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Autocrine/Paracrine IFN-ß Mediates the Lipopolysaccharide-Induced Activation of Transcription Factor Stat1 in Mouse Macrophages: Pivotal Role of Stat1 in Induction of the Inducible Nitric Oxide Synthase Gene¹

Jian Jun Gao^*, Michael B. Filla^*, Marion J. Fultz, Stefanie N. Vogel, Stephen W. Russell^* and William J. Murphy^2,*

^* Wilkinson Laboratory of the Kansas Cancer Institute and Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160; and Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have examined the role of Stat1 in the induction by LPS of the mouse inducible nitric oxide synthase (EC 1.14.13.39) gene. LPS induced both the tyrosine phosphorylation of Stat1 and the production of nitric oxide in a time- and dose-dependent manner. The phosphorylation of Stat1 elicited by LPS differed from that observed using IFN- or IFN-ß, in that LPS induced less phosphorylated protein and the time course of induction was much delayed (2–4 h compared with 30 min). Cycloheximide inhibited LPS-mediated Stat1 phosphorylation. In addition, cell culture supernatants derived from macrophages treated with LPS for 4 h could be transferred to naive macrophage cultures resulting in rapid (30 min), rather than delayed (4 h), phosphorylation of Stat1. Together, these results implicated an autocrine/paracrine effector protein(s) in the phosphorylation process. LPS stimulated phosphorylation of Stat1 in peritoneal macrophages derived from IFN--knockout mice, negating any possibility that IFN- was the mediator. By contrast, neutralizing Ig raised against mouse IFN-ß inhibited both the delayed LPS-mediated phosphorylation of Stat1 and the rapid induction of phosphorylation induced by supernatants from LPS-stimulated cultures. Collectively, these results show that LPS-induced IFN-ß production, Stat1 activation, and nitrite accumulation closely parallel one another, suggesting that indirect activation of transcription factor Stat1 by IFN-ß is a critical determinant of LPS-mediated inducible nitric oxide synthase gene expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mouse macrophages can be activated by LPS plus IFN- to produce the enzyme inducible nitric oxide synthase (iNOS).³ This, in turn, catalyzes the production of nitric oxide (NO), which helps mediate the cytotoxic function of macrophages against tumor cells and a variety of microbes (1, 2, 3). LPS, by itself, induces dose-dependent low level synthesis of iNOS, and these levels are synergistically augmented by the presence of IFN- (4, 5, 6, 7). IFN- alone fails to induce the production of iNOS by macrophages from certain strains of mice (7, 8, 9). The combination of LPS and IFN- has the effect of accelerating the kinetics of induction of iNOS gene transcription (7), mRNA accumulation (7), protein synthesis (10), and NO production (10, 11) compared with the kinetics of induction elicited by LPS alone.

It has been reported that the combination of LPS plus IFN- induces iNOS gene expression through the activation of transcription factors that bind to LPS and IFN response elements in the iNOS promoter. Among these elements, two B sites (12, 13, 14), an octamer (13, 15) (our unpublished observations), and two juxtaposed IFN-stimulated response elements (ISREs) (16, 17, 18) are functional in regulating iNOS gene expression. We have recently identified a sixth element, an IFN--activated site (GAS), that is also required for iNOS promoter activity (18). The iNOS GAS is bound by transcription factor Stat1 after stimulation of RAW 264.7 macrophages with IFN-, LPS, or LPS plus IFN- (18). IFN- or the combination of LPS plus IFN- induces GAS binding of Stat1 within 30 min of exposure. In contrast, LPS induction of GAS binding activity requires approximately 2 h and is quantitatively less than that induced by IFN- alone or LPS plus IFN- (18).

