Departments of 1 Physiology and Biophysics and 2 Medicine, Wright State University School of Medicine, Dayton 45435; and 3 Research Service, Dayton Veterans Affairs Medical Center, Dayton, Ohio 45428
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ABSTRACT |
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Mitogen-activated protein kinase (MAPK) isoform p42 is known to be active in exponentially growing cells at several points of the cell cycle. A high basal activity was present in three cell lines representative of immature myeloid cells tested: uHL-60, AML-14, and MPD. However, DMSO-induced differentiation of HL-60 cells (dHL-60) and subsequent expression of the neutrophilic phenotype occurred with a concomitant reduction on the basal level of MAPK activity. Simultaneously, extracellular stimuli like the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) induced a fast (<10 min) and robust response. In terms of MAPK activity, the more mature the cell was, the higher the corresponding activity, in the three differentiation series considered: AML-14 < 3D10; MPD < G-MPD; uHL-60 < dHL-60 < neutrophils. Interestingly, peripheral blood neutrophils expressed the highest (16-fold) MAPK activation level in response to GM-CSF. Finally, using the specific MAPK inhibitor PD-98059, we demonstrated that MAPK activation is needed for neutrophil chemotaxis toward interleukin-8 and its priming by GM-CSF. Since neutrophils are terminally differentiated cells, GM-CSF does not serve a purpose in proliferation, and it must trigger the recruitment of selective signal transduction pathways particular to that final stage that includes enhanced physiological functions such as chemotaxis.
mitogen-activated protein kinase; granulocyte/macrophage colony-stimulating factor; neutrophils; cell differentiation; leukemic cells
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INTRODUCTION |
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PROLIFERATION AND DIFFERENTIATION of myeloid progenitors is regulated by a family of hematopoietic growth factors, including granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF stimulates DNA synthesis and transcription in immature myelo-monocytic progenitor cells and acts on mature macrophages and granulocytes by priming them for their phagocytic functions. Specifically, at the site of infection or inflammation, GM-CSF enhances phagocytosis of invading pathogens by granulocytes and macrophages by increasing degranulation, adherence, alkalization of the cytosol, inhibition of migration, and the release of superoxide anions, platelet-activating factor, and arachidonic acid (reviewed in Ref. 12).
GM-CSF is one of the hematopoietic growth factors required for the proliferation and differentiation of myeloid-committed normal progenitor cells, but it fails to elicit differentiation in most transformed cells. To achieve differentiation in vitro, additional induction with an agent such as DMSO is needed (26). The mechanism by which DMSO induces maturation is still largely unknown. It might induce the acquisition of effector functions that are characteristic of mature neutrophils, such as key signal transduction molecule links (4, 19, 25).
The postreceptor signaling pathways utilized by GM-CSF are the subject
of intense study (1, 2, 5, 9, 11, 31). One of the earliest
events is tyrosine phosphorylation of the receptor -subunit
(GM-CSF · R
). Some important downstream events are
1) activation of the Ras (MAPK/ERK2) mitogen-activated
protein kinase/extracellularly regulated kinase 2 pathway, a common
signaling route for many hematopoietic growth factors, 2)
tyrosine phosphorylation and activation of p38 MAPK, an important
crosspoint in neutrophil signaling (18), and 3)
activation of p42 MAPK that leads to Ser/Thr phosphorylation of one of
its substrates, p90rsk (8), known to be
essential for cytostatic factor arrest (3). In the case of
hematopoietic models, it is well known that the expression and states
of various signaling molecules, such as src-like kinases,
change with myelo-monocytic differentiation. Thus there is reason to
anticipate that receptors may effect different signals depending on the
effector molecules downstream of them that depend on the
differentiation state of the cell.
This study addresses the cell signaling pathways that underlie the uniqueness of the neutrophil's functionality, compared with well-established cycling cells. Although GM-CSF has been shown from its discovery as being needed for DNA synthesis in progenitor cells, the focus of the present work is on how hematopoietic cells respond to the cytokine at several stages of differentiation. We provide evidence showing that neutrophils could have an enhanced functionality (such as chemotaxis) when primed by the cytokine as it occurs during the course of an infection. Upregulation of p42 MAPK might be at the crux of these events.
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MATERIALS AND METHODS |
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Materials and antibodies.
