STAT1 activation during monocyte to macrophage maturation: role of adhesion molecules

Eliana M. Coccia1,2, Nicoletta Del Russo1, Emilia Stellacci1, Ugo Testa3, Giovanna Marziali1 and Angela Battistini1

1 Laboratory of Virology,
2 Laboratory of Immunology, and
3 Laboratory of Hematology and Oncology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

Correspondence to: A. Battistini and E. Coccia


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
Human monocytes isolated from peripheral blood of healthy donors show a time-dependent differentiation into macrophages upon in vitro cultivation, closely mimicking their in vivo migration and maturation into extravascular tissues. The mediator(s) of this maturation process has not been yet defined. We investigated the involvement of signal transducers and activators of transcription (STAT) factors in this phenomenon and reported the specific, time-dependent, activation of STAT1 protein starting at day 0/1 of cultivation and maximally expressed at day 5. STAT1 activity was evident on the STAT binding sequences (SBE) present in the promoters of genes which are up-regulated during monocyte to macrophage maturation such as Fc{gamma}RI and ICAM-1, and in the promoter of the transcription factor IFN regulatory factor-1. Moreover, the effect of cell adhesion to fibronectin or laminin was studied to investigate mechanisms involved in STAT1 activation. Compared with monocytes adherent on plastic surfaces, freshly isolated cells allowed to adhere either to fibronectin- or laminin-coated flasks exhibited an increased STAT1 binding activity both in control and in IFN-{gamma}-treated cells. The molecular events leading to enhanced STAT1 activation and cytokine responsiveness concerned both Y701 and S727 STAT1 phosphorylation. Exogenous addition of transforming growth factor-ß, which exerts an inhibitory effect on some monocytic differentiation markers, inhibited macrophage maturation, integrin expression and STAT1 binding activity. Taken together these results indicate that STAT1 plays a pivotal role in the differentiation/maturation process of monocytes as an early transcription factor initially activated by adherence and then able to modulate the expression of functional genes, such as ICAM-1 and Fc{gamma}RI.

Keywords: cellular differentiation, signal transduction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
The STAT proteins (signal transducers and activators of transcription) are latent DNA binding factors, originally discovered in studies on transcriptional activation by IFNs, but suddenly recognized as a unifying paradigm in the signal transduction pathway of cytokines and growth factors. Binding of the ligand to its specific receptor triggers the phosphorylation of STAT proteins on specific tyrosine residues by members of the Janus kinase (JAK) family of protein tyrosine kinases (1). A maximal induction of different STAT proteins through the phosphorylation in serine residues has also been described (2). Activated STAT proteins homo- or hetero-dimerize, translocate into the nucleus where they bind to specific response elements on target genes and stimulate transcription. Thus far seven members of the STAT family are known to take part in the signaling pathways of specific cytokines (17). With the exception of STAT2, all STAT proteins bind with different affinities to >10 related sequences originally termed the IFN-{gamma} activated site (GAS), a regulatory element present in the promoter of IFN-{gamma}-inducible genes (6) and more recently referred to as SBE (STAT binding element). Although there is evidence that a number of genes are regulated by STAT proteins (18), the exact role of STAT proteins in cell differentiation and cell growth regulation remains to be elucidated. Recently, the first direct evidence that STAT5 and STAT3 play a key role in the regulation of cell proliferation and terminal differentiation of myeloid cells has been reported (912).

Human blood monocytes undergo differentiation in macrophages upon migration into extravascular tissue (13). This phenomenon can be well mimicked in cell culture; in fact, upon in vitro cultivation, monocytes adhere to the plastic surface and in a few days undergo a spontaneous, time-dependent differentiation process (14). During this process, the optimal expression of specific cell surface antigens characteristic of terminal maturation, such as transferrin receptor (CD71), leukocyte-ß2 integrin subfamily (CD11/CD18) and LPS receptor (CD14), is observed as well as a change in the secretory repertoire of these cells (13,14). Cell adherence of human monocytes to plastic culture tissue dishes results also in a rapid induction of multiple inflammatory mediator genes (15,16), while adherence to dishes coated with extracellular matrix components, such as fibronectin, laminin or collagen, results in a relatively selective pattern of gene induction (1720). The proteins that mediate many of the adhesive reactions of monocytes, such as transendothelial migration and cell adhesive interactions with the extracellular matrix, belong to the integrin family of cell surface receptors. It has recently been shown that binding of adhesive ligands to integrins also transduces signal into the cells through tyrosine phosphorylation (21).

