From the Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tumor necrosis factor- (TNF-
) gene is one
of the early response genes induced by phorbol 12-myristate 13-acetate
(PMA) in human HL-60 myeloid leukemia cells. In the present study, we
examined the role of the TNF-
autocrine loop in PMA-induced
macrophage differentiation and gene expression of 92- and 72-kDa
gelatinases (MMP-9 and MMP-2). In HL-60 cells, PMA inhibited cell
proliferation and induced cell adhesion and spreading, expression of
surface maturation marker OKM1 and phagocytic activity, as well as the expression of both gelatinases, which all characterize the macrophage phenotype. In contrast, TNF-
alone was only effective in inhibiting cell proliferation. Blocking the endogenous TNF-
activity with neutralizing anti-TNF-
antibodies abolished all these PMA-induced events with the exception of MMP-2 gene expression. Since
fibronectin (FN)-mediated cell adhesion and spreading are prerequisite
for both macrophage differentiation and MMP-9 gene
expression in HL-60 cells, we hypothesized that TNF-
might be
involved in modulating the expression of either the FN or its integrin
receptor genes. Whereas PMA substantially enhanced the steady state
mRNA and protein levels of both FN and
5
1 integrins, TNF-
alone had little
effect on the expression of these genes. However, anti-TNF-
antibodies blocked PMA-induced augmentation of both
5
and
1 integrin gene expression without affecting the
expression of the FN gene. Our results suggest that TNF-
may
regulate macrophage differentiation and critical matrix-degrading
activities of myeloid progenitor cells in an autocrine manner by
augmenting surface levels of the
5
1
integrin, thus promoting interactions with the extracellular matrix, a
key event for maturation and migration of these cells during
inflammation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tumor necrosis factor-
(TNF-
),1 an inflammatory
cytokine primarily produced by activated macrophages, participates in a
wide range of immunological processes which could be either beneficial or detrimental to the body (1). In addition to being a mediator of
activated macrophage function, TNF-
is known to be a feedback modulator of macrophage differentiation of myeloid progenitor cells.
The effects of TNF-
on proliferation and differentiation of myeloid
progenitor cells are bidirectional, depending on the differentiation
state and potential of the target cells as well as the hematopoietic
growth factors used to promote their differentiation. Whereas TNF-
acts synergistically with macrophage-colony stimulating factor (M-CSF)
to stimulate proliferation of bone marrow cells differentiating toward
the macrophage lineage (2), the cytokine inhibits the proliferation and
promotes macrophage differentiation of bone marrow progenitor cells in
the presence of stem cell factor or granulocyte-macrophage-colony
stimulating factor (GM-CSF) (3-5); TNF-
exerts these effects in
both an autocrine and paracrine manner. In addition, TNF-
has been
shown to be identical to a differentiation-inducing factor produced by
mitogen-stimulated peripheral blood monocytes and leukemic cell lines
that is capable of inducing monocyte-like characteristics in a number
of myeloid cell lines (6). However, how TNF-
-induced gene expression contributes to macrophage differentiation of myeloid progenitor cells
remains poorly characterized.
The 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases, which belong to the matrix metalloproteinase family, are the key proteinases governing the degradation of basement membrane (7). Both MMP-9 and MMP-2 cleave basement membrane collagen types IV and V as well as different types of gelatin (8-11). Although the two proteinases share structural and catalytic similarities, their gene expression is differentially regulated, partly due to the distinct structure of the regulatory elements and promoters in their genes (12-14). Both MMP-9 and MMP-2 are produced by human macrophages, and their proteolytic activities are thought to be necessary for various functions of monocytes and macrophages such as extravasation, migration, and tissue remodeling during chronic inflammatory conditions (15-18). Although a number of previous studies have shown that the production of these MMPs is markedly up-regulated during macrophage differentiation, the regulatory mechanisms mediating this event remain to be elucidated.