The promyelocytic leukemic (M2 myeloblast-derived) HL-60 cell line was
from American Type Culture Collection (Rockville, MD); the acute
myeloid leukemia cell line AML-14 and the more differentiated (eosinophilic) subline AML-14.3D10, as well as the MPD cell line (non-chronic myelogenous leukemia myeloproliferative disorder) were developed by the authors (21, 22); tissue culture
material was from Becton Dickinson (Oxnard, CA) and Costar (Cambridge, MA); GM-CSF and granulocyte colony-stimulating factor (G-CSF) were from
R&D Systems (Minneapolis, MN); cAMP-dependent protein kinase
inhibitor, calphostin, anti-rabbit IgG (agarose beads), differentiation kit, trypan blue, nitro blue tetrazolium (NBT) reduction kit, and myelin basic protein (MBP) were from Sigma (St.
Louis, MO); electrophoresis chemicals were from Bio-Rad Laboratories (Richmond, CA); [-32P]ATP (30 Ci/mmol) was from
Amersham (Arlington Heights, IL); PD-98059 was from BioMol (Plymouth
Meeting, PA); ion-exchange chromatography cellulose phosphate paper was
from Whatman (Hillsboro, OR); antiphosphotyrosine (anti-PY;
PY20 clone), anti-p42mapk, and anti-p90rsk
kinase antibodies, MAPK peptide substrate APRTPGGRR, and ribosomal S6
kinase (RSK) peptide substrate RRRLSSLRA were from Upstate Biotechnology (Lake Placid, NY).
Isolation of peripheral blood neutrophils. The isolation procedure was based on English and Andersen (7). Between 50 and 55 ml of blood were collected from the antecubital veins of healthy individuals (who signed an Institutional Review Board-approved consent form), using sodium citrate as anticoagulant. Blood was mixed with 15 ml of 6% dextran, allowed to settle, and the plasma and buffy coat were removed and spun down at 800 g for 5 min. The pellet was resuspended in 35 ml of saline and centrifuged again for 15 min at 10°C in a Ficoll-Histopaque discontinuing gradient. Neutrophils were recovered, and contaminating erythrocytes were lysed by hypotonic shock. Cells were washed, and the pellet was resuspended in Hanks' balanced salt solution (HBSS). No neutrophil aggregation (i.e., the hallmark for neutrophil activation) was observed by using this isolation procedure. Viability was >98% as per trypan blue exclusion.
Leukemic cell culture and induction of differentiation. HL-60, AML-14 and its subline AML-14.3D10, and pluripotent MPD cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 5 µg/ml gentamicin. Stock cultures were diluted with fresh medium every other day, with extra medium on weekends, so that cell density was maintained between 0.1-1.0 × 106/ml. Vented culture flasks were changed biweekly. Cultures were checked for mycoplasma contamination once a month. Cell extracts were prepared as indicated in the next section. HL-60 cells were induced to differentiate by incubation with 1.25% (vol/vol) DMSO for a maximum of 6 days (19), but normally 3-4 days sufficed to achieve the neutrophilic phenotype. Aliquots were sampled daily and assessed for differentiation. To induce differentiation of the MPD cell line to mature neutrophils, cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium continuously supplemented with 20 pM G-CSF for 5 days. Viability assays were routinely conducted with 0.4% trypan blue stain in cell preparations before all analyses. Average counts were uHL-60 = 1.8 × 106 cells/ml (viability ~95%); 4-day DMSO: dHL-60 = 1.4 × 106 cells/ml (viability ~93%); MPD = 1.2 × 106 cells/ml (viability ~95%); 5-day G-CSF: G-MPD = 1.6 × 106 cells/ml (viability ~92%).
Assessment of differentiation. In standard procedures with HL-60, at 80 h of DMSO incubation, ~75% of the cells matured to at least the myelocytic stage and acquired the ability to release superoxide anion in response to bacterial extract challenge. To ascertain that DMSO produced the desired neutrophilic phenotype in HL-60 cells, cells were assayed for 1) morphology of Wright-stained slides; 2) cytopreparations of NBT-treated preparations of cells stimulated with 5 µl of nonviable bacterial extracts. Positive cells contained formazan deposits as dark, irregularly shaped crystal inclusions in the cytoplasm and were (positive/total) 2/290 = 0.6% (uHL-60); 22/227 = 9.7% (dHL-60); and 137/468 = 29% (neutrophils); 3) flow cytometric analysis of surface expression of differentiation-related antigens was performed with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against CD11b, CD11c, CD13, CD14, CD16, or CD54.
