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5ß1 Integrin and Secreted Fibronectin Is Involved in Macrophage Differentiation of Human HL-60 Myeloid Leukemia Cells1
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, IL 60439
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 integrin gene expression was observed
in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or
macrophage-CSF-treated blood monocytes before the manifestation of
macrophage markers. After treatment of HL-60 cells and monocytes, newly
synthesized FN was released and deposited on the dishes. An HL-60 cell
variant, HL-525, which is deficient in the protein kinase Cß
(PKC-ß) and resistant to PMA-induced differentiation, failed to
express FN after PMA treatment. Transfecting HL-525 cells with a
PKC-ß expression plasmid restored PMA-induced FN gene expression and
macrophage differentiation. Untreated HL-525 cells (which have a high
level of the 5ß1 integrin) incubated on FN
differentiated into macrophages. The percentage of cells having a
macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525
cells, or by either PMA or macrophage-CSF in monocytes was reduced in
the presence of mAbs to FN and 5ß1
integrin. The integrin-signaling nonreceptor tyrosine kinase,
p72Syk, was activated in PMA-treated HL-60 and
FN-treated HL-525 cells. We suggest that macrophage differentiation
involves the activation of PKC-ß and expression of extracellular
matrix proteins such as FN and the corresponding integrins,
5ß1 integrin in particular. The
stimulated cells, through the integrins, attach to substrates by
binding to the deposited FN. This attachment, in turn, may through
integrin signaling activate nonreceptor tyrosine kinases, including
p72Syk, and later lead to expression of other
genes involved in evoking the macrophage
phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß transmembrane glycoprotein subunits that are
noncovalently linked (2). The ß associations determine the
ligand-binding specificities of the integrin heterodimers for various
ECM proteins (3). In some cases, two integrins that share a ligand
recognize different regions of the ligand molecule, as is true of the
5ß1 and 4ß1
FN receptors, that bind to the Arg-Gly-Asp-Ser (RGDS) and Leu-Asp-Val
(LDV) motif of FN, respectively (4). Alternatively, two distinct
integrins can bind to the same region of the same ligand, such as the
5ß1 and vß3
integrins, which both recognize the RGDS site of FN (5).
In addition to being adhesion receptors, it has become clear that
integrins are also signal-transducing receptors (6) that regulate cell
growth (7) and apoptosis (8), influence gene expression (9), and
modulate tumor behavior (10). Several reports have shown that binding
of adhesive ligands to integrins can induce protein tyrosine
phosphorylation in hemopoietic cells (11, 12, 13, 14). These changes in
tyrosine phosphorylation are likely caused by the activation of
nonreceptor tyrosine kinases, such as
p125FAK or p72Syk
(9, 11, 14, 15). Subsequent to such a tyrosine phosphorylation event,
there is a rapid induction of immediate-early genes, including
transcription factors such as c-fos, c-jun, IkB,
and MAD-6, as well as cytokines such as IL-1ß, IL-8, and
TNF- (16).
Macrophages secrete FN, bind FN, and migrate in response to FN
(17, 18, 19, 20). In addition, FN provides signals that lead to enhanced
TNF- production, respiratory burst activity, and phagocytosis of
foreign microorganisms (18, 21, 22, 23), and promote the
differentiation of blood monocytes into tissue macrophages
(24, 25, 26). The process of FN secretion and adhesion was shown to be
under the control of protein kinase C (PKC) (15, 27, 28).
The human HL-60 myeloid leukemia cell line is often used as a model system to study terminal differentiation in myeloid cells (29, 30). Activation of PKC by PMA is well known to cause the induction of macrophage markers, such as adhesion and spreading, in myeloid cells (31, 32, 33). It is clear that HL-60 cells and blood monocytes adhere and spread on plastic tissue culture plates in response to PMA treatment; however, the precise mechanism by which signals are transmitted from PMA to the intracellular machinery that controls cell adhesion and spreading, and thus differentiation, remains largely unknown.