In the present study we report that in the absence of growth factors and exogenously added cytokines, under strictly controlled endotoxin-free conditions, freshly isolated monocytes do not show any STAT activity. Starting at day 1 and progressively increasing until day 5–7 of cultivation, a predominant STAT1 activity appears. STAT1 binding activity is detected on the SBE sequences present in the promoters of some genes encoding markers of macrophage differentiation, such as Fc{gamma}RI and ICAM-1, and in the promoter of the transcription factor IFN regulatory factor (IRF-1). Interestingly, we demonstrate that monocytes allowed to adhere to fibronectin- and laminin-coated plates exhibit a prompt activation of STAT1 binding mediated by both tyrosine and serine phosphorylation. Moreover, exogenous addition of transforming growth factor (TGF)-ß, a multifunctional cytokine able to affect the spontaneous differentiation of monocytes (22), inhibits both integrin expression on the cell membrane and STAT1 binding activity. These results suggest that STAT1 may be an early transcription factor activated by adhesion molecules during the monocyte maturation process upon migration into extravascular tissues; this activation then regulates the transcription of genes specific of mature macrophage.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
Isolation and culture of monocytes/macrophages
Peripheral blood mononuclear cells were obtained from 18- to 40-year-old healthy donors by a three-step density centrifugation procedure on Ficoll and Percoll gradients (Pharmacia, Uppsala, Sweden). The light density fraction from the 42.5–50% interface was recovered, and depleted of CD19+ B and CD2+ T lymphocytes using magnetic beads (Dynal, Oslo, Norway). The recovered population routinely contained >97% CD14+ cells. Macrophages were obtained after 5–7 days of in vitro cultivation in medium alone. Only monocyte preparations containing >90% CD14+ cells were used. Substratum-coated cell culture dishes were prepared as previously reported (21). Briefly, tissue culture dishes were incubated overnight with 50 µg/ml fibronectin or laminin (Sigma, St Louis, MO) at 4°C; the dishes were then blocked with cell culture grade 0.1% BSA (Boehringer-Mannheim, Mannheim, Germany) and washed with PBS before use. Where indicated, monocytes were treated with 1 µg/ml of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma), 10 ng/ml of human recombinant IFN-{gamma}, 10 ng/ml of macrophage inflammatory protein (MIP)-1{alpha}, 100 ng/ml of tumor necrosis factor (TNF)-{alpha}, 5 ng/ml of TGF-ß or 10 ng/ml IL-10 (PrepoTech, Rocky Hill, NJ; endotoxin <0.1 ng/µg); 10 ng/ml of human recombinant IL-6 and granulocyte macrophage colony stimulating factor (GM-CSF; Genetics Institute, Cambridge, MA); and 1000 IU/ml of human recombinant IFN-{alpha} (IFN-{alpha}2ß, Intron A; Shering, Bloomfield, NJ). Anti-IL-6 and anti-IL-10 neutralizing antibodies were purchased from R & D Systems/British Biotechnology (Minneapolis, MN).

DNA electrophoretic mobility shift assay (EMSA)
Synthetic double-stranded oligonucleotides, prepared by a DNA synthesizer (Applied Biosystems, Foster City, CA), were end-labeled with [{gamma}- 32P]ATP by T4 polynucleotide kinase. Whole-cell extracts (10 µg) were prepared and analyzed by EMSA as described (23). For supershift analysis, whole-cell extracts (5 µg) were incubated with 3 µg of anti-STAT1, anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) or 1 µg of anti-STAT5 antibody (10). The oligonucleotide probes used were: human IRF-1 SBE (5'-GATCCATTTCCCCGAAAT GA3'), Fc{gamma}RI SBE (5'-TTCCTTTTCTGGGAAATACATCTC-3') and ICAM-1 SBE (5'-CGGGATCCGTTTCCCGGAAAGCAGCA-3').

Western blot assay
Either 30 or 70 µg of whole-cell extracts was denatured and separated on 10% SDS–PAGE. Proteins were transferred onto nitrocellulose paper, incubated with monoclonal anti-STAT1 antibodies (Signal Transduction Laboratory, Lexington, KY) and reacted with anti-rabbit horseradish peroxidase-coupled secondary antibody (Amersham, Little Chalfont, UK) using the enhanced chemiluminescence system. Rabbit antiserum specific for tyrosine- and serine-phosphorylated STAT1 isoforms were purchased from UBI (New York, NY) and used according to the manufacturer's instructions.

RNase protection experiments
Total RNA was isolated by the guanidium–cesium chloride method and analyzed as previously described (23). To obtain the pBS IRF-1 construct, the plasmid pUC IRF-1 was digested with SmaI and the 400 bp long fragment was cloned into the same sites of pBluescript/KS (Stratagene, La Jolla, CA). To generate the 32P-labeled 280 bp long antisense IRF-1 RNA probe, the plasmid (pBS IRF-1) was linearized with EcoRI and transcribed by T7 polymerase. The Fc{gamma}RI probe was obtained cloning a 267 bp long fragment excised by XbaI digestion from the plasmid p135 (24) into the same site of pBluescript/KS (Stratagene). The Fc{gamma}RI riboprobe was obtained by transcribing with T7 polymerase the plasmid linearized with PstI. The 434 bp long pTRI-GAPDH human antisense control template (Ambion, Austin, TX) was used as an internal standard to establish the relative amount of RNA loaded. The probe was synthesized by in vitro transcription from linear template using SP6 polymerase.

Flow cytometric analysis of surface antigens on monocytes/macrophages
Cells were incubated for 1 h at 4°C in the presence of an appropriate dilution of one of the following mAb: phycoerythrin (PE)-conjugated anti-CD54 (Becton Dickinson, Mountain View, CA), FITC-conjugated anti-CD64, FITC-conjugated anti-CD32 (Medarex, East Annandale, NJ); PE-conjugated anti-CD14, and PE-conjugated anti-VLA-4, anti-VLA-5 and anti-VLA-6 (PharMingen/Becton Dickinson, Bedford, CA). For negative controls the cells were incubated with mouse IgG of the same isotype labeled either with PE or FITC as previously described (14). Fluorescence data were analyzed by the Lysys II software program (Becton Dickinson), and the data are expressed in terms of the percentage of positive cells and of fluorescence intensity evaluated as relative fluorescence intensity (calculated as the difference of fluorescence intensity observed for cells incubated with each mAb minus the fluorescence intensity of the cells incubated with the appropriate negative control).