Preparation of cell extracts, immunoprecipitation, and Western blotting. Cells were washed in HBSS (to eliminate any potential autocrine cytokine production) and incubated with GM-CSF at 37°C. Aliquots were spun down for 30 s at 7,000 g, and pellets resuspended in 200 µl lysis buffer (12 mM Tris · HCl, pH 7.2, 0.75 mM NaCl, 100 µM sodium orthovanadate, 10 µM phenylmethylsulfonyl fluoride, 5 µg/ml each of aprotinin, pepstatin A, and leupeptin, and 0.12% Triton X-100) and sonicated on ice. Total cell lysates were centrifuged (for 2 min at 14,000 g at 4°C), and supernatants (~1 mg/ml protein) were saved for immunoprecipitation (16). Immune complexes were resuspended in a final volume of 60 µl with lysis buffer and mixed with 2× SDS sample buffer (1:1 vol/vol) for subsequent Western blotting or with kinase buffer for subsequent kinase assays. When required, bands from X-ray films derived from Western blots were scanned and area x optical density was calculated. When mobility shift measurements were required, the distance migrated by a particular band in Western blots was measured (in mm) and extrapolated in a SDS-polyacrylamide gel calibration curve for known molecular weight protein standards vs. distance.
In vitro kinase assays.
The enzymatic activities in whole lysates or immune complexes were
measured as described (23) with some modifications. The phosphoacceptor peptide, 1 mM APRTPGGRR [bearing the
phosphorylation site of amino acids 95-98 (PRTP) from
bovine MBP], was diluted in freshly prepared kinase buffer (13.4 mM
HEPES, pH 7.3, 25 mM MgCl2, 30 µM
Na2VO2, 5 µM p-nitrophenyl
phosphate, 2 mM EDTA, 2 µM cAMP-dependent kinase inhibitor, 5 µM
calphostin, 21 µCi [-32P]ATP/ml, and 68 mM unlabeled
ATP). The phosphotransferase reaction was initiated by mixing aliquots
(20 µl) of kinase buffer with the cell lysates or immunoprecipitates
at 1:1 (vol/vol) ratio for 20 min and was terminated by blotting 20 µl of the reaction mixture onto cellulose phosphate P81 papers. These
were dried and counted for radioactivity. Controls were run in
parallel, by which the reaction was carried over with all the
indicated reagents in the absence of the phosphoacceptor peptide and
radioactivity counts were subtracted from the experimental conditions.
In-gel renatured kinase assays.
MAPK activity was also measured by an "in-gel" kinase assay, as
described in (17), with some modifications. Briefly, 250 µg/ml MBP, to measure p42mapk, or 100 µg/ml RSK peptide
(RRRLSSLRA = amino acids 231-239 of human protein S6), to
measure p90rsk, was mixed with acrylamide in an SDS-PAGE
gel (1.5 mm thin). After electrophoresis, gels were freed of SDS by
incubation with 20% isopropanol in buffer A (100 mM
Tris · HCl, pH 8, and 5 mM 2-mercaptoethanol), then with
buffer A containing 6 M guanidine-HCl, and finally, in
buffer A containing 0.04% Tween 40 at 4°C for 6 h.
Renatured gels were overlaid with kinase buffer (20 mM
Tris · HCl, pH 8, 10 mM MgCl2, and 25 mCi
[-32P]ATP) and incubated at room temperature for 30 min. Gels were washed with 1% pyrophosphate in 5% TCA, dried, and
exposed to X-ray films. For the immunocomplex kinase assay, agarose
beads were resuspended in 30 µl of lysis buffer, boiled for 5 min,
spun down (15 s, 14,000 g), and supernatants were loaded in
the substrate-embedded SDS gels. When needed, the kinase of interest
was excised off the dried gel and counted for radioactivity.
Functional assay: chemotaxis. Neutrophils (5 × 105) in chemotaxis buffer (HBSS + 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA) were placed on the upper chambers of 6.5-mm Transwell dishes (Costar) that were separated from the lower chambers by a 5-µm pore nucleopore polycarbonate membrane. Interleukin-8 (IL-8) was added in 0.6 ml of chemotaxis buffer to the lower chamber. The dishes were incubated for 1 h at 37°C under a 5% CO2 atmosphere, and the cells that migrated to the lower chambers were counted on a microscope by using a hemocytometer. Viability at the end of the assay in both chambers remained >97 ± 2%, even in the presence of MAP or ERK kinase (MEK) inhibitor, ruling out a toxic effect. Maintenance of the experiment for 2 h proportionally increased the number of migrating cells, but viability decreased to >75 ± 3%.
Statistical analysis. The difference between means was assessed by the single factor analysis of variance test. Probability (P) of <0.05 was considered to indicate a significant difference. Several P values are given in the figure legends.
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RESULTS |
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The extent of MAPK tyrosyl phosphorylation in response to GM-CSF is
greater in mature than in undifferentiated leukemic cells.