Induction of the iNOS gene results from a signal transduction cascade that activates/induces the aforementioned transcription factors. Thus, we hypothesized that the reduced and delayed production of iNOS and NO by LPS stimulation alone is due to rate- and amplitude-limiting steps in the activation or induction of these transcription factors. Consistent with this hypothesis, it has previously been demonstrated that induction by LPS alone of iNOS protein production (19) and NO synthesis (the latter measured as nitrite accumulation) (20, 21) is dependent upon the prior autocrine/paracrine synthesis of IFN-ß. Because both type I and type II IFNs induce tyrosine phosphorylation of Stat1 (22), we predicted that activated Stat1 would be the limiting transcription factor in LPS-stimulated vs LPS- plus IFN--stimulated macrophages. The data presented here confirm that these hypotheses are correct; autocrine/paracrine IFN-ß is the LPS-induced feedback activator of Stat1, a transcription factor critical for LPS-mediated induction of the mouse iNOS gene.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, culture medium, and reagents

Female C57BL/6 wild-type mice were obtained from The Jackson Laboratory (Bar Harbor, ME). IFN--knockout mice were provided by Genentech (South San Francisco, CA) and obtained through the National Institutes of Health (Bethesda, MD) after backcross with the C57BL/6 strain. Each strain was used at the age of 6 to 8 wk. Mice were injected i.p. with 3 ml of 4% Brewer thioglycolate (Difco, Detroit, MI), and peritoneal macrophages were harvested 5 days later by lavage with cold HEPES-buffered (25 mM) RPMI 1640 growth medium (Life Technologies, Grand Island, NY). They were then seeded at 1 x 10⁷ cells/100-mm culture dish and incubated overnight in growth medium supplemented with 10% fetal clone I (HyClone, Logan, UT), 2 mM glutamine (JRH Biosciences, Lenexa, KS), 100 µg/ml streptomycin (Sigma, St. Louis, MO), and 100 U/ml penicillin (Apothecon, Princeton, NJ) at 37°C in a humidified, 5% CO₂ environment before stimulation.

Natural mouse IFN-ß and rabbit anti-mouse-IFN-ß antiserum were purchased from Lee BioMolecular Research Laboratory (San Diego, CA). Recombinant murine IFN- was obtained from Schering-Plough through the American Cancer Society (Atlanta, GA). The lipid A-rich fraction II of LPS, extracted from Escherichia coli O111:B4 with phenol, was obtained from List Biological Laboratories (Campbell, CA). Anti-Stat1 (N terminus) mAbs (used for Western blot analyses) were purchased from Transduction Laboratories (Lexington, KY). Anti-Stat1 84/91 polyclonal Abs (used for immunoprecipitation) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-Sepharose conjugate (used for Ig purification and immunoprecipitation), rabbit anti-mouse IgG-horseradish peroxidase Ab conjugate (used for Western blots), and normal rabbit serum (used for Ab neutralization and immunoprecipitation studies) were purchased from Sigma. Anti-phosphotyrosine (anti-P-tyr) Ab mixture (PY-Plus) was obtained from Zymed (South San Francisco, CA). Endotoxin was undetectable in culture medium or reagents, as determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA), at a sensitivity of 50 pg/ml.

Cell extract preparation and electrophoretic mobility shift assays (EMSAs)

After overnight culture, cells were washed twice and then incubated in medium alone or in medium that contained appropriate stimuli for the times indicated in the figures. Whole cell extracts were prepared as described by Andrews and Faller (23), with slight modification. Cells were washed twice with cold PBS and once with cold PBS containing 1 mM sodium orthovanadate, and lysed in high salt buffer that contained 20 mM HEPES (pH 7.9 with KOH), 1.5 mM MgCl₂, 0.2 mM EDTA (pH 8.0 with KOH), 420 mM KCl, 25% glycerol, 1 mM DTT, and the following protease/phosphatase inhibitors: 0.5 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate. Cell lysates were centrifuged (23), and supernatants from each lysate were aliquoted and frozen at -70°C until needed. Nuclear extracts were prepared by a modification of the method of Shapiro et al. (24). Cells were washed as described above and then washed once with hypotonic buffer (24) that contained the same protease/phosphatase inhibitors described above. Cells were scraped into 1 ml of hypotonic buffer plus protease/phosphatase inhibitors and disrupted with 10 strokes of a tight-fitting Dounce pestle (Kontes, Vineland, NJ). Immediately after breaking the cells, 0.1 vol of 75% sucrose were added with mixing. Nuclei were pelleted in an Eppendorf microcentrifuge (model 5415 C, Eppendorf, Fremont, CA) at maximal speed for 2 min at 4°C. The supernatant was discarded, and the nuclear pellet was resuspended in 60 µl of the high salt buffer used for whole cell extracts (see above). The nuclear suspension was incubated on ice for 30 min with periodic gentle mixing. Nuclear debris were removed by centrifugation, as described above, for 30 min. The supernatant was removed to a new tube, and the extract was aliquoted and stored frozen (-70°C) until needed. EMSAs and supershift assays were performed using nuclear extracts prepared in this manner, exactly as described by Gao et al. (18).