Cytokine assays (ELISA)
The concentration of cytokines (IFN-{gamma}, IL-6, GM-CSF and IL-10) in monocyte cell culture supernatants was evaluated by sensitive and specific immunoassays (R & D Systems/British Biotechnology). The detection threshold was 5 pg/ml for all four cytokines.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
Activation of STAT proteins during spontaneous in vitro monocyte/macrophage differentiation
Monocytes, isolated by centrifugation over Ficoll-Hypaque and further separated on a Percoll gradient, were seeded and proteins extracted after 1 and 5 days. The presence of activated STAT proteins was evaluated by EMSA using specific oligonucleotides corresponding to specific SBE sequences present in the promoters of IRF-1, ICAM-1 and Fc{gamma}RI (Fig. 1AGo). Freshly isolated monocytes (day 0/1) display very low binding activity for all these sequences, whereas at day 5 discrete complexes are formed between proteins present in cell extracts and DNA probes. Monocytes isolated by density gradient centrifugation exhibited an identical pattern of binding to the indicated SBE sequences (data not shown).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. STAT(s) binding activities to SBE sequences in monocytes/macrophages. (A) Whole-cell extracts were prepared from monocytes at day 1 and 5 of culture. Proteins (10 µg) were subjected to EMSA analysis with the indicated SBE sequences, described in Methods. (B) Supershift assays were performed, where indicated, after incubation of whole-cell extracts (5 µg) with anti-STAT(s)-specific antibodies. As control, the specific induction of STAT1 monocytes treated for 20 min with 10 ng/ml of IFN-{gamma} was shown. (C) Whole-cell extracts were prepared from monocytes unstimulated (–) or stimulated with the indicated cytokines for 20 min. Proteins (10 µg) were incubated with radiolabeled oligonucleotide containing the SBE consensus binding site present in the IRF-1 promoter.

 
To identify which STAT are activated during the spontaneous monocyte maturation, supershift experiments using specific STAT1, STAT3 and STAT5 antibodies were carried out by EMSA using IRF-1 SBE oligonucleotide. As positive control, whole-cell extracts were prepared from monocytes treated for 20 min with IFN-{gamma}, which is known to activate STAT1. As shown in Fig. 1Go(B), antibodies reacting with STAT1 protein were able to supershift the major DNA–protein complex both during monocyte to macrophage maturation and after IFN-{gamma} treatment. Conversely, the same complex was not affected by the addition of anti-STAT3- and anti-STAT5-specific antibodies.

Since macrophages are able to secrete a large array of cytokines and chemokines, we evaluated the rapid activation of specific STAT proteins after short-time treatment of freshly isolated monocytes with the indicated cytokines (Fig. 1CGo). EMSA experiments were performed using double-stranded radiolabeled IRF-1 SBE oligonucleotide. Moreover, we also evaluated the effects of LPS and IFN-{gamma}, both potent activators of monocyte/macrophages. As shown in Fig. 1Go(C), IFN-{gamma} and IFN-{alpha} rapidly activated a major band corresponding to STAT1 homodimers as already reported (25,26). IL-6, IL-10 and GM-CSF similarly induced a band with the same mobility but with a much lower intensity. Conversely, LPS, MIP-1{alpha} and TNF-{alpha} treatments were not effective in increasing the basal level of SBE binding activity. These results indicate that freshly isolated monocytes are able to respond to several cytokines with a rapid stimulation of STAT(s) activity.

To verify if cytokines present and/or produced in the cultures could be responsible for the STAT(s) progressive activation observed during in vitro monocyte maturation (Fig. 1AGo), the levels of the cytokines in the culture media were quantified using an ELISA assay at different days of culture. Results shown in Table 1Go indicate that the amounts of IFN-{gamma} and GM-CSF, detected at different days of culture, are in the range of few picograms, i.e. at the lowest limit of the test sensitivity. On the contrary, IL-6 and IL-10 are present at all days of culture at more elevated levels, i.e. 50–70 pg/ml and 0.5–1 ng/ml respectively. In the attempt to evaluate the involvement of these two cytokines in the progressive activation of STAT(s), anti-IL-6 and anti-IL-10 neutralizing antibodies were added to the cell cultures alone or in combination. Similarly, to exclude that the activation of STAT(s) observed in our system could be due to the presence of type I IFN, freshly isolated monocytes were seeded in the presence of polyclonal antibodies neutralizing human IFN-{alpha} and -ß (27), and cell extracts were analyzed by EMSA. The neutralization of these cytokines was not effective in inhibiting the spontaneous activation of STAT(s) observed at day 5 of culture (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1. Concentration of cytokines in monocyte culture mediuma
 