The present study was initiated to test the hypothesis that PMA-induced macrophage differentiation in HL-60 cells and peripheral blood monocytes may involve interaction between secreted FN and its integrin receptors; validation of this hypothesis would allow the delineation of the signaling role that PKC-ß and nonreceptor tyrosine kinase p72Syk play in this differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isotypic controls (IgG1, IgG2a, and IgG2b), were purchased from
Sigma (St. Louis, MO) as were dialyzed murine mAbs to human fibronectin
(FN-15, IgG1), an -naphthyl acetate esterase assay kit, RGDS and
Gly-Pro-Arg-Pro (GPRP) peptides, and Ficoll-Hypaque. mAbs to the human
ß1 (K20, IgG2a), 4 (HP211, IgG1), and
5 (SAM1, IgG2b) were from Immunotech (Westbrook, ME).
The mAb to focal adhesion kinase (p125FAK) was
from Transduction Laboratories (Lexington, KY) and Upstate
Biotechnology (Lake Placid, NY), and the mAb to
p72Syk was from Wako Chemicals (Richmond, VA).
Indocarcyanine-conjugated anti-murine goat Ig (CY3) was purchased
from Jackson ImmunoResearch (West Grove, PA), and the mAb to the human
Vß3 integrin mAb (23CG,
IgG1) was bought from PharMingen (San Diego, CA). The anti-human
ß46 integrin mAb was kindly provided by
Dr. S. Kennel of Oak Ridge National Laboratory. The macrophage-CSF
(M-CSF) was from Biosource International (Camarillo, CA), and plates
precoated with mouse laminin, collagen type I, or collagen type IV were
from Becton Dickinson (Bedford, MA). Human 5- and
ß1-chain integrin cDNAs were purchased from Life
Technologies (Grand Island, NY).
Cells and cell culture
The human myeloid HL-60 leukemia cell line was originally obtained from R. C. Gallo of the National Cancer Institute. The HL-525 cells were established in our laboratory and have been described previously (31). The cells were incubated in tissue culture plates with RPMI 1640 medium supplemented with 15% heat-inactivated FCS (Intergen, Purchase, NY), penicillin (100 µg/ml), streptomycin (100 µg/ml), and 2 mM L-glutamine (Life Technologies) at 37°C in a humidified atmosphere containing 8% CO2. Human peripheral blood leukocytes were obtained from heparinized whole venous blood. Monocytes were separated by Ficoll-Hypaque density gradient (1.077 g/ml) centrifugation. The mononuclear cells concentrated at the surface were collected and washed twice in RPMI 1640 medium. Monocytes (>95%) were then isolated by selective adherence in tissue culture dishes (Nunc, Naperville, IL) for 90 min at 37°C.
Stable transfection of cells
All transfections were performed as previously described (32), by electroporation. A Bio-Rad gene pulser apparatus with a capacitance extender in 0.4-cm gap electroporation cuvettes (Eppendorf Scientific, Madison, WI) was used. The pMV7-RP58 plasmid (kindly provided by Dr. I. B. Weinstein, Columbia University, New York, NY) contained both the full-length rat PKC-ß1 cDNA and the bacterial neomycin phosphotransferase (neo) gene that confers resistance to the antibiotic G418 (geneticin, Sigma). The pMV7 plasmid contained the neomycin gene only. For each transfection, 5 x 106 cells were mixed with 10 µg of supercoiled plasmid DNA and 0.2 ml of phosphate-buffered sucrose (272 mM sucrose, 7 mM Na2HPO4, pH 7.4) in a total volume of 0.5 ml. The cells were electroporated at 250 V and allowed to recover in 10 ml of serum-supplemented RPMI medium for 24 h before selection in a medium containing 0.5 mg/ml geneticin. The geneticin-resistant transfectants were obtained by limited dilution in 24-well plates and tested for PKC-ß expression and PMA inducibility of macrophage markers. The selected clones were maintained in a geneticin-containing medium.
