Autocrine Regulation of Macrophage Differentiation and 92-kDa Gelatinase Production by Tumor Necrosis Factor-alpha via alpha 5beta 1 Integrin in HL-60 Cells*

Bei Xie, Amale Laouar, and Eliezer HubermanDagger

From the Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Tumor necrosis factor-alpha (TNF-alpha ) 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-alpha 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-alpha alone was only effective in inhibiting cell proliferation. Blocking the endogenous TNF-alpha activity with neutralizing anti-TNF-alpha 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-alpha 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 alpha 5beta 1 integrins, TNF-alpha alone had little effect on the expression of these genes. However, anti-TNF-alpha antibodies blocked PMA-induced augmentation of both alpha 5 and beta 1 integrin gene expression without affecting the expression of the FN gene. Our results suggest that TNF-alpha may regulate macrophage differentiation and critical matrix-degrading activities of myeloid progenitor cells in an autocrine manner by augmenting surface levels of the alpha 5beta 1 integrin, thus promoting interactions with the extracellular matrix, a key event for maturation and migration of these cells during inflammation.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Tumor necrosis factor-alpha (TNF-alpha ),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-alpha is known to be a feedback modulator of macrophage differentiation of myeloid progenitor cells. The effects of TNF-alpha 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-alpha 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-alpha exerts these effects in both an autocrine and paracrine manner. In addition, TNF-alpha 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-alpha -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-alpha gene is one of the early response genes induced by PMA during this process (23). In this study, we explored the role of TNF-alpha 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-alpha was as effective as PMA in inhibiting cell replication, it could not induce the other three differentiation markers. However, neutralizing anti-TNF-alpha antibodies inhibited all four PMA-induced macrophage markers, indicating that TNF-alpha is an autocrine factor critical for PMA-induced macrophage differentiation in HL-60 cells. In addition, treatment with anti-TNF-alpha antibodies abolished PMA-induced MMP-9 gene expression without affecting the PMA-induced expression of MMP-2, suggesting that TNF-alpha does not mediate all PMA-induced gene expression in HL-60 cells. We demonstrate herein that one of the mechanisms whereby TNF-alpha modulates PMA-induced macrophage differentiation and MMP-9 gene expression is through augmenting the gene expression of the surface adhesion molecule alpha 5beta 1 integrin. Our results suggest that TNF-alpha 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Phorbol 12-myristate 13-acetate (PMA) was purchased from Chemicals for Cancer Research (Eden Prairie, MN). Tumor necrosis factor-alpha (TNF-alpha ) (>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 beta 1 (K20, IgG2a) and alpha 5 (SAM1, IgG2b) integrin were obtained from Immunotech (Westbrook, ME). Both monoclonal (mouse) and polyclonal (goat) anti-TNF-alpha 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-alpha 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-alpha in the presence or absence of 10 µg/ml preimmune IgG or anti-TNF-alpha 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.

To examine the phagocytic activity, the cells (7 × 104 cells/well) were plated in eight-well Lab-Tak chamber slides (Nunc, Inc., Naperville, IL) and treated as described above. After treatment, the cells were incubated for 18 h with 2 × 106 sterilized and opsonized 1.72-µm (diameter) Fluoresbrite beads (Polysciences, Inc., Warrington, PA) (25). Thereafter, the medium was rapidly aspirated, and the cells fixed with 4% paraformaldehyde in PBS for 20 min prior to permeabilization for 20 min with 10 µg/ml L-alpha -lysophosphatidylcholine (Sigma). The cells were then stained for 10 min at room temperature with 0.1 µg/ml 4,6-diamidino-2-phenylindole (Boehringer Mannheim), followed by three washes in PBS and a 15-min incubation in hydroethidine working solution (Polysciences, Inc.). Following three final washes in PBS, the cells were mounted with phosphate-buffered gelatol (Becton Dickinson, Sunnyvale, CA) and analyzed by a Micro-Tome Mac Digital Confocal microscope system (VayTek, Inc., Fairfield, IA) attached to a Leitz Orthoplan fluorescence microscope. The cells were considered positive if they engulfed >= 20 beads/cell.

