing chemokines using transwell plates with 3 µm polycarbonate membranes. Shown are chemotaxis responses from G-CSF-induced 32Dcl3 cells versus ATRA-induced EPRO cells to recombinant mouse chemokines (A) KC and (B) MIP-2. Also shown are responses of NB4 cells to fMLP and two recombinant human chemokines, IL-8 and GRO-{alpha} (C). Data shown are given as averages ± SD from triplicate samples analyzed in parallel and are representative of at least three independent experiments. Shown above each dataset are fold differences between medium alone versus medium containing chemokine and corresponding P values generated by the Student’s t-test.

 
Murine and human myeloid cell lines demonstrate significant phagocytosis
To test for phagocytosis by human and mouse myeloid cell lines, uninduced or differentiated cells were incubated with fluorescein-labeled OZ particles and were observed under a fluorescent microscope for the number of particles engulfed by each cell. Most cells showed few adherent particles, and internalized particles could be distinguished from those adhering to the external surface by carefully observing cells rolling past the field of view. As a positive control for these studies, neutrophils from murine or human peripheral blood were also tested. At least 98% of all murine and human peripheral blood neutrophils were observed to engulf OZ particles, and each cell engulfed an average of three to four particles (mouse) or seven to eight particles (human) per cell. For all cell lines tested, cells incubated on ice failed to show appreciable levels of phagocytosis (data not shown). Differentiated forms of each cell line demonstrated high levels of phagocytosis when incubated at 37°C, and levels observed in differentiated MPRO cells approached that of mouse peripheral blood neutrophils (89% vs. 98%, respectively, Fig. 6 ). The average number of particles engulfed by each cell line, shown in parentheses, was similar between mouse cell lines and mouse peripheral blood neutrophils, whereas the human leukemic cell lines engulfed, on average, approximately half as many particles as human peripheral blood neutrophils (three to four vs. seven to eight, respectively). Uninduced cells displayed low levels of phagocytosis, and one to two particles were engulfed per positive cell.



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  		Figure 6. Phagocytosis of OZ particles by differentiated cell lines. Uninduced or differentiated cells were assessed for phagocytic responses using fluorescein-labeled OZ with human or mouse serum. Phagocytosis was measured by counting the number of engulfed particles per cell under a fluorescent microscope. Graphed are the average percentages of cells ± SD containing at least one zymosan particle from at least three independent inductions, and the average number of particles ± SD per positive cell is shown in parentheses above each plot.

 
Mac-1 expression is up-regulated rapidly in EPRO cells but not in other myeloid models
Mac-1 is a surface heterodimeric (CD11b/CD18) adhesion molecule of the ß2 integrin family, which mediates adhesion of activated neutrophils to the vascular endothelium and subsequent transmigration through vessel walls. This process is facilitated by increased expression of Mac-1 on the cell surface during neutrophil activation. Studies on the subcellular localization of Mac-1 have indicated that this up-regulation occurs when neutrophils undergo exocytosis, where intracellular pools of Mac-1 localized to secretory vesicle and secondary granule membranes are contributed to the plasma membrane during vesicle fusion [43 44 45 ]. Thus, increased Mac-1 expression can indicate not only neutrophil activation but also the capacity of cells to degranulate [32 ]. As a final test of neutrophil function, we examined whether the differentiated cell lines demonstrate increased Mac-1 expression upon activation. Similar to previous results from our laboratory on MPRO cells [31 ], EPRO cells showed increased cell-surface Mac-1 expression after ATRA-induced differentiation (Fig. 7A , uninduced vs. induced). Furthermore differentiated EPRO cells activated with PMA showed a rapid (within 5 min; data not shown) and marked increase (approximately fourfold after 15 min) in Mac-1 expression. Similar results were observed in MPRO cells and normal mouse neutrophils (data not shown). In contrast, neither differentiated 32Dcl3 nor NB4 cells demonstrated any further increase in Mac-1 expression upon PMA stimulation (Fig. 7B and 7C) . A PMA-stimulated increase in Mac-1 expression was observed by differentiated HL-60 cells; however, levels of increase were substantially less than that observed in EPRO cells (Fig. 7D) .