Differentiation markers
To determine the percentage of adherent and spread cells, we
inoculated 5 x 105 cells in 0.5 ml of medium into
each well of a 24-well tissue culture plate in the presence or absence
of PMA. For some experiments, we precoated the surface of the wells
with either FN or BSA by overnight incubation at room temperature with
0.5 ml of PBS solution containing protein at 20 µg/ml. The
nonspecific sites were blocked with 1% BSA in PBS for 30 min. The
wells were then washed with 3 µM MnCl2, inoculated with
the cells, and incubated for different time intervals. The percentage
of cell attachment and spreading was determined as previously described
(34). The percentage of attached cells was up to 20% higher than that
of spread cells. Nonspecific esterase activity was determined using the
-naphthyl acetate esterase assay kit as prescribed by the
manufacturer. Phagocytosis was detected as described previously (32),
by the ability of cells to ingest 1.7-µm-diameter Fluoresbrite beads
(Polysciences, Warrington, PA) that had been sterilized and opsonized
by incubation in 70% ethanol for 20 min and then in RPMI supplemented
with 20% PBS (not heated) for 18 h at 37°C; cells were
considered positive if they engulfed 20 beads/cell. Lysozyme activity
was determined as previously described (35). To examine the blocking
effect of mAbs, peptides, or other kinase inhibitors on differentiation
induction, cells were incubated with the mAb or peptide for 20 min
before as well as during treatment with the inducers.
Immunofluorescence
The immunostaining procedures were conducted at 4°C by using either 96-microwell plates or tissue culture chamber slides (Nunc). The cells were washed twice with PBSA (PBS containing 1% BSA and 0.1% NaN3) and incubated for 45 min with the appropriate primary mAb under saturating conditions. The cells were then washed twice with PBSA and incubated for an additional 45 min with the secondary Ab CY3. After a wash with PBSA, the slides were mounted with phosphate-buffered Gelvatol (Becton Dickinson, Sunnyvale, CA). Fluorescence was examined by using a Vaytek digital confocal microscope. To examine the blocking effects of the protein kinase inhibitors, cells were incubated with the inhibitors 20 min before as well as during treatment with the inducers.
RT-PCR analysis
RNA was purified by centrifugation through a CsCl cushion as previously described (36). cDNA was synthesized from total cellular RNA using SuperScript II reverse transcriptase (Life Technologies) under the conditions recommended by the supplier. The reverse transcriptase reaction used 2 µg of total RNA and either 100 ng of oligo(dT) primer or 2 pmol of a gene-specific primer. PCR amplification used the Tfl polymerase (Epicentre Technologies, Madison, WI) under conditions recommended by the supplier. The FN template primers, F1F/F2R nucleotides 3945–3966 and 4325–4346; 396-bp product) and F5F/F6R (nucleotides 3981–4001 and 4706–4727; 746-bp product), were derived from the human sequence (GenBank accession number X02761). The combination of primer F1F and F6R resulted in a 782-bp PCR product. The template primers for human glyceraldehyde-3-phosphate dehydrogenase, G1F/G2R (nucleotides 19–39 and 713–734; 715-bp product), were derived from the human sequence (GenBank accession number X01677). One set of cycle parameters was used for all primers (denaturation at 94°C for 50 s; annealing at 63°C for 1 min; extension at 73°C for 1 min), with the total number of cycles (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) tailored to the specific primer pair. For all reactions, various amounts of the reverse transcriptase reaction were used to ensure correspondence between the amount of amplified product and the input cDNA. For the FN amplification reactions, at least three independent primer pairs were used for each set of reverse transcriptase products to validate the amplification pattern.