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-alpha 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-alpha , and GAPDH were obtained from American Type Culture Collection (Rockville, MD), and those for alpha 5 and beta 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Autocrine Regulation of PMA-induced Macrophage Differentiation by TNF-alpha -- To assess the role TNF-alpha 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-alpha -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-alpha activities by neutralizing anti-TNF-alpha 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-alpha loop.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Manifestation of differentiation markers in HL-60 cells treated for 24 h with 10 ng/ml TNF-alpha or 3 nM PMA in the presence or absence of 10 µg/ml anti-TNF-alpha antibodies or preimmune IgG
The results shown represent the mean ± S.D. of 3-6 independent experiments performed.


View larger version (179K):
[in this window]
[in a new window]
 
Fig. 1.   Inhibitory effect of anti-TNF-alpha antibodies on PMA-induced cell adhesion and spreading in HL-60 cells. The cells (2 × 105 cells/ml) were seeded in plastic tissue culture dishes and treated with 10 ng/ml TNF-alpha or 3 nM PMA for 24 h in the absence or presence of 10 µg/ml neutralizing anti-TNF-alpha antibodies. a, untreated cells; b, TNF-alpha ; c, PMA; d, PMA + anti-TNF-alpha . × 320. Note that TNF-alpha reduced cell number but did not affect cell morphology. Treatment with anti-TNF-alpha antibodies abolished PMA-induced cell adhesion and spreading and resumed cell proliferation inhibited by PMA.

To confirm further the role of TNF-alpha in PMA-induced HL-60 differentiation, we analyzed three additional differentiation markers; the results are summarized in Table I. TNF-alpha at 10 ng/ml was as potent as PMA at 3 nM in inhibiting HL-60 cell proliferation at 24 h after treatment. Neutralizing anti-TNF-alpha antibodies (10 µg/ml) were able to largely reverse both the PMA- and the TNF-alpha -induced growth inhibition. In addition, treatment with PMA resulted in approximately 85% HL-60 cells exhibiting reactivity with the OKM1 mMAb, which is characteristic of mature human monocytes, macrophages, and granulocytes (29). Addition of anti-TNF-alpha antibodies, but not the IgG control, to PMA-treated cells reduced cell reactivity with the OKM1 mAb to 5% or less, which is similar to the basal level observed in untreated or TNF-alpha -treated cells. Similarly, anti-TNF-alpha antibodies caused a more than 70% reduction in PMA-induced phagocytic activity, a primary function of mature macrophages, whereas exogenous TNF-alpha had no effect compared with the untreated cells. Based on these results, we conclude that TNF-alpha is an autocrine regulator of PMA-induced macrophage differentiation in HL-60 cells but in itself it is insufficient to induce a macrophage phenotype. Thus, additional factor(s), which have yet to be identified, are required to cooperate with TNF-alpha to generate sufficient signals for macrophage differentiation in HL-60 cells.

Dissociated Regulation of PMA-induced Gene Expression of 92- and 72-kDa Gelatinases by TNF-alpha -- 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-alpha (10 ng/ml) only weakly induced MMP-2 and MMP-9 gene expression as well as MMP-9 proenzyme secretion. Anti-TNF-alpha 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-alpha autocrine loop, PMA-induced MMP-2 gene expression is independent of the endogenous TNF-alpha activity.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of anti-TNF-alpha antibodies on PMA-induced MMP-2 and MMP-9 gene expression in HL-60 cells. The cells (4 × 105 cells/ml) were either untreated (Control) or treated with 10 ng/ml TNF-alpha or 3 nM PMA in the absence or presence of 10 µg/ml preimmune IgG or anti-TNF-alpha antibodies in serum-supplemented RPMI 1640 medium. At 24 h, cells were harvested for RNA isolation. RNA samples (20 µg/lane) were resolved on a 1.2% agarose gel containing 2.2 M formaldehyde as described under "Experimental Procedures." The blots were hybridized to radiolabeled cDNAs for MMP-2 (A) or MMP-9 (B) and autoradiographed for 3 days or 6 h, respectively. Hybridization to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to demonstrate equal loading of the RNA samples. C, cells were treated as above. After incubation for 24 h, cells were replaced with serum-free medium and incubated for additional 24 h prior to collection of conditioned media for analysis of gelatinolytic activity of MMP-9 by gelatin zymogram as described under "Experimental Procedures." The gelatinolytic activity of MMP-9 proenzyme is visualized as a clear band against a dark background.