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  		Figure 7. Increased cell-surface Mac-1 expression in PMA-stimulated EPRO cells. Differentiated cells were stimulated with PMA for 15 min and analyzed for increased Mac-1 expression on the plasma membrane by flow cytometry. Shown are differentiated (A) EPRO cells, (B) 32Dcl3 cells, (C) NB4 cells, and (D) HL-60 cells stained for Mac-1 expression prior to (Induced) or after (Induced + PMA) PMA stimulation. Cells unstained or stained with a PE-conjugated isotype control showed background levels of fluorescence. Also shown are Mac-1 expression levels in uninduced EPRO, NB4, and HL-60 cells, which were consistently less than those observed in induced cells. Similar results were observed in uninduced 32Dcl3 cells (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The activation of mature granulocytes at sites of infection causes a complex series of functional responses critical to the ability of neutrophils to locate, engulf, and destroy invading pathogens. The importance of each neutrophil functional response in humans is exemplified by the symptoms of patients harboring mutations that specifically affect the expression of genes critical to neutrophil function. Chronic granulomatous disease, for example, is caused by genetic mutations affecting the expression of any one of the NADPH oxidase subunit genes (recently reviewed in refs. [3 , 28 ]) rendering patients susceptible to catalase-positive (e.g., Staphylococcus aureus) bacterial infections. The correct temporal expression of adhesion molecules such as CD11b/CD18 (Mac-1) is also vital to neutrophil function, as evident by the effects of leukocyte adhesion deficiency, an autosomal, recessive disease often caused by a single mutation in the gene encoding the ß2 subunit of Mac-1 [46 ]. Mature neutrophils derived from peripheral blood are short-lived, however, and are refractory to genetic manipulation. Myeloid cell lines that can be induced to undergo neutrophil maturation, therefore, provide attractive models for studies aimed at dissecting the molecular control of neutrophil development.

Our interest in using myeloid cell lines to study the molecular mechanisms governing neutrophil functional activation led us to investigate the functional capacity of human and mouse myeloid cell line models. Previous studies have demonstrated that the two human myeloid models, NB4 and HL-60 cells, acquire the ability to produce a respiratory burst and undergo phagocytosis [7 8 9 10 11 12 ]. As these cell lines fail to express secondary granule proteins, however, their usefulness as models of normal neutrophil maturation is questionable. Furthermore, cytochrome b558 and Mac-1, protein complexes normally localized to the membranes of specific granules and secretory vesicles, are mis-localized to the plasma membrane in HL-60 cells [9 ].

In addition to human models of neutrophil maturation, a mouse model has also been shown to perform several functional responses. The murine factor-dependent cell line 32Dcl3 has previously been shown to perform phagocytosis and intraphagosomal degranulation [25 ] and more recently was shown to undergo increased adhesion on G-CSF-induced differentiation [26 ], but our initial studies [27 ] and those performed by Guchhait et al. [26 ] demonstrate that these cells completely lack a respiratory burst. This suggests that the 32Dcl3 cell line is not an ideal model of neutrophil maturation.

We have further investigated the functional responses of these myeloid models upon neutrophil differentiation and have compared responses to those of differentiated EPRO and MPRO cells. Upon differentiation, EPRO and MPRO cells display rapid changes in morphology coincident with up-regulation of lactoferrin and increased cell-surface Mac-1 expression, hallmarks of normal neutrophil maturation (Figs. 1B and 7A) [31 ]. We have now demonstrated that both cell lines also produce a respiratory burst, as evident by oxidation of luminol and DHR upon activation (Figs. 2 and 3) . The decreased peak level of respiratory burst in response to OZ in EPRO and MPRO cells is consistent with previous reports indicating that neutrophils stimulated with OZ only generate intracellular ROS, whereas PMA stimulation provides intracellular and extracellular reactive oxygen production [37 , 47 , 48 ].