Human HL-60 myeloid leukemia cells retain the ability to differentiate
along the monocyte, macrophage, or granulocyte pathway (19). This cell
line, therefore, serves as a useful model system for studying the
critical cellular events involved in these differentiation processes.
Phorbol 12-myristate 13-acetate (PMA) induces HL-60 cells to
differentiate toward the macrophage lineage (20-22), and the TNF-
gene is one of the early response genes induced by PMA during this
process (23). In this study, we explored the role of TNF-
as an
autocrine regulator in PMA-induced HL-60 differentiation. We examined
four characteristic macrophage markers induced by PMA: (i) inhibition
of cell replication; (ii) cell adhesion and spreading; (iii)
manifestation of the surface maturation marker OKM1 (CD11b); and (iv)
phagocytic activity. Although TNF-
was as effective as PMA in
inhibiting cell replication, it could not induce the other three
differentiation markers. However, neutralizing anti-TNF-
antibodies
inhibited all four PMA-induced macrophage markers, indicating that
TNF-
is an autocrine factor critical for PMA-induced macrophage
differentiation in HL-60 cells. In addition, treatment with
anti-TNF-
antibodies abolished PMA-induced MMP-9 gene
expression without affecting the PMA-induced expression of
MMP-2, suggesting that TNF-
does not mediate all
PMA-induced gene expression in HL-60 cells. We demonstrate herein that
one of the mechanisms whereby TNF-
modulates PMA-induced macrophage differentiation and MMP-9 gene expression is through
augmenting the gene expression of the surface adhesion molecule
5
1 integrin. Our results suggest that
TNF-
may act in an autocrine manner to enhance macrophage
differentiation and matrix-degrading capability via promoting
interactions of myeloid progenitor cells with extracellular matrix
proteins.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
Phorbol 12-myristate 13-acetate (PMA) was
purchased from Chemicals for Cancer Research (Eden Prairie, MN). Tumor
necrosis factor- (TNF-
) (>1.0 × 108 units/mg)
was purchased from Boehringer Mannheim. Gelatin and a mouse monoclonal
antibody (mAb) to human CD11b (OKM1) (IgG1) and mAb to
human FN (FN-15, IgG1), which was dialyzed before use, were
purchased from Sigma. Mouse mAbs to human
1 (K20,
IgG2a) and
5 (SAM1, IgG2b)
integrin were obtained from Immunotech (Westbrook, ME). Both monoclonal
(mouse) and polyclonal (goat) anti-TNF-
neutralizing antibodies were
purchased from R & D Systems (Minneapolis, MN) and were found equally
effective in our study. Mouse IgG1 and goat IgG were also
purchased from R & D Systems and used as a control. The experiments
presented in this study were conducted by using polyclonal anti-TNF-
antibodies and goat IgG.
Cells and Cell Culture-- The human HL-60 myeloid leukemia cell line was originally obtained from R. C. Gallo (National Cancer Institute). The cells were cultured and maintained in Petri dishes in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 15% heat-inactivated fetal bovine serum (Intergen Co., Purchase, NY), penicillin (100 units/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM) (Life Technologies, Inc.) in a humidified atmosphere containing 8% CO2 at 37 °C. All treatments were carried out in tissue culture dishes or cluster plates in serum-supplemented RPMI 1640 medium.
Differentiation Markers--
To determine the differentiation
markers, HL-60 cells were seeded at 2-4 × 105
cells/ml and treated for 24 h with 3 nM PMA or 10 ng/ml (>103 units/ml) TNF- in the presence or absence
of 10 µg/ml preimmune IgG or anti-TNF-
antibodies. The number of
cells was determined by hemocytometer chamber counting; the percentage
of cell adhesion and spreading was determined as described previously
(24), and the percentage of cells exhibiting a cell surface maturation
antigen OKM1 was determined by indirect immunofluorescent staining with the OKM1 mAb.