Purification of Abs

Rabbit Ig against mouse IFN-ß and normal rabbit Ig were purified by protein A-Sepharose column chromatography. After purification, Igs were sterilized by filtration through a Millex-GV filter (Millipore, Bedford, MA) and assayed to determine the concentration of protein. Either normal (control) or anti-IFN-ß IgG was then used for IFN-ß neutralization studies, as indicated in Results.

Western blot analysis

Proteins for analysis were separated via SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were incubated overnight at 4°C in blocking buffer (1% BSA, 10 mM Tris-HCl (pH 7.5), and 100 mM NaCl), blotted with anti-Stat1 (N terminus) mAbs or anti-P-Tyr Abs in blocking buffer that contained 0.1% Tween-20 for 1 h, washed five times in wash buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.5% Tween 20) for 6 min each time, and blotted for 1 h with a rabbit anti-mouse IgG-horseradish peroxidase conjugate in wash buffer that contained 5% nonfat milk. Each membrane was then washed five times in wash buffer for 6 min each. Specific binding of anti-Stat1 (N terminus) or anti-P-Tyr was detected using the Renaissance chemiluminescence detection kit (DuPont, Boston, MA). Autoradiograms were scanned, where indicated, with a Molecular Dynamics (Sunnyvale, CA) Personal Densitometer SI and analyzed with the ImageQuaNT software package (Molecular Dynamics) using a Windows NT (Microsoft, Redmond, WA) format.

To strip and reprobe PVDF membranes, they were incubated in a solution containing 7 M guanidine hydrochloride, 50 mM glycine (pH 10.8), 0.05 mM EDTA, 0.1 M KCl, and 20 mM ß-ME for 30 min at room temperature. Membranes were then washed twice with water for 10 min each, blocked, reprobed as indicated in Figure 2, and then developed as described above.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Immunoprecipitation and Western blot analysis of LPS-induced Stat1 phosphorylation. Nuclear extracts were prepared from peritoneal macrophages that either were left untreated (M) or were treated with IFN- () for 30 min, with LPS (L) for 4 h, or with IFN-ß (ß) for 30 min at the concentrations used in Figure 1. Immunoprecipitation with anti-Stat1 polyclonal Abs was performed with aliquots of each extract as described in Materials and Methods. The immunoprecipitated proteins were separated by SDS-PAGE (7.5% gel) followed by transfer to a PVDF membrane. The resultant blot was first probed with anti-P-Tyr Abs (upper panel). The membrane was then stripped and reprobed with anti-Stat1 mAb (lower panel). The methods for blotting, stripping, and reprobing of membranes are described in Materials and Methods.

Nitrite concentrations were measured in cell culture supernatants using the Greiss reagent in a previously described colorimetric assay (25). Absorbance (at 570 nM) was determined using a Dynatech MR700 plate reader (Chantilly, VA). Sodium nitrite, dissolved in RPMI 1640, was used to generate a standard concentration curve. Cell culture supernatants were assayed for antiviral activity (IFN-ß) by the plaque reduction method using monolayers of vesicular stomatitis virus-infected mouse L-929 cells. The assay was performed as previously described (26), except that 96-well tissue culture dishes were used. Each supernatant dilution was assayed in triplicate. IFN activity is expressed in terms of National Institutes of Health reference units, where 1 U of activity is defined as the amount of IFN that causes a 50% reduction in viral plaques. Mouse IFN-ß (Lee BioMolecular Research Laboratory) was used as the reference standard.