Differentiated NB4 and HL-60 cells also exhibited respiratory burst activity in response to PMA or OZ (Fig. 2D) ; however, levels were consistently less than that observed in human neutrophils. Studies on human neutrophils have demonstrated that cytochrome b558 is primarily expressed in the membranes of secondary granules and to a lesser extent, secretory vesicles [49 ]. Upon stimulation, these organelles fuse with the plasma membrane, thereby translocating cytochrome b558 to the cell surface, whereupon cytosolic subunits associate with membrane-bound cytochrome b558 to form functional NADPH complexes [2 , 50 , 51 ]. In contrast to normal neutrophils, cytochrome b558 is restricted to the plasma membrane in HL-60 and NB4 cells [8 , 10 , 21 , 51 ]. The different levels of peak chemiluminescence between these leukemic cell lines and peripheral blood neutrophils may therefore result from a mis-localization of NADPH oxidase complexes, which may affect the level of ROS produced upon stimulation.

We note that PMA-stimulated levels of chemiluminescence were significantly different between mouse and human cells. This difference may reflect qualitative differences between human and mouse neutrophils, which is supported by previous studies demonstrating that several important antimicrobial proteins expressed in human neutrophils do not appear to be expressed in mouse neutrophils. For example, although human neutrophils express high levels of defensins and bactericidal/permeability-increasing protein (BPI), expression of these antimicrobial peptides is lacking in mouse neutrophils, although an ortholog of BPI has been identified recently in murine bone marrow [52 , 53 ]. As MPO is primarily responsible for the excitation of luminol, our observed differences in levels of luminol-ECL may reflect quantitative differences in MPO expression by the human versus mouse cell lines. Alternatively, these differences might reflect different levels of NADPH oxidase expression between cell types: Our Western analysis of gp91phox expression indicated significant differences in protein levels of gp91phox in differentiated human cell lines as compared with levels in ATRA-induced EPRO and MPRO cells (Fig. 4C) . However, we did not notice consistent differences in the levels of PMA-stimulated DHR oxidation between the differentiated mouse cell lines and differentiated NB4 or HL-60 cells. Nonetheless, results from both assays indicate significant respiratory burst activity is generated from EPRO or MPRO cell lines, whereas 32Dcl3 cells completely lack a respiratory burst.

Our analyses of NADPH oxidase expression in 32Dcl3 cells demonstrate that protein expression of the gp91phox subunit is absent following G-CSF-induced differentiation, explaining the lack of respiratory burst activity in these cells. The molecular basis of the absent protein expression, however, remains unclear. We were able to detect a gene product by RT-PCR in long-term induced cells after 30 cycles of amplification, but this product failed to reveal any mutations that might disrupt the binding site of the anti-gp91phox antibody (generated from the carboxyl terminus of the full-length protein). It is possible that the gp91phox gene is mutated such that transcriptional activation is mostly blocked, or the mutation causes transcript instability and premature RNA degradation. Such a mutation might be caused by a retroviral insertion, as 32Dcl3 cells were derived from bone marrow of a Friend murine leukemia virus-infected mouse [22 ], and our laboratory has previously demonstrated that the evi-1 locus contains a genomic sequence encoding the murine leukemia virus envelope (env) gene [54 ]. To address this issue, we performed a preliminary Southern blot analysis of the gp91phox gene locus in 32Dcl3 cells as compared with MPRO cells but were unable to detect a disruption of the gp91phox locus (data not shown). Therefore, an exact determination of the mutation affecting gp91phox expression in 32Dcl3 cells will require more in-depth analyses beyond the scope of this investigation.