Immunoprecipitation and in vitro kinase activity
Pellets containing 109 cells were lysed in buffer (1% Triton X-100; 0.1% SDS, 1% deoxycholate sodium, 50 mM NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM sodium o-vanadate) for 30 min (37). Nuclei and cellular debris were removed by centrifugation at 16,000 x g for 15 min. Supernatants were incubated with anti-p72Syk mAb for 2 h at 4°C, and the immunocomplex was recovered by further incubation with 50 µl of protein G-Sepharose for 1 h at 4°C. Immunoprecipitates were washed in 1 ml of lysis buffer and divided into two aliquots. One was subjected to an in vitro kinase assay, and the other was subjected to immunoblotting analysis. For immunoblotting analysis, immunoprecipitates were resuspended in Laemmli’s sample buffer, boiled, and subjected to SDS-PAGE (7.5% acrylamide). Proteins were transferred onto polyvinylidene fluoride membranes. Immunoblots were incubated with 1% BSA in TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) for 30 min. Then anti-p72Syk mAb was added for 1 h. After being washed in TBST, the immunoblots were incubated with alkaline phosphatase-conjugated anti-IgG for 30 min, washed twice in TBS buffer (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl), and then incubated with alkaline phosphatase substrate. When color reaction developed to the desired intensity, the immunoblots were washed in deionized water and photographed. For in vitro kinase activity, one-half of each immunoprecipitate was incubated with an equal volume of kinase buffer (25 mM HEPES (pH 7.5), 10 mM MnCl2, and 5 mM MgCl2) containing 0.5 µCi of [32P]ATP and 0.2 mg/ml histone for 30 min. Proteins were then resuspended in sample buffer for SDS-PAGE and separated on 12% acrylamide gels. p72Syk activity was measured by the phosphorylation of histone (32P-labeled histone).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined FN gene expression during macrophage differentiation
of HL-60 cells treated with PMA because this ECM protein has been
reported to be produced by blood macrophages (18, 19, 20). An attempt to
assess FN mRNA levels by standard Northern hybridization to total RNA
or poly(A)-enriched RNA yielded inconsistent results. To circumvent
this problem, we introduced semiquantitative RT-PCR analysis. By using
this approach with three different sets of primers (including one that
codes for RGDS), we observed barely detectable FN-specific
amplification products in untreated HL-60 cells (Fig. 1
A). FN steady-state mRNA as
assessed by RT-PCR was induced at 4 h, and its level was further
increased at 24 h after 3 nM PMA treatment. We also examined FN
gene expression in cells from an HL-60 variant, HL-525. These cells,
unlike the parental cells, are resistant to PMA-induced macrophage
differentiation (31, 32). Contrary to what was observed in the HL-60
cells, no FN gene expression was observed in either untreated or
PMA-treated HL-525 cells (Fig. 1A).
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B). Treatment with 3 nM PMA caused
the cells to display an increase of FN immunostaining as early as
4 h. Prolonging this treatment to 2 days, which caused the HL-60
cells to acquire a macrophage phenotype (32), resulted in scattered FN
immunostaining across the surface of the cells. A noteworthy
observation was the abundance of speckled FN immunostaining in the
intercellular spaces of the culture dishes containing HL-60 macrophages
but not in those containing control cells (Fig. 1B). Little
or no immunostaining with the FN mAb was observed in untreated or
PMA-treated HL-525 cells, or with the isotypic control Abs in untreated
or in PMA-treated HL-60 and HL-525 cells. These results indicate that PMA treatment of HL-60 cells results in increased FN gene expression and cell surface manifestation of FN within 4 h after PMA treatment, a time frame that precedes the expression of the macrophage phenotype. These results also show that PMA treatment causes the release and deposition of FN.
Induction of macrophage differentiation in HL-525 cells by exogenous FN
Our analysis of FN gene expression suggested that FN might be
involved in the signaling pathway of PMA-induced macrophage
differentiation in HL-60 cells. It was therefore of interest to
determine whether supplying FN exogenously would promote macrophage
differentiation in the FN-deficient HL-525 cells. For this reason, we
cultured HL-525 cells on FN-precoated dishes in the presence and
absence of 30 nM PMA. The presence of this ECM protein was in itself
sufficient to cause >75% of the HL-525 cells to manifest macrophage
markers such as cell adherence and spreading (Fig. 1C), and
PMA treatment further increased this percentage to 90%. No significant
cell attachment and spreading was observed on insolubilized FN when
HL-60 cells were used or when HL-60 and HL-525 were cultured on dishes
precoated with BSA or other ECM proteins such as laminin or collagen
type I or type IV (data not shown). Taken together, our results
implicate FN in the induction of macrophage differentiation and
indicate that exogenous FN can substitute for the insufficient FN gene
expression needed for such a differentiation in the PMA-resistant
HL-525 cells.