Indirect Immunofluorescence--
The cells (7 × 104 cells/well) were seeded in eight-well Lab-Tek chamber
slides and treated for 24 h with 3 nM PMA in the
absence or presence of 10 µg/ml preimmune IgG or anti-TNF-
antibodies. After treatment, the cells were rinsed with PBS and
incubated for 30 min at room temperature with a blocking solution
containing 1% bovine serum albumin and 1% normal goat serum (Sigma)
in PBS, followed by a 2-h incubation with the appropriate primary mAb at saturating concentrations. The cells were then washed twice with PBS
and incubated for an additional 45 min with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch, West Grove, PA). Following three washes in PBS, the
slides were mounted with phosphate-buffered gelatol. Fluorescence was
examined using the Micro-Tome Mac Digital Confocal microscope described above.
Gelatin Zymography-- The cells (1.5 × 105 cells/ml) were treated in serum-supplemented RPMI 1640 medium as indicated in the figure legends. After treatment, the cells were replaced with serum-free medium containing the appropriate antibodies and incubated for an additional 24 h prior to collection of conditioned media. Gelatin zymography analysis was performed on 7.5% SDS-polyacrylamide gels containing 1 mg/ml gelatin (Sigma) as described previously (26). Gelatinolytic activity was visualized as clear zones with Coomassie Brilliant Blue R-250 staining.
RNA Isolation and Northern Analysis--
Total RNA was
purified by centrifugation through a cesium chloride cushion as
described by Chirgwin et al. (27). Northern blot
analysis was performed as described previously (28). Hybridizations were performed with radiolabeled probes at 60 °C for 18-24 h in 0.5 M sodium phosphate, pH 7.2, 2 mM EDTA, 1%
bovine serum albumin, 7% SDS. The blots were washed at 60 °C for 30 min once in 1× SSPE (10 mM sodium phosphate, pH 7.4, 150 mM sodium chloride, 1 mM EDTA), 0.1% SDS, and
once in 0.1× SSPE, 0.1% SDS, followed by autoradiographing in the
dark at 80 °C. Human cDNA probe for MMP-9 was
kindly provided by Dr. W. Stetler-Stevenson, National Institutes of
Health, Bethesda. Human cDNA probes for MMP-2, TNF-
, and GAPDH were obtained from American Type Culture Collection (Rockville, MD), and those for
5 and
1
integrins were from Life Technologies, Inc. The mRNA level of a
specific gene relative to that of GAPDH was determined by using a HP
ScanJet 4c Scanner (Hewlett-Packard).
RT-PCR Analysis-- Total RNA was purified as described above. cDNA was synthesized from total cellular RNA using SuperScriptTM II reverse transcriptase (Life Technologies, Inc.) under the conditions recommended by the supplier. The reverse transcriptase (RT) reaction used 1-2 µg of total RNA and either 100 ng of oligo(dT) primer or 2 pmol of a gene-specific primer. Polymerase chain reaction (PCR) amplification used Tfl polymerase (Stratagene) under conditions recommended by the supplier. The FN template primers, F1F/F2R (nucleotides (nt) 3945-3966 and 4325-4346; 396-bp product) and F5F/F6R (nt 3981-4001 and 4706-4727; 746 bp product) were derived from the human sequence (GenBankTM accession number X02761). The combination of primer F5F and F2R resulted in a 365-bp PCR product and was used for this study. The template primers for human GAPDH, G1F/G2R (nt 19-39 and 713-734; 715-bp product), were derived from the human sequence (GenBankTM accession number X01677). One set of cycle parameters was used for all primers (denaturation at 94 °C, 50 s; annealing at 63 °C, 1 min; extension at 73 °C, 1 min) with the total number of cycles (25-40) tailored to the specific primer pair. For all reactions, various amounts of RNA samples and the RT reaction were used to ensure correspondence between the amount of amplification product and the input of RNA samples. For the FN amplification reactions, at least three independent primer pairs were used for reverse transcriptase reaction to validate the amplification pattern.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Autocrine Regulation of PMA-induced Macrophage Differentiation by
TNF---
To assess the role TNF-
plays in regulating macrophage
differentiation induced by PMA in HL-60 cells, we first examined its effect on PMA-induced cell adhesion and spreading, which are the hallmarks of macrophage differentiation (19). Whereas more than 90%
HL-60 cells exhibited cell adhesion and spreading after a 24-h PMA
treatment, TNF-
-treated cells remained in suspension and
demonstrated no apparent morphological changes as compared with the
untreated HL-60 cells (Table I and Fig.