Effect of adhesion on STAT1 activation
In order to identify the mechanisms involved in STAT1 activation during monocyte maturation, the effect of cell adhesion on the phosphorylation state and binding activity of STAT1 was evaluated. Cells were seeded on either fibronectin- or laminin-coated dishes for 12 h and, where indicated, cells were also treated with IFN-{gamma} for 30 min. EMSA was performed on whole-cell extracts using the IRF-1-SBE oligonucleotide. As shown in Fig. 2Go(A), adherence to fibronectin and laminin resulted in a slight but consistent increase in STAT1 binding activity; when monocytes cultivated on fibronectin- or laminin-coated dishes were exposed to IFN-{gamma}, there was a 2-fold increase in STAT1 binding activity over the levels observed in cells treated with IFN-{gamma} alone. Shorter times of incubation with fibronectin or laminin were not sufficient to induce STAT1 binding activity (data not shown). Since phosphorylation on both serine and tyrosine residues is responsible for full STAT1 activity (28), we investigated whether these two post-translational modifications could account for the activation of STAT1 binding activity observed in monocytes grown on fibronectin- or laminin-coated plates. The experiments shown in Fig. 2Go(B) demonstrate a clear phosphorylation of STAT1 at the level of both tyrosine (Y701) and serine (S727) in response to the monocyte interaction with laminin and fibronectin. As expected, in both cell culture conditions the IFN-{gamma}-mediated S727 and Y701 STAT1 phosphorylation was more pronounced as compared to the levels observed in cells treated with IFN-{gamma} alone and allowed to adhere to uncoated plastic dishes. Interestingly, the analysis of STAT1 content on cell extracts from freshly isolated monocytes and from mature macrophages showed a clear increase (3-fold) in STAT1 protein content at day 5 of culture as compared to that observed at day 1 (Fig. 2BGo, bottom panel).




View larger version (45K):
[in this window]
[in a new window]
 
Fig. 2. Effects of adhesion molecules on STAT1 activation. (A) Whole-cell extracts were prepared from monocytes allowed to adhere to either untreated or laminin- and fibronectin-coated tissue culture dishes for 12 h. Where indicated, the cells were treated with 10 ng/ml of IFN-{gamma} for another 20 min. Whole-cell extracts (10 µg) were subjected to EMSA with IRF-1 SBE oligonucleotide. (B) Western blot analysis of phosphorylation status and protein content of STAT1. Whole-cell extracts (30 µg) obtained from cell conditions described in (A) were analyzed for the relative amount of tyrosine-phosphorylated STAT1 by staining with anti-pY701-STAT1 antiserum. To detect the p701-STAT1, a higher amount of proteins (70 µg) was used for extracts from control (–), and laminin- and fibronectin-treated cells. The same extracts (30 µg) were also stained with anti-pS727-STAT1 antibody which detects only the slower migrating STAT1 isoform of 91 kDa. STAT1 content was evaluated incubating the blot with anti-STAT1 antibodies.

 
STAT1 activation during the monocyte to macrophage maturation correlates with the increased expression of a specific set of STAT1-target genes

The expression of maturation-associated genes such as Fc{gamma}RI (CD64) and ICAM-1 (CD54) containing a SBE on their promoters was then evaluated during in vitro maturation of monocytes to macrophages. In addition, the expression of another gene regulated by SBE sequences, i.e. IRF-1, a transcription factor important for the induction of genes expressed in activated macrophages, such as MHC I, iNOS and IFN-ß (2931), was investigated. Total RNA was extracted from monocytes/macrophages at day 1 and at day 5 of culture respectively, and analyzed by RNase protection with specific riboprobes for Fc{gamma}RI and IRF-1 (Fig. 3A and BGo). An increase in both IRF-1 and Fc{gamma}RI mRNAs was observed at day 5 with respect to day 1 of culture. The protein level on the cell membrane of Fc{gamma}RI (CD64) and of another marker of monocyte maturation, such as ICAM-1 antigen (CD54), was then examined by flow cytometric assay (Fig. 3CGo). The percentage of cells reacting with anti-CD64 and anti-CD54 remained virtually unmodified during monocyte to macrophage in vitro maturation, whereas a progressive and significant increase in the fluorescence intensity was observed at day 5. As control, the stimulation of the surface expression of these two antigens was also determined after addition of both IFN-{gamma} and GM-CSF. IFN-{gamma} greatly stimulated fluorescence intensity of both antigens, as expected, whereas GM-CSF did not further increase the ICAM-1 and Fc{gamma}RI expression.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Spontaneous STAT1 activation correlates with increased expression of IRF-1 and macrophage-associated antigens. (A and B) Total RNA (10 µg) was extracted at day 1 and 5 of culture, and analyzed by RNase protection assay with specific riboprobes for IRF-1, Fc{gamma}RI and GAPDH mRNAs as described in Methods. (C) Expression of Fc{gamma}RI (CD64) and ICAM-1 (CD54) on the cell membrane. Monocytes were grown either under standard conditions (control) or in the presence of GM-CSF or IFN-{gamma} and processed for membrane fluorescence.

 
Furthermore, the effect of interactions between integrin receptors and their ligands on Fc{gamma}RI and ICAM-1 expression was evaluated by flow cytometry experiments. The results obtained (data not shown) indicate that both fibronectin and laminin elicited a moderate, but significant, enhancement of Fc{gamma}RI and ICAM-1 expression at day 1 and 2 of culture compared to the expression of these markers on cells cultured on uncoated plastic dishes.