Essential role of secreted FN and 5ß1
integrin in macrophage differentiation
Because FN has been reported to bind to a large degree to
ß1 and ß3 integrins (reviewed in Refs. 2,
5, and 38), it was of interest to determine whether HL-60 and HL-525
cells manifest these receptors on their cell surfaces. For comparison
and as a control, we also tested for 6ß4
integrin, which binds laminin but not FN (39). Immunostaining of viable
cells showed that HL-60 and HL-525 cells exhibit barely detectable
immunofluorescence when reacted with Mabs specific to
vß3 FN-binding integrin (2, 5, 38), as
well as to 6ß4, a laminin-binding integrin
(39). PMA treatment yielded only some immunostaining for these Ags
(Table I). Immunostaining with mAbs to
5, ß1, and 4 integrin
chains, which was also barely detectable in HL-60 cells, was intense on
HL-525 cells (Table I, Fig. 2). PMA
treatment caused both cell lines to display an increase in
5 and ß1 and a decrease in
4 immunostaining (Table I, Fig. 2). Moreover, the
similar intensity of 5 and ß1
immunostaining in PMA-treated HL-60 cells and untreated HL-525 cells
(Table I, Fig. 2) can explain the ability of HL-525 cells to adhere to
and differentiate on FN-precoated dishes, a result that suggests
a role for the FN-binding integrin,
5ß1, in macrophage differentiation.
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5, and ß1 integrin
chains and the RGDS peptide attenuated the manifestation of macrophage
markers; the combination of the anti-ß1 and
anti-FN mAbs reduced the manifestation of several differentiation
markers by >80% (Table II) (cell
spreading, nonspecific esterase activity, phagocytosis, and lysozyme
activity). Moreover, this Ab combination also abated the inhibited cell
proliferation induced in HL-60 cells by PMA and in HL-525 cells by FN.
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vß3
integrin that binds to FN through an RGDS site but does not belong to
the ß1 integrin family. This mAb reduced the
manifestation of macrophage markers in the PMA-treated HL-60 cells and
FN-treated HL-525 cells (Table II) by only 
20%, a result we
attribute to the low abundance of this integrin on these cell lines
(Table I). The mAb to the 4 chain integrin, which does
not bind to the RGDS motif of FN, was ineffective in altering
macrophage differentiation in either HL-60 or HL-525 cells (Table II),
implicating RGDS-dependent cell adhesion and spreading in the
macrophage differentiation process. As a negative control, we used the
mAb to the 6ß4 integrin, which does not
bind to FN (2); this mAb failed to affect these markers in treated
cells (Table II). Control Abs and control peptide GPRP had no effect on
the expression of the macrophage markers or on cell proliferation. All
mAbs and peptides, when used alone or in combination, did not affect
the replication or expression of maturation markers in untreated HL-60
cells and HL-525 cells cultured on dishes precoated with BSA (Table II). These results indicate that the acquisition of a macrophage
phenotype in HL-60 cells involves the production, release, and
deposition of FN and its binding to the
5ß1 integrin.
To ensure that the involvement of an FN/ß1-integrins
interaction is not restricted to macrophage differentiation in the
HL-60 cell system, we included human peripheral blood monocytes in our
study. Our results showed that a 2-day stimulation of blood monocytes
by either of two macrophage inducers, PMA or M-CSF (9, 14, 33),
increased the amount of speckled FN immunostaining across the cells and
in the intercellular spaces (Fig. 3A). As was the case for
PMA-treated HL-60 cells, the addition of anti-FN or
anti-ß1 mAbs reduced the number of spread cells by
60% in PMA- and M-CSF-treated monocytes (Fig. 3B). Taken
together, our findings indicate that an interaction between deposited
FN and ß1 integrins is also involved in PMA- or
M-CSF-induced macrophage differentiation in blood monocytes.