1, a-c). Blocking the endogenous TNF-
activities by neutralizing anti-TNF-
antibodies prevented PMA-induced cell adhesion and spreading (Table I
and Fig. 1d), indicating that PMA-induced cell adhesion and
spreading in these cells involve the autocrine TNF-
loop.
|
|
Dissociated Regulation of PMA-induced Gene Expression of 92- and
72-kDa Gelatinases by TNF---
The induction of matrix-degrading
proteinases such as the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases
is associated with macrophage differentiation (15-17). PMA treatment
(3 nM) resulted in the induction of both MMP-2
and MMP-9 gene expression (Fig. 2, A and B), with
the level of MMP-2 steady state mRNA being much lower than that of
the MMP-9 steady state mRNA (3 days' exposure of the
autoradiograph film for MMP-2 versus 6-h exposure for
MMP-9). Accordingly, MMP-2 secretion was barely detectable by gelatin zymogram (data not shown), whereas secretion of MMP-9 proenzyme was
abundant (Fig. 2C). Compared with PMA, TNF-
(10 ng/ml)
only weakly induced MMP-2 and MMP-9 gene
expression as well as MMP-9 proenzyme secretion. Anti-TNF-
antibodies abolished PMA-induced MMP-9 but not
MMP-2 gene expression (Fig. 2, A and
B). The effect of the antibody treatment on secretion of the
MMP-9 proenzyme (Fig. 2C) mirrored that on its mRNA.
These findings suggest that while PMA-induced MMP-9 gene
expression is mediated through the TNF-
autocrine loop, PMA-induced
MMP-2 gene expression is independent of the endogenous
TNF-
activity.
|
TNF- Autocrine Loop Is Essential but Not Sufficient for
PMA-induced Gene Expression of
5
1
Integrin--
Since we found previously that both PMA-induced
macrophage differentiation2
and MMP-9 gene expression (30) required FN-mediated cell
adhesion and spreading, and anti-TNF-
antibodies blocked this
process, we suspected that TNF-
might be the intermediary that
controls PMA-induced gene expression of FN or its surface integrin
receptors. To confirm this hypothesis, we examined the expression of
the FN and
5
1 integrin genes in PMA- and
TNF-
-treated HL-60 cells, and we determined the effect of
anti-TNF-
antibodies on these events. We did not include the
4
1 integrin in this study because this
integrin was not involved in FN-mediated cell adhesion and spreading
induced by PMA or M-CSF in both HL-60 and human peripheral blood
monocytes.2 Expression of both
5 and
1 integrin genes was examined by Northern blotting
analysis. The FN gene expression was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR), because its expression was induced in low abundance and Northern blotting analysis
yielded inconsistent results. Three sets of FN primers were tested, and
the results shown in Fig. 3A
represent amplification of a single 365-bp FN fragment. Various amounts
of RNA samples and the RT reaction were used to ensure that
amplification of the 365-bp FN fragment corresponded to the input of
RNA samples. PMA (3 nM) induced a 5-fold increase in the
level of FN steady state mRNA (Fig. 3A), which was
followed by cell surface display and extracellular deposition of the FN
protein (Fig. 4). Similarly, PMA also
enhanced the steady state level of
1 integrin mRNA
by 4-fold and
5 integrin mRNA by 6-fold (Fig.