The expression of surface antigens in both undifferentiated (uHL-60)
and differentiated HL-60 (dHL-60) cells was first investigated to
confirm that DMSO had induced cell maturation. Upregulation was
observed in nearly all surface antigens studied by flow
cytometry (Fig. 1). Noticeable changes in
CD11b (14, 27) and CD13 (zinc metalloproteinase) were
present, and equivalent levels were expressed in both progenitors and
mature myelomonocytic cells as expected. The assayed
integrins, intercellular adhesion molecules, immunoglobulin fragment
Fc, and lipopolysaccharide (LPS) receptor, as representatives of granulocyte extravasation, phagocytosis, and antibody-dependent cell-mediated cytotoxicity, confirmed the presence of the neutrophilic phenotype in dHL-60 cells.
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The highest MAPK enzymatic activity after stimulation occurs in
neutrophils while differentiated leukemic cells have an intermediary
activity.
Results from immunoblots and immunoprecipitates are indicative of
a conformational change of the protein due to phosphorylation, and
consequently, an increase in enzymatic activity could be expected. To
fully assess MAPK enzymatic activity with confidence, we used all
contemporary methodology that exists; e.g., Western blotting and study
of shift mobility, in vitro 32P[ATP] incorporation,
immunokinase assay with specific immunocomplex preparations, and in-gel
renatured kinase assay in precleared/immunoprecipitated samples. The
first approach was done initially by detecting in vitro 32P
incorporation to the specific MAPK substrate APRTPGGRR. Cell lysates
were prepared from an array of myeloid leukemic cells, both
undifferentiated and induced to mature, including the eosinophilic cell
line AML-14 and its relatively more mature subline AML-14.3D10, uHL-60,
dHL-60, and HL-60 treated with all-trans-retinoic acid (aHL-60) (Fig. 5). Two points were noted.
1) The most immature cells (AML-14, MPD, and 3D10) had the
highest basal level of kinase activity. This was also true for fresh,
exponentially growing uHL-60 cells, in agreement with Okuda et al.
(20), in that transformed, immortalized cultured cells had
a high operating level of the Ras/MAPK pathway, even in the absence of
growth factors. 2) Basal level was increased in response to
GM-CSF stimulation, but at very different rates in the series
considered. The incorporation of 32P into the
phosphoacceptor substrate followed the order AML-14 MPD < 3D10
uHL-60 < dHL-60
aHL-60 < G-MPD < neutrophils.
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p90rsk Activation follows the p42 MAPK pattern in HL-60
cells.
We next studied the response of one of the MAPK substrates, the
ribosomal p90rsk, to GM-CSF challenge. The presence of
p90rsk was detected in leukemic cell lines with the
antibody recognizing a band of the proper molecular mass
(i.e., 88-90 kDa) (Fig. 8). As with
MAPK, GM-CSF caused a shift in relative electrophoretic mobility of
p90rsk higher in dHL-60 than in uHL-60 cells. In blots such
as the one presented in Fig. 6, mass of p90rsk was
measured. The presence of phosphorylation was confirmed by an in-gel
renatured kinase method (Fig. 8B). Because an increase in
band intensity correlated with an elevated enzymatic activity of the
kinase, p90rsk from dHL-60 cells clearly responded to
GM-CSF stimulation better than uHL-60. Finally, the effect was
dependent on the dose (Fig. 8C) being more pronounced in
dHL-60/neutrophils than in uHL-60. The pattern for p90rsk
activation was uHL-60 < dHL-60 Neut (neutrophils not
being higher than dHL-60, as indicated in Ref. 16).
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MAPK upregulation is related to cell function in neutrophils.
Results from the above experiments suggested a blockage in the
transduction of signals between the binding of GM-CSF and the activation of the kinase in uHL-60 cells but upregulation in
neutrophils. To ascertain a physiological implication of this effect,
we looked at chemotaxis toward a well-defined and potent neutrophil
stimulus, IL-8. Cell migration toward 50 or 500 nM IL-8 was inhibited
by the MEK inhibitor PD-98059 in a dose-dependent manner (Fig.
9A), with an IC50
of ~10 µM. Additionally, the role of GM-CSF for MAPK in more mature
cells was underlined by the finding that chemotaxis was augmented by
GM-CSF preincubation. This priming effect was also abrogated by
PD-98059 (Fig. 9B).