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Since the HL-525 cells exhibited markedly diminished PKC-ß gene
expression (Fig. 4A), it is
possible that this PKC isoenzyme is critical for FN gene expression. To
test for this possibility, we transfected the HL-525 cells with an
expression plasmid containing the full-length PKC-ß cDNA and the
neomycin gene that confers geneticin resistance. As a control, the
HL-525 cells were transfected with a plasmid containing only the
neomycin gene. Our findings showed that PKC-ß gene expression in
HL-525 cells transfected with PKC-ß expression plasmid was restored
to levels similar to those of HL-60 cells (Fig. 4A). These
cells also regained susceptibility to PMA-induced FN gene expression
(Fig. 4B) and macrophage differentiation, as manifested by a
significant increase in adherent and spread cells, in phagocytizing
cells, and in lysozyme activity after PMA treatment (Fig. 4C). No restoration of PMA-induced FN gene expression and
macrophage differentiation was observed in HL-525 cells transfected
with the control vector (Fig. 4, B and
C). These results implicate the activation of PKC-ß in FN
gene expression during PMA-induced macrophage differentiation in the
HL-60 cell system.
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Because the activation of nonreceptor tyrosine kinases such as
p125FAK and p72Syk has
been implicated in FN-evoked integrin-mediated signal transduction (11, 15), we examined the role of these kinases in macrophage
differentiation in HL-60 and HL-525 cells. The
p125FAK and p72Syk
kinases were selected because they are found in some myeloid cell types
(9, 11, 12, 13). By using specific mAbs, we detected the
p72Syk but not the
p125FAK kinase, with the level of p72
Syk protein varying only slightly among
untreated and treated HL-60 and HL-525 cells (Fig. 5). p72Syk
activation was observed within 15 min after treatment of HL-60 cells
with 30 nM PMA and after incubation of HL-525 cells on the dishes
precoated with FN (Fig. 5). Interestingly, treatment with 30 nM PMA did
not activate p72Syk in the treated HL-525
cells (Fig. 5), indicating that this event may require PKC-ß
activity. To further investigate the role that PKC-ß plays in
activating of p72Syk, we examined the activation
of this tyrosine kinase in PKC-ß-transfected HL-525 cells. Results
were inconsistent due to the high reversion frequently of these cells
after six cell passages (32).
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Taken together, our results support the hypothesis that
PMA-induced HL-60 macrophage differentiation occurs in a coherent
sequence of events initiated by the activation of PKC-ß. This
activation evokes a series of chronological changes in gene expression
(depicted in Fig. 6), leading to the
development of a macrophage phenotype. Induction of FN steady-state
mRNA was observed within 15 min and was maintained for up to 24 h
(Fig. 6A). Up-regulation of 5 and
ß1 integrin gene expression, appeared to begin at 4
h and steadily increased thereafter for up to 24 h after PMA
treatment (Fig. 6B). Activation of
p72Syk tyrosine kinase, which was also observed
at 4 h, decreased markedly at 24 h after PMA treatment (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 integrin are essential steps in the
signaling pathway leading to acquisition of the macrophage phenotype. Previously, it was found that macrophages secrete FN (17, 18, 19, 20). Although much is known about the role of FN in inflammation and phagocytosis (21, 22, 23), little is known about the influence that this released ECM protein may have on early stages of macrophage differentiation. Here we provide explicit evidence that FN is one of the key elements in the macrophage differentiation signaling cascade.