Immunoprecipitation

Peritoneal macrophages were cultured overnight (2 x 10⁷ cells/150-mm culture dish), washed twice with culture medium, and then incubated with medium that contained appropriate stimuli for the times indicated in Figure 2. Cells were then washed three times with cold PBS, incubated in cold lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 µg/ml PMSF, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate) with shaking for 10 min, and disrupted by repeated aspiration/ejection through a 23-gauge needle. Cellular debris were removed by centrifugation in an Eppendorf microcentrifuge (1 min, 4°C, maximum speed), and the supernatant was precleared by adding 1 µg of normal rabbit Ig with 20 µl of protein A-Sepharose conjugate followed by a 10-min incubation at 4°C and another brief centrifugation. Two micrograms of polyclonal anti-Stat1 84/91 Abs were added to the cellular lysate, which was then incubated overnight at 4°C. After this, 100 µl of protein A-Sepharose conjugate was added, and the incubation was continued for approximately 4 h at 4°C. The immunoprecipitate was collected after a brief centrifugation. The pelleted beads were washed four times with cold lysis buffer, resuspended in 100 µl of SDS-sample buffer, and boiled for 3 min. After another brief centrifugation, an aliquot was used for Western blot analysis, as described above. Remaining sample was stored at -70°C for future use.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LPS-induced, delayed activation of Stat1 in mouse macrophages

Previous results from our laboratory (18) demonstrated a delayed induction of the binding of Stat1 to oligonucleotides that mimic either the iNOS or IFN regulatory factor (IRF-1) GAS binding sites. Because the activation of Stat1 is accompanied by a mobility shift in SDS-polyacrylamide gels (27), we sought to determine, by Western blot analysis, the kinetics of Stat1 activation in response to treatment of C57BL/6 peritoneal macrophages with IFN-, IFN-ß, or LPS. Figure 1 shows a Western blot that investigates these kinetics. Both IFN- and IFN-ß induced the rapid appearance (within 30 min) of the slower migrating form of activated Stat1 (A and B). Although 100-fold more IFN-ß than IFN- was used on a per unit basis, conversion of Stat1 to its activated form was more complete with IFN- than with IFN-ß (compare the 0.5 h lanes, A and B). Levels of activated Stat1 declined after 0.5 h of treatment and were not detectable 4 h poststimulation.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Time courses of Stat1 phosphorylation induced by IFN-, IFN-ß, and LPS. Peritoneal macrophages were stimulated with IFN- (100 U/ml), IFN-ß (10,000 U/ml), or LPS (100 ng/ml) for the times indicated in the figure. Cultures incubated in medium only are indicated by M. Fifty micrograms of each whole cell extract was electrophoresed on a SDS-polyacrylamide gel and then immunoblotted as described in Materials and Methods. Phosphorylated Stat1 is revealed as a band (indicated by pStat1) that migrates more slowly than does the unphosphorylated Stat1 (27). In Figures 1 through 6 and 8, the 84-kDa Stat1ß band has been cropped for the sake of clarity. The phosphorylation of Stat1ß parallels that of Stat1 in all experiments described in these figures. The apparent molecular masses of Stat1 and its phosphorylated form are 91 and 92 kDa, respectively.

In addition to these changes in Stat1 activation, the abundance of Stat1 protein itself was also affected by the stimuli used in Figure 1. Each of these agents increased, by approximately 50%, the level of Stat1 protein found in whole cell extracts (compare lanes M with lanes 12 in A–C; densitometric scanning data not shown). The significance of the stimulatory effect of these agents on Stat1 production, as it may relate to secondary gene induction, is currently not known.

LPS induces the tyrosine phosphorylation of Stat1

The activation of Stat1 and the SDS-PAGE mobility shift that accompanies such are the results of tyrosine phosphorylation of residue Y⁷⁰¹ of this protein (28). Because tyrosine phosphorylation of Stat1 is essential for its transcriptional activity (22, 27, 28), we sought to determine whether LPS also induced tyrosine phosphorylation of Stat1. To this end, immunoprecipitation and Western blot experiments were performed. Cellular lysates were first immunoprecipitated with anti-Stat1 polyclonal Abs. Next, electrophoretically separated proteins were blotted and reacted sequentially with the anti-P-Tyr Ab mixture and then with the anti-Stat1 mAbs (see Materials and Methods). The results of such experiments are shown in Figure 2. Untreated macrophages contained no detectable tyrosine-phosphorylated Stat1 (Fig. 2, top panel, lane M). However, IFN-, LPS, and IFN-ß each induced the appearance of a new band that reacted with the anti-P-Tyr Abs (Fig. 2, top panel, lanes , L, and ß, respectively). In this experiment treatment with the IFNs was performed for 30 min, while LPS treatment was conducted for 4 h. As expected, LPS stimulated consistently less tyrosine-phosphorylated Stat1 than that induced by IFN- and required a longer period of stimulation before it was detectable. These results validated an assumption made in Figure 1 that the Stat1 mobility shift caused by LPS treatment was the result of tyrosine phosphorylation of Stat1 similar to that observed with the IFNs, but with different kinetics.