    Effect of TGF-ß on monocyte maturation and STAT1 activity
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
TGF-ß, among its pleiotropic effects on a variety of cell types, exerts both immunosuppressive and proinflammatory properties (22). In this respect it is able to inhibit the expression of some monocyte differentiation markers such as CD14, CD16 and CD89 (32,33). We thus tested if the expression of the STAT1-dependent markers, i.e. ICAM-1 and Fc{gamma}RI, was also inhibited in monocytes by the TGF-ß treatment. In monocytes grown for 5 days in the presence of TGF-ß, the fluorescence intensity, as assessed by flow cytometric analysis (Fig. 4AGo) of both Fc{gamma}RI and ICAM-1 antigens, was markedly decreased as compared to that observed in control culture. On the contrary, the fluorescence intensity of the Fc{gamma}RII was not modulated both during monocyte to macrophage differentiation, as well as in TGF-ß-treated cells, indicating a specific effect of the cytokine for macrophage differentiation markers. In Fig. 4Go (right side of panel A), a representative experiment of Fc{gamma}RI immunodetection in monocytes grown for 5 days either in the absence or in the presence of 5 ng/ml of TGF-ß is shown. This inhibition correlates with an impairment in the STAT1 binding to the SBE sequence present in the Fc{gamma}RI, ICAM-1 and IRF-1 promoters in cell extracts derived from TGF-ß-treated monocytes for 5 days compared with those obtained in control cultures (Fig. 4BGo). The analysis of STAT1 content by Western blot on cell extracts from monocytes treated or not with TGF-ß for 5 days clearly showed that TGF-ß treatment also decreases STAT1 protein expression (data not shown). Since TGF-ß treatment is able to affect both the STAT1 binding activity and content, we tested if short IFN-{gamma} treatment of monocytes treated for 5 days with TGF-ß was able to restore full STAT1 activity. As shown in Fig. 4B, Goa prompt activation of STAT1 binding to the IRF-1 SBE sequence was obtained at a similar extent both in the control and TGF-ß-treated cells, indicating that TGF-ß did not impair the transduction pathway leading to STAT1 activation by IFN-{gamma}.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. TGF-ß inhibits STAT1 expression and binding activity. Monocytes were grown either under standard conditions (control) or in the presence of 5 ng/ml of TGF-ß. (A) After 5 days cells were processed for membrane fluorescence using anti-CD54 (ICAM-1), anti-CD64, (Fc{gamma}RI) or anti-CD16 (Fc{gamma}RII) mAb as described in Methods. On the right side of the panel, a representative flow cytometric analysis using anti-CD64 mAb is shown. (B) Cells were treated with control medium or TGF-ß for 5 days and for an additional 30 min with IFN-{gamma}, where indicated. Whole-cell extracts (10 µg) were incubated with radiolabeled oligonucleotide containing the SBE sequence present in the Fc{gamma}RI, ICAM-1 and IRF-1 promoters, and the complexes formed were analyzed by EMSA.

 
To further characterize the inhibitory mechanism exerted by TGF-ß on STAT1 binding activity we analyzed if TGF-ß affects the expression of integrins which were shown (Fig. 2Go) to mediate, at least in part, STAT1 activation in monocytes. In Fig. 5Go the flow cytometric analysis of VLA-4, VLA-5 and VLA-6 on macrophages untreated or treated for the indicated times with TGF-ß is shown. The results of this analysis indicate that the constitutive expression of the examined integrins was down-modulated following TGF-ß treatment.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. TGF-ß inhibits integrin expression. Freshly isolated monocytes were grown with medium alone or with 5 ng/ml of TGF-ß for the indicated days. The cells were processed for membrane fluorescence using anti-VLA-4, anti-VLA-5 and anti-VLA-6 as described in Methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 
Monocytes isolated from peripheral blood are committed cells not yet fully differentiated and undergo terminal maturation to macrophages after migration in tissues. These cells also spontaneously mature in vitro in the absence of any added cytokine or growth factor, thus representing a useful experimental model to understand the molecular events underlying the in vivo monocyte to macrophage maturation. Here we provide evidence that STAT1 transcription factor plays a pivotal role in the terminal maturation of monocytes: STAT1 activity, in fact, is absent or very low in freshly isolated cells, but is spontaneously activated during the monocytic maturation process. The time-dependent induction of STAT1 activity well correlates with the enhanced expression of some markers of macrophage differentiation. Conversely, inhibition of this maturation process by TGF-ß affects STAT1 activation and expression. The absence of STAT1 activity in monocytes as well as in macrophages treated with TGF-ß is not the result of a lack of protein and/or its inability to be activated since a short incubation period with IFN-{gamma}, promptly allows its binding to DNA (Figs 1B and 4BGoGo).

In the attempt to identify the signals leading to STAT1 activation, the presence of several cytokines was measured in the supernatants by using specific and sensitive immunoassays (Table 1Go). Both IFN-{gamma} and GM-CSF are present at very low levels at all days of culture (Table 1Go). Conversely, IL-6 and IL-10, which are known to be secreted by macrophages (34,35), are present throughout the culture period, IL-10 production being consistent and increasing over days (Table 1Go). To test if the STAT1 activation observed was due to these cytokines, we used neutralizing antibodies to IL-6 and IL-10, added both separately and together. The addition of these neutralizing antibodies did not inhibit the maturation of monocytes as well as the progressive activation of STAT1. Similarly, the addition of polyclonal antibodies neutralizing human IFN-{alpha} and -ß did not affect the STAT1 activation (data not shown).