3B), with concomitant increases in surface expression of the
respective protein (Fig. 4). TNF-
alone failed to affect expression
of FN or
5 or
1 integrin gene. Blocking
the endogenous TNF-
activity with anti-TNF-
antibodies had little
or no effect on PMA-induced FN steady state mRNA and protein levels
(Fig. 3A and Fig. 4), demonstrating that PMA-induced FN
expression requires neither the autocrine TNF-
loop nor cell
adhesion and spreading. In contrast, treatment with anti-TNF-
antibodies resulted in a substantial reduction in PMA-induced
1 and
5 gene expression (Fig.
3B and Fig. 4). Accordingly, surface levels of both
1 and
5 proteins stimulated by PMA were
substantially inhibited by anti-TNF-
antibodies (Fig. 4). Taken
together, our results have shown that TNF-
is an autocrine mediator
for PMA-induced augmentation of
5
1
integrin gene expression, but TNF-
alone is insufficient and
necessitates additional factor(s) to stimulate
5
1 gene expression. Our findings were
further supported by the temporal sequence of expression of the FN,
TNF-
, and
5
1 integrin genes induced by
PMA. As shown in Fig. 5A, PMA
induced an early and transient expression of the TNF-
gene. Peak
induction of the TNF-
steady state mRNA was observed at 2 h
after PMA treatment, which was maintained for up to 4 h. The
TNF-
steady state mRNA levels then dropped markedly at 8 h
and became nearly undetectable at 24 h after treatment. The
induction of TNF-
gene expression was followed by augmented
expression of the
1 and
5 genes, which started at 4-8 h after addition of PMA and steadily increased thereafter for up to 24 h (Fig. 5A). On the other hand,
induction of the FN gene expression occurred within 30 min after
addition of PMA, and the FN steady state mRNA levels increased
steadily for at least 24 h after PMA treatment (Fig.
5B). Therefore, induction of FN gene expression occurs
before that of TNF-
, which is consistent with our conclusion that
PMA-induced FN expression is independent of the autocrine TNF-
activities.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have shown that a TNF- autocrine loop is
required, but not sufficient, for macrophage differentiation induced by
PMA in HL-60 cells. Consistent with our finding, several previous studies have established TNF-
as a competence factor which primes the progenitor cells at early stages of macrophage differentiation. TNF-
acts in concert with other hematopoietic growth factors to
inhibit cell proliferation and promote maturation, and this action may
be achieved in either an autocrine or paracrine fashion. TNF-
was
found to be expressed in all colonies of bone marrow progenitor cells
induced to differentiate toward the macrophage lineage by M-CSF or
GM-CSF, regardless of the differentiation stages, suggesting an
important role of this molecule during macrophage differentiation (31).
Blocking the endogenous TNF-
activity during GM-CSF-induced
macrophage differentiation in bone marrow progenitor cells resulted in
increased cell proliferation, suggesting the involvement of an
autocrine mechanism in which TNF-
expression signals the onset of
differentiation and the cessation of proliferation (4); there seemed to
be a time window during which the differentiating cells were responsive
to the endogenous production of TNF-
, since blocking TNF-
expression was effective on day 3 of differentiation but not on other
days. Similarly, in a study using neonatal cord blood-derived stem
cells (5), it has been found that there is a window of sensitivity
related to the priming effects of TNF-
; stem cells pretreated with
TNF-
for up to 3 days responded to the differentiating effects of
GM-CSF, and after 5 days of TNF-
pretreatment, GM-CSF was unable to
promote maturation. Although the cellular events triggered by TNF-
during macrophage differentiation in these progenitor cells remain to
be clarified, our findings in HL-60 cells provide hints that one of
these events may be the stimulation of gene expression of surface
adhesion molecules such as
5
1 integrin,
thus promoting interactions of immature cells with the marrow
microenvironment. In accordance with this hypothesis, synthesis and
surface expression of the
5
1 integrin are
stimulated by M-CSF during macrophage differentiation of bone marrow
precursor cells (32), and mature monocytes constitutively express an
abundance of this integrin (33), suggesting that appearance of this
integrin is an event accompanying macrophage differentiation. Indeed,
we found that blocking the endogenous TNF-
activity with
neutralizing anti-TNF-
antibodies failed to affect PMA- or
M-CSF-induced cell adhesion and maturation in human peripheral blood
monocytes,3 suggesting that
TNF-
acts at differentiation stages preceding the monocytic
maturation.