TNF-alpha Autocrine Loop Is Essential but Not Sufficient for PMA-induced Gene Expression of alpha 5beta 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-alpha antibodies blocked this process, we suspected that TNF-alpha 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 alpha 5beta 1 integrin genes in PMA- and TNF-alpha -treated HL-60 cells, and we determined the effect of anti-TNF-alpha antibodies on these events. We did not include the alpha 4beta 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 alpha 5 and beta 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 beta 1 integrin mRNA by 4-fold and alpha 5 integrin mRNA by 6-fold (Fig. 3B), with concomitant increases in surface expression of the respective protein (Fig. 4). TNF-alpha alone failed to affect expression of FN or alpha 5 or beta 1 integrin gene. Blocking the endogenous TNF-alpha activity with anti-TNF-alpha 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-alpha loop nor cell adhesion and spreading. In contrast, treatment with anti-TNF-alpha antibodies resulted in a substantial reduction in PMA-induced beta 1 and alpha 5 gene expression (Fig. 3B and Fig. 4). Accordingly, surface levels of both beta 1 and alpha 5 proteins stimulated by PMA were substantially inhibited by anti-TNF-alpha antibodies (Fig. 4). Taken together, our results have shown that TNF-alpha is an autocrine mediator for PMA-induced augmentation of alpha 5beta 1 integrin gene expression, but TNF-alpha alone is insufficient and necessitates additional factor(s) to stimulate alpha 5beta 1 gene expression. Our findings were further supported by the temporal sequence of expression of the FN, TNF-alpha , and alpha 5beta 1 integrin genes induced by PMA. As shown in Fig. 5A, PMA induced an early and transient expression of the TNF-alpha gene. Peak induction of the TNF-alpha steady state mRNA was observed at 2 h after PMA treatment, which was maintained for up to 4 h. The TNF-alpha steady state mRNA levels then dropped markedly at 8 h and became nearly undetectable at 24 h after treatment. The induction of TNF-alpha gene expression was followed by augmented expression of the beta 1 and alpha 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-alpha , which is consistent with our conclusion that PMA-induced FN expression is independent of the autocrine TNF-alpha activities.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of anti-TNF-alpha antibodies on alpha 5beta 1 integrin and fibronectin gene expression in PMA- or TNF-alpha -treated HL-60 cells. The cells were either untreated (Control) or treated for 24 h with 10 ng/ml TNF-alpha or 3 nM PMA in the absence or presence of 10 µg/ml preimmune IgG or anti-TNF-alpha antibodies. After incubation, the cells were harvested for RNA isolation. A, FN mRNA levels were analyzed by RT-PCR as described under "Experimental Procedures." A single 365-bp fragment was detected. Amplification of GAPDH mRNA was used as a quantitative control for RNA samples and amplification efficacy. Various amounts of RNA samples and the RT reaction were used to ensure that the amount of the amplification product corresponded to that of RNA samples used. The results were reproducible with RNA samples isolated from three independent treatments. B, RNA samples (20 µg/lane) were resolved on a 1.2% agarose gel containing 2.2 M formaldehyde and subject to Northern blot analysis as described. The blot was hybridized sequentially to radiolabeled cDNAs for beta 1 and alpha 5 integrins. GAPDH hybridization was used to demonstrate the equal loading of RNA samples.