Looking for other inducers of STAT1 binding activity in macrophages, we considered the possibility that adherence per se could represent the primary stimulus. Leukocyte activation associated with the initiation of inflammation or immune response is a complex and incompletely understood process. Rapid and dynamic changes in monocyte cell adhesion are associated with arrest and extravasation, and also with increased responsiveness to cytokines and amplification of signal transduction pathway (36). Since the proteins that mediate many of the adhesive reactions of monocytes belong to the integrin family, we analyzed the effects of their ligands, such as fibronectin and laminin, on STAT activation. Results in Fig. 2Go show that adhesion is able to directly activate STAT1 binding activity, through the induction of STAT1 phosphorylation both in tyrosine and serine residues. As far as we aware, this observation represents the first evidence for STAT1 activation triggered by adhesion.

Previous studies using various cell types have shown that a number of intracellular signaling pathways may be stimulated upon ligand binding and clustering of integrins including activation of PI, pKC ras/MAP pathways (21,3638). Interestingly, there is evidence that the activities of both STAT1 and STAT3 are maximally induced by cytokine/growth factor-stimulated serine phosphorylation (39) and several studies indicate that ERK/MAPK may phosphorylate the serine residue of STAT1. However, even if indirect evidence in different systems suggests a cross-talk between JAK–STAT and the ras/MAP (2,40), the specific mechanisms and molecules involved have not yet been characterized. The identification of specific protein kinases responsible for STAT1 activation triggered by adhesion in monocytes is currently under investigation.

With the exception of some IFN-stimulated genes, the cellular genes dependent on cytokine-activated STAT proteins are poorly defined. STAT1 is the prototype of the STAT family whose activation has been shown to mediate transcriptional effects of IFN-{gamma} (25,26,41,42). Even if the specific biological effects that result from STAT1 activation in vivo are not yet defined, the potential role of STAT1 in transcription during monocyte maturation and activation is suggested by the finding that it binds to the SBE elements of genes functionally expressed in macrophages and/or directly activated by IFN and inflammatory cytokines. Included in this set are genes encoding the high-affinity receptor for IgG (Fc{gamma}RI), adhesion molecules such as ICAM-1 and the transcription factor IRF-1 (6,41). This pattern of expression is compatible with a role of activated monocytes and macrophages in antibody-dependent cellular cytotoxicity reactions and in promoting communication with lymphocytes or other cells.

The involvement of STAT1 in monocytic differentiation is confirmed by the experiments with TGF-ß which is known to reduce the MHC class II mRNA (43), to down-regulate CD89 and CD14 expression on human peripheral blood monocytes (32,33), and to have a general inhibitory effect on monocytic maturation. Similarly, we show that TGF-ß is able to impair STAT1 activity. An inhibitory effect of TGF-ß on several cytokine-induced JAK–STAT pathways has also been reported (4446). This inhibition resulted in some cases in hypophosphorylation of both JAK and STAT involved, and has been ascribed either to the activation of a phosphatase activity or to a direct effect of TGF-ß on the cytokine receptor. Our results, in accord with what was observed in rat astrocytes (47), show that STAT1 phosphorylation induced by IFN-{gamma} is not impaired in TGF-ß-treated cells. Instead, we observed that TGF-ß down-modulates receptors for fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) (Fig. 5Go). This inhibition can thus be considered a new mechanism responsible for the inhibitory effects exerted by TGF-ß on STAT1 activation and macrophage maturation.

In conclusion, our studies shed light on the molecular mechanisms underlying the maturation process of circulating monocytes, suggesting the essential role of the STAT1 as an early transcription factor activated by the adhesion events which leads to the activation of specific functional gene expression when monocytes are recruited at the site of inflammation. Elucidation of other specific transcription factors responsible for integrin-regulated gene expression may help in uncovering the mechanism involved in these events.


    Acknowledgments
 
We thank Dr T. Taniguchi for the generous gift of the IRF-1 plasmid, Dr T. Decker for the antibodies anti-STAT5, Dr B. Seed for Fc{gamma}RI plasmid and Dr M. Capobianchi for rabbit polyclonal antibodies neutralizing type I IFN. We thank Eleonora Benedetti and Roberto Orsatti for technical assistance, and Stefania Mochi for oligonucleotide preparation. The editorial assistance of Sabrina Tocchio is also gratefully acknowledged. We also thank Eugenio Morassi for preparing drawings. This work was supported in part by grants from the Italy–USA program on `Therapy of Tumors' and the Istituto Superiore di Sanità (Progetto Nazionale Tubercolosi and Special Project on AIDS). E. S. was supported by a post-doctoral fellowship from Istituto Superiore di Sanità, Italy.