Cycloheximide (CHX) inhibits the LPS-induced phosphorylation of Stat1

The delay in LPS-induced tyrosine phosphorylation of Stat1 raised the possibility that an autocrine/paracrine mediator of Stat1 activation might be induced by LPS. If the proposed autocrine/paracrine mediator were a protein factor, then its induction should be ablated by the protein synthesis inhibitor, CHX. Thus, macrophage cultures were preincubated for 30 min in the presence or the absence of CHX, after which cultures were either stimulated with 100 ng/ml LPS or left untreated for 2 h (in the continued presence of CHX where appropriate). The CHX concentration used was previously determined to inhibit >95% of cellular protein synthesis (data not shown). As shown in Figure 3, CHX did not affect the Stat1 detected in untreated macrophages (compare lane 2 with lane 1). However, the LPS-induced activation of Stat1 (lane 3) was completely abolished by treatment with CHX (lane 4). Thus, protein synthesis is required for Stat1 phosphorylation to develop, supporting the hypothesis that autocrine/paracrine protein(s) mediates LPS-induced Stat1 phosphorylation. In addition, the LPS-induced increase in Stat1 protein accumulation, observed in Figure 1C after 0.5 to 2 h of stimulation, was also inhibited by CHX treatment (compare the band intensity observed in lane 4 with that observed in lane 3).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. The effect of CHX on LPS-induced phosphorylation of Stat1. Peritoneal macrophages were preincubated for 30 min with medium alone (lanes 1 and 3) or with medium containing 3 µg/ml of CHX (C; lanes 2 and 4). Medium was then replaced with medium alone (M; lane 1), medium containing CHX (M/C; lane 2), medium containing 100 ng/ml LPS (L; lane 3), or medium containing LPS plus CHX (L/C; lane 4). Cultures were subsequently incubated for an additional 2 h. Parallel cultures treated in the same manner showed a 95% level of cellular exclusion of trypan blue, indicating no acute toxic effects of these agents, either alone or in combination, during the 2-h treatment period. Thirty micrograms of nuclear extract prepared from each culture were subjected to SDS-PAGE and immunoblotted as described in Materials and Methods.

The culture supernatant from LPS-treated cells induces rapid, rather than delayed, Stat1 phosphorylation

In an approach complementary to the CHX experiments, we asked whether supernatants from LPS-stimulated macrophage cultures, when transferred to naive cultures, could induce rapid (30 min), rather than delayed (4 h), activation of Stat1. As shown in Figure 4, when a primary macrophage culture was treated for 4 h with 100 ng/ml LPS and the culture supernatant was then transferred to a fresh, secondary culture of macrophages, phosphorylation of Stat1 in this secondary culture was observed within 30 min (Fig. 4, lane 3). The observed result was similar to that seen when macrophage cultures were treated with IFN- alone for 30 min (compare lanes 2 and 3) or with LPS alone for 4 h (compare lane 3 with lane 4 of Fig. 1C). Treatment of secondary macrophage cultures for 4 h with supernatants from LPS-treated primary cultures showed results similar to those obtained with a 30-min treatment (Fig. 4, lane 4).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. The effect of culture supernatant derived from primary cultures treated with LPS on the phosphorylation of Stat1 in secondary cell cultures. Peritoneal macrophages were incubated for the times indicated at the top of the figure with medium (lane 1), medium containing 100 U/ml IFN- (lane 2), or with the cell culture supernatants (lanes 3 and 4) from peritoneal macrophages that had been stimulated with 100 ng/ml LPS for 4 h. Thirty micrograms of nuclear extract prepared from each culture were subjected to SDS-PAGE and immunoblotted as described in Materials and Methods.