View larger version (98K):
[in this window]
[in a new window]
 
Fig. 4.   Immunofluorescent staining of cell surface alpha 5beta 1 integrin and fibronectin in HL-60 cells treated with PMA in the presence or absence of anti-TNF-alpha antibodies. The cells were either untreated (Control) or treated for 24 h with 3 nM PMA in the absence or presence of 10 µg/ml anti-TNF-alpha antibodies. After incubation, the cells were reacted with mAb to FN, beta 1 or alpha 5 integrin at 1:30 dilution without fixation and permeabilization for 2 h at 25 °C, then washed with PBS and reacted with goat anti-mouse IgG fluorescein isothiocyanate conjugate. Immunofluorescence was examined under a digital confocal microscope. Note that surface levels of both beta 1 and alpha 5 integrins are distinctly enhanced by PMA treatment and nearly diminished to the basal level in the presence of anti-TNF-alpha antibodies, whereas FN staining remained unaffected by the anti-TNF-alpha antibodies.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Time-course study of steady state mRNA levels of TNF-alpha , alpha 5beta 1 integrin, and fibronectin in PMA-treated HL-60 cells. The cells were either untreated (C) or treated with 3 nM PMA for the various times as indicated. A, Northern blot analysis of total RNA samples (20 µg/lane) for TNF-alpha , beta 1 and alpha 5 integrins. GAPDH hybridization was used to demonstrate equal loading of RNA samples. B, RT-PCR analysis of mRNA levels of FN and GAPDH. Various amounts of RNA samples and the RT reaction were used to ensure that the levels of the amplification product corresponded to the input of RNA samples.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we have shown that a TNF-alpha 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-alpha as a competence factor which primes the progenitor cells at early stages of macrophage differentiation. TNF-alpha 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-alpha 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-alpha 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-alpha 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-alpha , since blocking TNF-alpha 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-alpha ; stem cells pretreated with TNF-alpha for up to 3 days responded to the differentiating effects of GM-CSF, and after 5 days of TNF-alpha pretreatment, GM-CSF was unable to promote maturation. Although the cellular events triggered by TNF-alpha 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 alpha 5beta 1 integrin, thus promoting interactions of immature cells with the marrow microenvironment. In accordance with this hypothesis, synthesis and surface expression of the alpha 5beta 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-alpha activity with neutralizing anti-TNF-alpha antibodies failed to affect PMA- or M-CSF-induced cell adhesion and maturation in human peripheral blood monocytes,3 suggesting that TNF-alpha 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 alpha 5beta 1 integrin receptor and apparently does not involve the alpha 4beta 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-alpha antibodies causes down-regulation of alpha 5beta 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-alpha 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-alpha antibodies (34), suggesting that TNF-alpha is a key molecule in controlling the critical matrix-degrading activities during macrophage differentiation. The MMP whose expression escapes the control of TNF-alpha 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-alpha 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-alpha 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.