    Abbreviations
 
EMSAelectrophoretic mobility shift assay
SBESTAT binding element
GM-CSFgranulocyte macrophage-CSF
IRFIFN regulatory factor
JAKJanus kinase
MIPmacrophage inflammatory protein
PEphycoerythrin
STATsignal transducers and activators of transcription
TGFtransforming growth factor
TNFtumor necrosis factor

    Notes
 
Transmitting editor: T. Taniguchi

Received 6 October 1999, accepted 18 March 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Effect of TGF-{beta} on...
 Discussion
 References
 

  1. Schindler, C. and Darnell, J. E., Jr. 1995. Transcriptional responses to polypeptide ligands: the JAK–STAT pathway. Annu. Rev. Biochem. 64:621.[ISI][Medline]
  2. Stancato, L. F., Yu, C.-R., Petricoin, E. F., III and Larner, A. C. 1998. Activation of Raf-1 by interferon {gamma} and oncostatin M requires expression of the Stat1 transcription factor. J. Biol. Chem. 273:18701.[Abstract/Free Full Text]
  3. Decker, T., Lew, D. J., Mirkovitch, J. and Darnell, J. E., Jr. 1991. Cytoplasmatic activation of GAF, an IFN{gamma}-regulated DNA-binding factor. EMBO J. 10927.[Abstract]
  4. Mui, A. L., Wakao, H., O'Farrell, A., Harada, N. and Miyajima, A. 1995. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologous. EMBO J. 14:1166.[Abstract]
  5. Zhong, Z., Wen, Z. and Darnell, J. E., Jr. 1994. Stat3 and Stat4: members of the family of signal transducers and activators of transcription. Proc. Natl Acad. Sci. USA 91:4806.[Abstract]
  6. Decker, T., Kovarik, P. and Meinke, A. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J. Interferon Cytokine Res. 17:121.[ISI][Medline]
  7. Wegenka, U. M., Buschmann, J., Lutticken, C., Heinrich, P. C. and Horn, F. 1993. Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Mol. Cell. Biol. 13:276.[Abstract]
  8. Harroch, S., Gothelf, Y., Watanabe, N., Revel, M. and Chebath, J. 1993. Interleukin-6 activates and regulates transcription factors of the interferon regulatory factor family in M1 cells. J. Biol. Chem. 268:9092.[Abstract/Free Full Text]
  9. Mui, A. L., Wakao, H., Kinoshita, T., Kitamura, T. and Miyajima, A. 1996. Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation. EMBO J. 15:2425.[Abstract]
  10. Meinke, A., Barahmand-Pour, F., Wohrl, S., Stoiber, D. and Decker, T. 1996. Activation of different Stat5 isoforms contributes to cell-type-restricted signalling in response to interferons. Mol. Cell. Biol. 16:6937.[Abstract]
  11. Nakajima, K., Yamanaka, Y., Nakae, K., Kojima, H., Ichiba, M., Kiuchi, N., Kitaoka, T., Fukada, T., Hibi, M. and Hirano, T. 1996A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. EMBO J. 15:3651.[Abstract]
  12. Chakraborty, A., White, S. M., Schaefer, T. S., Ball, E. D., Dyer, K. F. and Tweardy, D. J. 1996. Granulocyte colony-stimulating factor activation of Stat3 alpha and Stat3 beta in immature normal and leukemic human myeloid cells. Blood 88:2442.[Abstract/Free Full Text]
  13. Werb, Z. 1987. Phagocytic cells: chemotaxis and effector functions of macrophages and granulocytes. In Sites, D. P., Stobe, J. P. and Wells, J. V., eds, Basic and Clinical Immunology, p. 96. Appleton-Century-Crofts, Norwalk, CT.
  14. Gessani, S., Testa, U., Varano, B., Di Marzio, P., Borghi, P., Conti, L., Barberi, T., Tritarelli, E., Martucci, R., Seripa, D., Peschle, C. and Belardelli, F. 1993. Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. J. Immunol. 151:3758.[Abstract/Free Full Text]
  15. Fuhlbrigge, R. C., Chaplin D. D., Kiely J. M. and Unamme, E. R. 1987. Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide. J. Immunol. 138:3799.[Abstract/Free Full Text]
  16. Courtneidge, S. A., Dhand, R., Pilat, D., Twarmley, G. M., Waterfield, M. D. and Roussel, M. F. 1993. Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor. EMBO J. 12:943.[Abstract]
  17. Haskill, S., Johnson, C., Eierman, D., Becker, S. and Warren, K. 1988. Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes. J. Immunol. 140:1690.[Abstract/Free Full Text]
  18. Sporn, S. A., Eierman, D. F., Johnson, C. E., Morris, J., Martin, G., Ladner, M. and Haskill, S. 1990. Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J. Immunol. 144:4434.[Abstract/Free Full Text]
  19. Eierman, D. F., Johnson, C. E. and Haskill, S. J. 1989. Human monocyte inflammatory mediator gene expression is selectively regulated by adherence substrates. J. Immunol. 142:1970.[Abstract/Free Full Text]
  20. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P. and Baldwin, A. S., Jr. 1991. Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. Cell 65:1281.[ISI][Medline]
  21. Lin, T. H., Yurochko, A., Kornberg, L., Morris, J., Walker, J. J., Haskill, S. and Juliano, R. L. 1994. The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes. J. Cell. Biol. 126:1585.[Abstract]
  22. Koli, K. and Keski-Oja, J. 1996. Transforming growth factor-ß system and its regulation by members of the steroid-thyroid hormone superfamily. Cancer Res. 70:63.