In addition to respiratory burst activity, EPRO and MPRO cells display phagocytosis comparable with that observed in murine peripheral blood neutrophils and chemotaxis in response to two different chemokines, KC and MIP-2. 32Dcl3 cells also demonstrate phagocytosis and chemotaxis, and fold increases in chemoattractant-stimulated migration were similar to those observed in EPRO and MPRO cells. However, the levels of migration in buffer alone or buffer containing a chemoattractant were substantially less in 32Dcl3 cells as compared with those observed in EPRO cells (Fig. 5) . These results may suggest that differentiated 32Dcl3 cells harbor a defect in the actin filamentous network that mediates cell migration, a notion supported by the recent finding by Guchhait et al. [26] showing reduced shape changes in response to KC by differentiated 32Dcl3 cells as compared with murine neutrophils. We also found that the NB4 and HL-60 cell lines, both of which displayed significant levels of phagocytosis, failed to show significant migration in response to multiple chemotactic agents. As previously mentioned, there are conflicting data as to the capacity of these leukemic cell lines to perform chemotaxis. One report does show that DMSO-induced HL-60 cells migrate in response to the chemoattractant N-formyl-nle-leu-phe-nle-tyr-lys [14 ], but we have not confirmed this result with either human cell line. In addition, Hauert et al. [14] show HL-60 cells express undetectable levels of the IL-8 receptor CXCR2 (IL-8RB) suggested to be the primary receptor responsible for IL-8 response in normal human neutrophils [55 ]. To our knowledge, chemotaxis of NB4 cells has not been tested previously, and our results suggest that similar to HL-60 cells, this cell line fails to respond significantly to multiple chemoattractants.

Finally, our studies on EPRO and MPRO cells demonstrate rapid, increased cell-surface expression of Mac-1 during PMA stimulation (Fig. 7) , providing further evidence that these cells display complete, functional activation. Despite the ability to migrate and engulf particles, 32Dcl3 cells did not exhibit increased PMA-stimulated Mac-1 expression, further suggesting that this cell line is functionally defective. We were not surprised to find a lack of PMA-stimulated, increased Mac-1 expression in NB4 cells, as they lack peroxidase-negative granules, where Mac-1 is primarily stored. It is somewhat surprising to see a small shift in Mac-1 expression in HL-60 cells upon PMA stimulation, as HL-60 cells also lack secondary granules. However, ectopically expressed neutrophil gelatinase-associated lipocalin in these cells has been shown to be mis-localized to primary granules [9 ]. Therefore, the small increase in Mac-1 expression might reflect aberrant localization of the integrin to primary granules, which also fuse with the plasma membrane during activation and the release of MPO.

In conclusion, we have demonstrated that EPRO and MPRO cells display multiple functional responses upon differentiation, suggesting these promyelocytes provide reliable cell line models of murine neutrophil differentiation and functional activation. In contrast, 32Dcl3 cells fail to acquire the capacity for respiratory burst activity and appear to have a defect in secondary granule formation despite their ability to express multiple secondary granule proteins. Future studies to address where these granule proteins are localized within differentiated 32Dcl3 cells might reveal aberrant localization to other vesicles, such as that observed in HL-60 cells overexpressing neutrophil gelatinase [9 ]. The functional responses of the EPRO and MPRO cell lines also paralleled several of the responses observed in the human leukemic models, and despite different levels of respiratory burst activity, as assessed by the oxidation of luminol, both mouse models demonstrated significant chemotaxis toward multiple chemokines. As EPRO cells are derived from multipotent EML cells, the EML/EPRO line offers an attractive system for study of the full sequence of gene expression changes and functional maturation associated with murine granulocyte differentiation.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Awards R01-DK53471 and P01-HL63357 (N. B.) and K01-DK60565 (P. G.). We thank Dr. Rocco Carbone for expert assistance with flow cytometry and Dr. Kimberly Lezon-Geyda and Sharon Lin for technical assistance.

Received October 6, 2004; revised January 5, 2005; accepted January 6, 2005.


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