The autocrine/paracrine mediator of LPS-induced Stat1 activation is IFN-ß

LPS is known to induce production of several cytokines by mouse macrophages (29). A number of these could interact with the producer macrophages and cause activation of transcription factor Stat1 (30). The prototypic activators of Stat1 are IFN- and IFN-ß. Because the production of both IFN- (31) and IFN-ß (20, 21, 32, 33, 34, 35, 36) by mouse macrophages can be induced by LPS, we wanted to determine whether either was the mediator of LPS-induced Stat1 activation.

The potential role of IFN- was tested by examining the ability of LPS to stimulate Stat1 phosphorylation in macrophages from IFN--knockout mice. LPS treatment of peritoneal macrophages isolated from both wild-type and IFN--knockout C57BL/6 mice resulted in equivalent levels of Stat1 phosphorylation (Fig. 5, compare lanes 2 and 4). Therefore, production of IFN- was not required for LPS-induced phosphorylation of Stat1.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Analysis of phosphorylation of Stat1 in IFN--knockout macrophages. Peritoneal macrophages from IFN--knockout (GKO) or wild-type C57BL/6 (BL6) mice were incubated with medium alone (lanes 1 and 3) or with medium containing 100 ng/ml LPS (lanes 2 and 4) for 4 h. Thirty micrograms of nuclear extract prepared from each culture were subjected to SDS-PAGE and immunoblotted as described in Materials and Methods.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. The effect of neutralizing anti-IFN-ß Abs on LPS-induced Stat1 phosphorylation. Six culture dishes of peritoneal macrophages were prepared as described in Materials and Methods. Two sets of two were preincubated for 30 min with either control IgG (C; lanes 3 and 4) or anti-IFN-ß IgG (ß; lanes 5 and 6). The remaining set of two was preincubated with medium alone. After preincubation, one culture dish from each set was supplemented with 100 ng/ml LPS (L, lanes 2, 4, and 6), while the other was left alone (M, lanes 1, 3, and 5). Incubation of all six cultures was then continued for 4 h. Additionally, culture supernatants from peritoneal macrophage cultures treated for 4 h with 100 ng/ml LPS were supplemented with control IgG (C; lanes 7 and 9) or anti-IFN-ß IgG (ß; lanes 8, 10, and 11) and transferred to fresh macrophage monolayers followed by incubation for the times indicated above the figure. For lanes 10 and 11, secondary cell cultures were each treated identically, except that the culture represented in lane 11 was additionally treated with IFN- during the last 30 min of stimulation to demonstrate that the anti-IFN-ß Abs themselves had no deleterious effect on the activation of Stat1. Thirty micrograms of nuclear extract prepared from each culture was subjected to SDS-PAGE and immunoblotted as described in Materials and Methods.

To confirm and extend the link between the activation of Stat1 by LPS-induced autocrine/paracrine IFN-ß and induction of the iNOS gene, we performed EMSAs using an oligonucleotide containing the iNOS gene’s GAS and the nuclear extracts used in lanes 7 and 8 of Figure 6. Nuclear extracts, prepared from secondary macrophage cultures that were treated with primary culture supernatant for 30 min in the presence of control Ig, contained an iNOS GAS binding activity (Fig. 7, lane 4, the position of the specific binding complex is marked by the filled arrowhead) that was not detected in extracts from untreated macrophage cultures (lane 1). This complex was similar to that observed in extracts prepared from IFN--treated cultures (lane 2), although binding induced by IFN- was greater than that induced in the supernatant transfer experiment, as expected. In contrast with these EMSAs, nuclear extracts prepared from secondary macrophage cultures, which had been treated for 30 min with primary culture supernatant that additionally contained anti-IFN-ß-Ig, showed little specific DNA-binding complex formation (lane 6).