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DISCUSSION |
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We show here that MAPK activation in response to GM-CSF increases
with the maturational state of the cells. The ability of GM-CSF to
induce tyrosine phosphorylation and activation of the p42 isoform of
MAPK was studied in three well-defined cell line models and found to
directly correlate with maturation stage. Even though the isoform
p42mapk was present (and at a high basal level) in
exponentially growing, undifferentiated HL-60 cells, only a marginal
increase in tyrosine phosphorylation and enzyme activity was observed
on cell stimulation with GM-CSF. A maximal response was seen in mature
neutrophils. These results were replicated in other GM-CSF
receptor-expressing cell line models: the blastlike cell line AML-14,
its more differentiated (expressing the eosinophil phenotype) subline
AML-14.3D10, and the recently described pluripotent MPD cell line
(22), representative of non-CML MPD that differentiates
into mature granulocytes with continuous exposure to G-CSF.
Quantitatively, the order of MAPK activation was AML-14 MPD < 3D10
uHL-60 < dHL-60
aHL-60 < G-MPD < neutrophils.
A higher phosphorylation activity in the more differentiated cells is in agreement with previous studies demonstrating p93fes and p60src (30), tyrosine phosphorylation (26), and MAP2 kinase (24) activation. Recently, a low response to GM-CSF was linked to low Janus kinase 2 levels in HL-60 cells, although this could not be extrapolated to CD34+ cells (29). In the present study, we observed that in spite of MAPK being maximally activated by GM-CSF in neutrophils, one of its main substrates, p90rsk, reached its maximum of activation only in dHL-60 cells. This indicates that pathways other that the classical ones leading to cell division must exist, especially for terminally differentiated cells.
While in stem, cycling cells, MAPK is important for the exit
G0 G1 and for the spindle formation;
in mature granulocytes, a massive response of MAPK to short stimulation
by GM-CSF must have a different functionality. This
stimulation can still be related to microtubule assembly. Precisely,
the first name proposed for MAPK was "microtubule-associated protein
kinase" for its ability to phosphorylate microtubule-associated
proteins, or MAPs. It is likely that in neutrophils, an activation of
these or related proteins can lead to increased cell movement events in
the infection site, namely, chemotaxis and phagolysosome formation. Our
results (Fig. 9) point to the direction of MAPK being important in
neutrophil chemotaxis and the role of GM-CSF for MAPK activation in
more mature cells. Still, other pathways could act in parallel. Thus, while the activation of the MAPK pathway is necessary for insulin-like growth factor-I-induced mitogenesis, the activation of
phosphatidylinositol 3-kinases (PI 3-kinases) is very important for
cell migration and integrin activation (15). cAMP-induced
activation of the sodium taurocholate cotransport may be mediated via
the PI 3-kinases/protein kinase B signaling pathway requiring
intact actin filaments (28). Also, and equally important,
the differential effect of stimuli in a particular cell type should be
kept in mind. For example, GM-CSF, LPS, or tumor necrosis factor-
(TNF-
) stimulate cellular functions that are not induced by phorbol
12-myristate 13-acetate or ionomycin (32).
A role for the "classic" MAPK (ERK2, p42mapk) in the cell cycle has been recently demonstrated (10, 13). MAPK is a key player in cycling cells. Its activity was elevated in mitosis of HeLa cells, but only in interphase did cells respond to epidermal growth factor, as if mitotic cells were sheltered from perturbing signals from the exterior. As differentiation reaches a terminal phase, the cyclic pattern changes to a steady state in which MAPK is available for activation only when cells engage in their physiological microtubule-related functions, migration and phagocytosis, both needed in the inflammation site and primed by cytokines such as GM-CSF.
Thus the presence of a highly stimulatable MAPK activity in neutrophils is not by any means a leftover of mitotic, hematopoietic stem cells from which neutrophils derive. Rather, this enzyme might be regulated differently and uniquely. We are currently continuing our work in the direction of finding the precise connection between the two cell signaling pathways presented herein and the motility-related neutrophil functionality.
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ACKNOWLEDGEMENTS |
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We thank Dr. Peter K. Lauf for critical reading of this manuscript and helpful suggestions.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-056653 and American Heart Association Grant 9806283 (to J. Gómez-Cambronero), by Veterans Affairs Merit Review grants, by a Medical Innovation Program from Wright State Univ. School of Medicine (to C. C. Paul and M. A. Baumann), and by the American Cancer Society, Ohio Division, and the Ohio State Board of Regents Research Challenge Program (to M. A. Baumann).
Address for reprint requests and other correspondence: J. Gómez-Cambronero, Dept. of Physiology and Biophysics, Wright State Univ. School of Medicine, 3640 Colonel Glenn Hwy., Dayton, OH 45435.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 March 2000; accepted in final form 15 August 2000.
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