During macrophage differentiation, one of the major changes in gene
expression is the induction of matrix metalloproteinases (MMPs) such as
92- and 72-kDa gelatinases (MMP-9 and MMP-2, respectively) (15-17).
Production of these enzymes is thought to be critical for extravasation
and migration of monocytes and macrophages through the extracellular
matrix (18). We have shown in a separate report (30) that
differentiation-associated MMP-9 gene expression in HL-60
cells and in human peripheral blood monocytes requires FN-mediated cell
adhesion and spreading; signaling of the FN-induced MMP-9 gene expression is channeled through the
5
1 integrin receptor and apparently does
not involve the
4
1 receptor. Dependence of PMA-induced MMP-9 gene expression on FN/integrin-mediated
cell adhesion and spreading is further confirmed by our finding in this
study; treatment with neutralizing anti-TNF-
antibodies causes
down-regulation of
5
1 integrin gene
expression, resulting in substantial inhibition of cell adhesion and
spreading as well as of MMP-9 gene expression. The autocrine
regulation of TNF-
is not restricted to the MMP-9 gene
expression. In U937 cells, PMA-induced gene expression of interstitial
collagenase (MMP-1) is also significantly reduced by anti-TNF-
antibodies (34), suggesting that TNF-
is a key molecule in
controlling the critical matrix-degrading activities during macrophage
differentiation. The MMP whose expression escapes the control of
TNF-
is MMP-2, providing another example of dissociated regulation
of this MMP and other members of the MMP family. It is intriguing that
PMA induces MMP-2 gene expression in HL-60 (this study) and
U937 cells (16), although its promoter does not contain an AP-1 site
(12). Since we noted an 8-h lag phase for PMA-induced MMP-2
gene expression,3 it is possible that induction of MMP-2 is
not directly mediated by PMA but rather by factors induced by PMA.
In summary, we have shown in the present study that TNF- acts as a
feedback regulator of PMA-induced macrophage differentiation in HL-60
cells. The cytokine also plays a role in controlling the gene
expression of matrix-degrading proteinases such as MMP-9 during
PMA-induced macrophage differentiation. Our findings suggest that
during inflammatory responses, TNF-
may cooperate with other hematopoietic factors to promote maturation of myeloid progenitor cells
and their migration through the extracellular matrix by modulating the
gene expression of integrins, the key cell surface receptors for matrix
macromolecules.
![]() |
FOOTNOTES |
---|
* This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under Contract W-31-109-ENG-38.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.
To whom correspondence and requests for reprints should be
addressed: Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Ave., Argonne, IL 60439-4833. Tel.: 630-252-3819; Fax: 630-252-3853.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; FN, fibronectin; GM-CSF,
granulocyte-macrophage-colony stimulating factor; M-CSF,
macrophage-colony stimulating factor; MMP-2, 72-kDa type IV
collagenase/gelatinase; MMP-9, 92-kDa type IV collagenase/gelatinase; RT-PCR, reverse transcriptase-polymerase chain reaction; PMA, phorbol
12-myristate 13-acetate; PBS, phosphate-buffered saline; bp, base
pair(s); mAb, monoclonal antibody; nt, nucleotide.
2 A. Laouar, C. B. H. Chubb, F. R. Collart, and E. Huberman, manuscript in preparation.
3 B. Xie and E. Huberman, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|