View larger version (47K): [in this window] [in a new window]   FIGURE 7. The effect of culture supernatant derived from primary cultures treated with LPS and supplemented with either control or anti-IFN-ß IgG on induction of iNOS GAS binding activity. The nuclear extracts used in lanes 4 and 6 of Figure 6 were used for these experiments. These extracts were prepared from secondary macrophage cultures that had been treated with culture supernatant derived from primary cultures treated with LPS for 4 h. The transferred supernatants were additionally supplemented with either control IgG (C; lanes 4 and 5) or anti-IFN-ß IgG (ß; lanes 6 and 7). In addition, nuclear extracts prepared from untreated peritoneal macrophages (lane 1) and those treated with 100 U/ml IFN- (lanes 2 and 3) were employed as negative and positive controls, respectively. All extracts were used for EMSA (lanes 1, 2, 4, and 6) or for supershift analysis (lanes 3, 5, and 7) as described in Materials and Methods.

Figure 7 further shows that the specific DNA binding complexes detected in lanes 2 and 4, could be supershifted by adding anti-Stat1 mAbs to the EMSA reaction. The binding of this anti-Stat1 Ab appeared to increase the interaction of Stat1 with the iNOS GAS-containing oligonucleotide and to sharpen the resultant band that was seen upon autoradiography (see Fig. 7) (18). Thus, the small amount of residual GAS binding activity observed in lane 6 was more readily detected in the supershift experiment shown in lane 7. The supershift experiments confirmed that the EMSA activity detected in this figure was, in fact, due to the binding of transcription factor Stat1 to the iNOS GAS.

The activation of Stat1, production of IFN-ß, and accumulation of nitrite parallel one another in a LPS dose-dependent fashion

The data presented here suggest that the production of autocrine/paracrine IFN-ß, the activation of transcription factor Stat1, and the elaboration of NO in LPS-stimulated peritoneal macrophage are coupled components of a LPS-induced regulatory cascade. To investigate this further, we performed the LPS dose-response experiment shown in Figure 8. Peritoneal macrophage cultures were treated with the indicated doses of LPS for 4 h. Cell culture supernatants were then assayed for the presence of IFN bioactivity, while nuclear extracts prepared from these same cultures were assayed for Stat1 activation. A parallel set of macrophage cultures was treated for 24 h, followed by the assay of culture supernatants for nitrite. The upper portion of Figure 8 shows that the activation of Stat1 was dependent upon the dose of LPS used; 1 ng/ml LPS elicited barely detectable levels of phosphorylated Stat1, while levels increased using 10 ng/ml and increased yet again using 100 ng/ml. The graph in the bottom portion of Figure 8 illustrates that the production of IFN-ß and nitrite increased in parallel with the accumulation of phosphorylated Stat1 when each was plotted as a function of the LPS concentration used.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Dose-responsive production of IFN-ß, activation of Stat1, and accumulation of nitrite in peritoneal macrophages stimulated with LPS. Peritoneal macrophages were incubated with LPS at the concentrations indicated in the figure or with medium alone (lane M). After 4 h culture supernatants were removed and assayed for the presence of IFN-ß. Whole cell extracts were prepared from treated macrophages, and extracts were subjected to Western blot analysis using anti-Stat1 mAbs (top portion). Parallel cultures were set up in triplicate in 96-well culture plates and treated identically, except that treatment was allowed to continue for 24 h. Supernatants from these cultures were then assayed for the accumulation of nitrite. The graph at the bottom of the figure shows a plot of nitrite and IFN-ß concentrations obtained as well as a plot of densitometric scanning data for phosphorylated Stat1 (from Western blots). The nitrite and IFN-ß data points are each averaged from three independent experiments, while the phosphorylated Stat1 densitometric scanning data are averaged from two of these three experiments. The actual values ± SEM for each point are as follows: nitrite: 10 ng/ml LPS, 1.9 ± 0.4 µM; 100 ng/ml LPS, 6.3 ± 1.5 µM; IFN-ß: 10 ng/ml LPS, 40.2 ± 5.9 U/ml; 100 ng/ml LPS; 106.0 ± 13.0 U/ml; Stat1: 1 ng/ml LPS, 0.03 ± 0.01 OD; 10 ng/ml LPS, 0.36 ± 0.07 OD; 100 ng/ml LPS, 0.84 ± 0.02 OD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The autocrine/paracrine production of IFN-ß has been shown to affect macrophage activation for nitrite accumulation both negatively and positively (19, 20, 21, 37). Both exogenously added and endogenously produced IFN-ß (the latter induced with poly(I-C)) inhibited IFN--induced nitrite accumulation in peritoneal macrophages from C3H/HeSlc mice (37). Conversely, other reports (19, 20, 21) demonstrated a positive effect of autocrine/paracrine IFN-ß on LPS-induced production of iNOS protein (19) and nitrite accumulation (20, 21). In these studies, anti-IFN-ß Abs nearly eliminated LPS-induced nitrite accumulation in peritoneal macrophages derived from C3HeB/FeJ mice (20) and significantly inhibited LPS-induced nitrite accumulation and iNOS production in RAW 264.7 (19, 20) and J774 (21) mouse macrophage cell lines. The contrasting effects of IFN-ß on LPS- vs IFN--induced nitrite accumulation most likely result from the different fates of Stat1 in IFN-- vs IFN-ß-stimulated cells. In IFN--treated cells, phosphorylated Stat1 exists almost exclusively as GAS-binding homodimers. In IFN-ß-treated cell cultures Stat1 is apportioned between the transcription factor complex, ISGF-3 (which contains Stat1, Stat2, and ISGF-3 (also called p48)), and complexes of phosphorylated Stat1 homodimers. Consequently, levels of Stat1 homodimers are lower in cells stimulated with IFN-ß than in those stimulated with IFN- (38) (J. J. Gao and M. J. Fultz, unpublished observations). Thus, IFN--induced nitrite accumulation would be expected to be diminished by IFN-ß treatment because Stat1 homodimers would be diminished by the IFN-ß-induced formation of ISGF-3. Conversely, autocrine/paracrine IFN-ß positively affects LPS-mediated induction of nitrite accumulation because it generates Stat1 homodimers, despite their lower concentration relative to that induced by IFN-, that are essential for gene activation.