In our initial experiments, we determined the level of gene expression and cell surface manifestation of FN in HL-60 cells. Whereas untreated cells exhibited little or no FN-specific RT-PCR amplification products, treatment with PMA markedly increased FN gene expression at as early as 15 min and in a time-dependent manner. Moreover, we found that increased FN gene expression was followed by FN secretion and deposition on the tissue culture dishes. Next, we examined the ability of blood monocytes to produce and secrete FN. Treatment with either PMA or M-CSF caused the cells to differentiate into macrophages and to display an increased level of FN, which was detected not only across the cells but also in the intercellular spaces. Additional support for an association between macrophage differentiation and FN came from our study with an HL-60 variant, HL-525 cells, which, unlike the parental cells, are resistant to PMA-induced macrophage maturation (31, 32, 40). Contrary to results found in HL-60 cells, little to no FN gene expression was observed in PMA-treated HL-525 cells. This result raised the possibility that FN might play a critical role in inducing macrophage differentiation. If so, one might speculate that supplying FN exogenously would, in itself, promote macrophage differentiation in the FN-deficient HL-525 cells. This notion was successfully proved, in that incubating HL-525 cells on dishes precoated with FN but not with laminin or collagen resulted in the manifestation of macrophage markers, including cell spreading, lysozyme production, phagocytosis, and nonspecific esterase activity. These findings implicate secreted FN as a signaling protein in the macrophage differentiation pathway.
It is well established that interactions between cells and ECM proteins
are often mediated by integrin receptors (2, 5). By using
anti-5 and anti-ß1 mAbs, we found
that whereas HL-60 cells displayed weak immunostaining of
5ß1 integrin, HL-525 cells exhibited an
intense immunostaining of this adhesion molecule. Because expression of
integrins is reportedly regulated by various growth factors and other
stimuli (34, 41), it was of interest to examine whether integrin cell
surface manifestation was affected by treatment with PMA. We found that
PMA induced HL-60 and untreated HL-525 cells to display an increase in
5ß1 integrin immunostaining. Moreover, the
intensity of 5ß1 integrin immunostaining
in PMA-treated HL-60 cells was similar to that in untreated HL-525
cells. This result can explain why HL-525 cells adhere and
differentiate on dishes precoated with FN, while HL-60 cells do so only
after treatment with PMA. We are currently extending the study reported
here to investigate the factors that account for the difference between
the HL-60 and HL-525 cell surface manifestations of the integrins.
One of the major observations we want to communicate is that the
interaction of FN with FN-binding integrins is intimately involved in
the induction of the macrophage differentiation process. We
demonstrated this with two experimental approaches. In one approach, we
tested the ability of anti-FN and anti-FN-binding integrin mAbs
to block macrophage differentiation in HL-60 cells and peripheral blood
monocytes. We assumed that these Abs would prevent the interaction of
the integrins with the deposited FN and that if this interaction was
involved in macrophage differentiation, then the presence of the mAbs
would inhibit such differentiation. Our studies indicated that the mAbs
to FN and 5ß1 integrin (which bind to the
RGDS motif on FN (4)) were able to reduce the percentage of cells
showing macrophage phenotypes in induced HL-60 cells and monocytes but
that the mAb to 4ß1 integrin (which binds
to the LDV motif on FN (4)) was not. Our results also showed a decrease
in HL-60 cell differentiation when the RGDS peptide was used. Other
investigators have found that synthetic peptides containing the RGDS
motif promote cell adhesion when immobilized on suitable substrates and
inhibit cell adhesion to tissue culture dishes precoated with FN (17).
Taken together, our results indicate that induction of macrophage
differentiation involves the interaction of
5ß1 integrin with, most likely, the RGDS
site of the deposited FN.
In another approach we used HL-525 cells, which exhibit little or no
PKC-ß nor FN gene expression. We showed that when these cells are
transfected with PKC-ß cDNA they regain PKC-ß expression and
susceptibility to PMA-induced differentiation, as well as FN gene
expression. Our results suggest that PKC-ß plays an essential role in
FN gene expression during PMA-induced macrophage maturation. We also
found that macrophage differentiation induced in HL-60 by PMA and in
HL-525 by FN was inhibited by anti-5 or
anti-ß1 integrins, anti-FN mAbs, and RGDS
peptide. These lines of evidence further support the hypothesis that
the interaction of FN and its 5ß1 receptor
is involved in attaining of a macrophage phenotype. This conclusion is
similar in concept to that proposed some years ago by Utsumi et al.
(42), who found that adhesion of immature thymocytes to thymic stromal
cells through FN-ß1 integrin interaction induces T
lymphocyte differentiation.