  23. Marziali, G., Perrotti, E., Ilari, R., Testa, U., Coccia, E. M. and Battistini, A. 1997. Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation. Mol. Cell. Biol. 17:1387.[Abstract]
  24. Allen, J. M. and Seed, B. 1989. Isolation and expression of functional high-affinity Fc receptor complementary DNAs. Science 243:378.[ISI][Medline]
  25. Pine, R., Canova A. and Schindler, C. 1994. Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN alpha and IFN gamma, and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Abstract]
  26. Larner, A. C., David, M., Feldman, G. M., Igarashi, K., Hackett, R. H., Webb, D. S., Sweitzer, S. M., Petricoin, E. F., III and Finbloom, D. S. 1993. Tyrosine phosphorylation of DNA binding proteins by multiple cytokines. Science 261:1730.[ISI][Medline]
  27. Capobianchi, M. R., Mattana, P., Mercuri, F., Conciatori, G., Ameglio, F., Ankel, H. and Dianzani, F. 1992. Acid lability is not an intrinsic property of interferon-alpha induced by HIV-infected cells. J. Interferon Res. 12:431.[ISI][Medline]
  28. Kovarik, P., Stoiber, D., Novy, M. and Decker, T. 1998. Stat1 combines signals derived from IFN-gamma and LPS receptors during macrophage activation. EMBO J. 17:3660.[Abstract/Free Full Text]
  29. Hobart, M., Ramassar, V., Goes, N., Urmson, J. and Halloran, P. F. 1997. IFN regulatory factor-1 plays a central role in the regulation of the expression of class I and II MHC genes in vivo. J. Immunol. 158:4260.[Abstract]
  30. MacMicking, J., Xie, Q.-W. and Nathan, C. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15 :323.[ISI][Medline]
  31. Taniguchi, T., Harada, H. and Lamphier, M. 1995. Regulation of the interferon system and cell growth by the IRF transcription factors. J. Cancer Res. Clin. Oncol. 121:516.[ISI][Medline]
  32. Reterink, T. J., Levarht, E. W., Klar-Mohamad, N., Van Es, L. A. and Daha, M. R. 1996. Transforming growth factor-beta 1 (TGF-beta 1) down-regulates IgA Fc-receptor (CD89) expression on human monocytes. Clin. Exp. Immunol. 103:161.[ISI][Medline]
  33. Hamon, G., Mulloy, R. H., Chen, G., Chow, R., Birkenmaier, C. and Horn, J. K. 1994. Transforming growth factor-beta 1 lowers the CD14 content of monocytes. J. Surg. Res. 57:574.[ISI][Medline]
  34. Ray, A., Tatter, S. B., Santhanam, U., Helfgott, D. C., May, L. T. and Sehgal, P. B. 1989. Regulation of expression of interleukin 6. Molecular and clinical studies. Ann. NY Acad. Sci. 557:353.[ISI][Medline]
  35. Mosmann, T. R. 1994. Properties and functions of interleukin-10. Adv. Immunol. 56:1.[ISI][Medline]
  36. McCarthy, J. B., Vachhani, B. V., Wahl, S. M., Finbloom, D. S. and Feldman, G. M. 1997. Human monocyte binding to fibronectin enhances IFN-gamma-induced early signaling events. J. Immunol. 159:2424.[Abstract]
  37. Li, Y. Q., Kobayashi, M., Yuan, L., Wang, J., Matsushita, K., Hamada, J. I., Kimura, K., Yagita, H., Okumura, K. and Hosokawa, M. 1998. Protein kinase C mediates the signal for interferon-gamma mRNA expression in cytotoxic T cells after their adhesion to laminin. Immunology 93:455.[ISI][Medline]
  38. Clark, E. A. and Brugget, J. S. 1995. Integrins and signal transduction pathways: the road taken. Science 268:23.[ISI]
  39. Wen, Z., Zhong, Z. and Darnell, J. E., Jr. 1995. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241.[ISI][Medline]
  40. Winston, L. A. and Hunter, T. 1996. Intracellular signalling: putting JAKs on the kinase MAP. Curr. Biol. 6:668.[ISI][Medline]
  41. Boehm, U., Klamp, T., Groot, M. and Howard, J. C. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15:749.[ISI][Medline]
  42. Coccia, E. M., Marziali, G., Stellacci, E., Perrotti, E., Ilari, R., Orsatti, R. and Battistini, A. 1995. Cells resistant to interferon-beta respond to interferon-gamma via the Stat1–IRF-1 pathway. Virology 211:113.[ISI][Medline]
  43. Czarniecki, C. W., Chiu, H. H., Wong, G. H., McCabe, S. M. and Palladino, M. A. 1988. Transforming growth factor-beta 1 modulates the expression of class II histocompatibility antigens on human cells. J. Immunol. 140:4217.[Abstract/Free Full Text]
  44. Bright, J. J. and Sriram, S. 1998. TGF-ß inhibits IL-12-induced activation of Jak–STAT pathway in T lymphocytes. J. Immunol. 161:1772.[Abstract/Free Full Text]
  45. Bright, J. J., Kerr, L. D. and Sriram, S. 1997. TGF-ß inhibits IL-2-induced tyrosine phosporylation and activation of Jak-1 and Stat 5 in T lymphocytes. J. Immunol. 159:175 .[Abstract]
  46. Pazdrak, K., Justement, L. and Alam, R. 1995. Mechanism of inhibition of eosinophil activation by transforming growth factor-ß. J. Immunol. 155:4454.[Abstract]
  47. Panek, R. B., Yi-Ju, L. and Benveniste, E. N. 1995. TGF-ß suppression of IFN-{gamma}-induced class II MHC gene expression does not involve inhibition of phosphorylation of JAK1, JAK2, or signal transducers and activators of transcription, or modification of IFN-{gamma} enhanced factor X expression. J. Immunol. 154:610.[Abstract/Free Full Text]