Dagger 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-alpha , tumor necrosis factor-alpha ; 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Tracey, K. J., and Cerami, A. (1993) Annu. Rev. Cell Biol. 9, 317-343[CrossRef]
  2. Branch, D. R., Turner, A. R., and Guilbert, L. J. (1989) Blood 73, 307-311[Abstract]
  3. Rusten, L. A., Smeland, E. B., Jacobsen, F. W., Lien, E., Lesslauer, W., Loetscher, H., Dubois, C. M., and Jacobsen, S. E. (1994) J. Clin. Invest. 94, 165-172[Medline] [Order article via Infotrieve]
  4. Witsell, A. L., and Schook, L. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4754-4758[Abstract]
  5. Santiago-Schwarz, F., Divaris, N., Kay, C., and Carsons, S. E. (1993) Blood 82, 3019-3028[Abstract]
  6. Takeda, K., Iwamoto, S., Sugimoto, H., Takuma, T., Kawatani, N., Noda, M., Masaki, A., Morise, H., Arimura, H., and Konno, K. (1986) Nature 323, 338-340[Medline] [Order article via Infotrieve]
  7. Stetler-Stevenson, W. G. (1990) Cancer Metastasis Rev. 9, 289-303[Medline] [Order article via Infotrieve]
  8. Hibbs, M. S., Hoidal, J. R., and Kang, A. H. (1987) J. Clin. Invest. 80, 1644-1650[Medline] [Order article via Infotrieve]
  9. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He, C. S., Bauer, E. A., and Goldberg, G. I. (1988) J. Biol. Chem. 263, 6579-6587[Abstract/Free Full Text]
  10. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I. (1989) J. Biol. Chem. 264, 17213-17221[Abstract/Free Full Text]
  11. Murphy, G., Ward, R., Hembery, R. M., Reynolds, J. J., Kuhn, K., and Tryggvason, K. (1989) Biochem. J. 258, 463-472[Medline] [Order article via Infotrieve]
  12. Huhtala, P., Chow, L. T., and Tryggvason, K. (1990) J. Biol. Chem. 265, 11077-11082[Abstract/Free Full Text]
  13. Huhtala, P., Tuuttila, A., Chow, L. T., Lohi, J., Keski-Oja, J., and Tryggvason, K. (1991) J. Biol. Chem. 266, 16485-16490[Abstract/Free Full Text]
  14. Sato, H., and Seiki, M. (1993) Oncogene 8, 395-405[Medline] [Order article via Infotrieve]
  15. Campbell, E. J., Cury, J. D., Shapiro, S. D., Goldberg, G. I., and Welgus, H. G. (1991) J. Immunol. 146, 1286-1293[Abstract/Free Full Text]
  16. Welgus, H. G., Campbell, E. J., Cury, J. D., Eisen, A. Z., Senior, R. M., Wilhelm, S. M., and Goldberg, G. I. (1990) J. Clin. Invest. 86, 1496-1502[Medline] [Order article via Infotrieve]
  17. Watanabe, H., Nakanishi, I., Yamashita, K., Hayakawa, T., and Okada, Y. (1993) J. Cell Sci. 104, 991-999[Abstract/Free Full Text]
  18. Saren, P., Welgus, H. G., and Kovanen, P. T. (1996) J. Immunol. 157, 4159-4165[Abstract]
  19. Collins, S. J. (1989) Blood 70, 1233-1244[Abstract]
  20. Huberman, E., and Callaham, M. F. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1293-1297[Abstract]
  21. Rovera, G., Santoli, D., and Damsky, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2779-2783[Abstract]
  22. Lotem, J., and Sachs, L. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5158-5162[Abstract]
  23. Horiguchi, J., Spriggs, D., Imamura, K., Stone, R., Leubbers, R., and Kufe, D. (1989) Mol. Cell. Biol. 9, 252-258[Medline] [Order article via Infotrieve]
  24. Bauvois, B., Rouillard, D., Sanceau, J., and Wietzerbin, J. (1992) J. Immunol. 148, 3912-3919[Abstract/Free Full Text]
  25. Blair, O. C., Carbone, R., and Santorelli, A. C. (1986) Cytometry 7, 171-177[Medline] [Order article via Infotrieve]
  26. Morodomi, T., Ogata, Y., Sasaguri, Y., Morimatsu, M., and Nagase, H. (1992) Biochem. J. 285, 603-611[Medline] [Order article via Infotrieve]
  27. Chirgwin, J. M., Przybyla, A. E., MacDonald, A. E., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299[Medline] [Order article via Infotrieve]
  28. Glesne, D. A., Collart, F. R., and Huberman, E. (1991) Mol. Cell. Biol. 11, 5417-5425[Medline] [Order article via Infotrieve]
  29. Foon, K. A., Schroff, R. W., and Gale, R. P. (1982) Blood 60, 1-19[Medline] [Order article via Infotrieve]
  30. Xie, B., Laouar, A., and Huberman, E. (1998) J. Biol. Chem. 273, 11576-11582[Abstract/Free Full Text]
  31. Witsell, A. L., and Schook, L. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1963-1967[Abstract]
  32. Shima, M., Teitelbaum, S. L., Holers, V. M., Ruzicka, C., Osmack, P., and Ross, F. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5179-5183[Abstract]
  33. Hemler, M. E. (1990) Annu. Rev. Immunol. 8, 365-400[CrossRef][Medline] [Order article via Infotrieve]
  34. Callaghan, M. M., Loris, R. M., Rammohan, C., Lu, Y., and Pope, R. M. (1996) J. Leukocyte Biol. 59, 125-132[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.