Evidence from several laboratories suggests that integrins are capable
of transmitting signals from the ECM to the cell interior (11, 12, 13, 15).
Many reports demonstrate that ligation of integrins leads to enhanced
activation of nonreceptor tyrosine kinases (11, 15). Although much
attention has been paid to p125fak, it is not
the only nonreceptor tyrosine kinase that is activated by cell adhesion
(2, 9). For monocytic cells, evidence for whether these cells express
p125FAK is contradictory (9, 11, 14). In our
experiments, neither untreated nor PMA-stimulated HL-60 or HL-525 cells
cultured in dishes precoated with FN or not expressed detectable
p125FAK protein. Recently, integrin ligation in
monocytes was found to lead to the activation of
p72Syk, a member of the ZAP70 family of tyrosine
kinase (9). Moreover, cross-linking of phagocytic Fc
receptors on
macrophages or cultured monocytes was shown to induce a rapid
phosphorylation of p72Syk, suggesting that this
enzyme may play a role in the phagocytic process (43). In our
experimental cell system, the interaction of integrins (most likely
5ß1) with secreted FN could activate
p72Syk. This possibility was investigated
through an in vitro kinase assay on p72Syk
immunoprecipitates. We found that this tyrosine kinase is present in
HL-60 and HL-525 cells and that its protein level varies only to a
limited degree between these two cell lines and between untreated and
treated cells. We also found that p72Syk was
activated in HL-525 and HL-60 cells under conditions that bring about a
macrophage phenotype, specifically, after treatment with FN and PMA,
respectively. A noteworthy observation was that
p72Syk activity was induced 4 h after PMA
treatment and decreased 24 h after PMA treatment in HL-60 cells,
suggesting that this enzyme is activated during the induction of
differentiation for only a limited time frame that is downstream of FN
gene expression. This result can explain why in a previous study, HL-60
cells stimulated with PMA for 24 to 72 h did not display
p72Syk activation (12). At present, we cannot
corroborate that p72Syk activation is indeed a
downstream signaling protein of FN that is involved in the pathway
leading to macrophage phenotype. For this reason, we simply raise the
possibility that this kinase has properties that make it a probable
candidate for this function.
We suggest that a pathway leading to macrophage differentiation in
human progenitor cells is followed in a chronological and coherent
sequence that involves the activation of PKC; the production, release,
and deposition of FN and the up-regulation of
5ß1 gene expression. The stimulated cells,
through their integrins (5ß1 in
particular), adhere to the deposited FN (most likely through its RGDS
site). This adhesion, in turn, leads to the activation, through
integrin signaling, of downstream kinases (6, 44), including
p72Syk, and later to the expression of genes
involved in evoking the macrophage phenotype.
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Acknowledgments |
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6ß4 integrin, Drs. M.
Bhattacharyya and D. Glesne for critical review of the
manuscript, and K. Nobles for secretarial expertise. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Eliezer Huberman, Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4833. E-mail address:
3 Abbreviations used in this paper: ECM, extracellular matrix; FN, fibronectin; RGDS, Arg-Gly-Asp-Ser; PKC, protein kinase C; M-CSF, macrophage-CSF.
Received for publication June 8, 1998. Accepted for publication August 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin associates with activated insulin and PDGF ß receptors and potentiates the biological activity of PDGF. EMBO J. 16:5600.[Medline]
5ß1 expression negatively regulates cell growth: reversal by attachment to fibronectin. Mol. Biol. Cell 6:725.[Abstract]
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and transforming growth factor ß1 differently regulate fibronectin and laminin receptors of human differentiation monocytic cells. J. of Immunol. 148:3912.[Abstract]
6ß4 integrin: distinct ß4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J. 14:4470.[Medline]
X/ß2) and VLA-4 (CD49d/CD29; 4/ß1) integrins during myeloid cell differentiation. Eur. J. Immunol. 24:41.[Medline]
subunit of Fc receptors, p72Syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269:3897.This article has been cited by other articles:
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