The activation of Stat1 correlated well with the observed activation of mouse macrophages for nitrite accumulation, implicating Stat1 as essential for controlling the ultimate level of iNOS gene induction. Concentrations of LPS <10 ng/ml stimulated little or no nitrite accumulation and, similarly, stimulated little or no IFN-ß production or Stat1 activation. On the other hand, 10 and 100 ng/ml LPS induced progressively more nitrite accumulation, IFN-ß production, and Stat1 activation. The combination of LPS and IFN- induces maximal Stat1 activation, as judged by the GAS binding activity of Stat1 homodimers (18), and, consequently, maximal nitrite accumulation (7). This contrasts sharply with the activation of transcription factor NF-B by LPS, where as little as 1 ng/ml is sufficient to achieve maximal NF-B binding to synthetic oligonucleotides yet induces no nitrite accumulation (14).

The information reported here, along with that accumulated in many publications investigating transcriptional induction of the mouse macrophage iNOS gene, have prompted us to propose the gene activation model outlined in Figure 9. When both LPS and IFN- are used as coinducers, the former stimulus effects the release of transcription factor NF-B from its inhibitor protein I-B. LPS also activates, without need for prior protein synthesis, the IRF-1 gene (39), which was shown previously to be necessary for iNOS gene induction (16, 17, 18). IFN-, on the other hand, stimulates the tyrosine phosphorylation of Stat1, allowing the predominant formation of homodimers. Stat1 homodimers also participate in trans activation of the IRF-1 gene by binding the IRF-1 gene’s GAS, leading to increased abundance of the transcription factor IRF-1 (40). The accumulation of these three transcription factors (NF-B, IRF-1, and Stat1) in the nucleus leads to their binding, along with one of the constitutively present octamer binding factors (our unpublished observations), to their cognate sequence elements upstream of the iNOS gene. This, in turn, leads to the initiation of iNOS gene transcription.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Model of iNOS gene activation in mouse macrophages stimulated with either LPS plus IFN- or LPS alone. The model is described in the text. Each figure shows a schematic representation of a mouse macrophage with plasma membrane, cytoplasm, and nucleus labeled accordingly. Ellipses represent receptors for IFN-ß (ßR), IFN- (R), and LPS (LR). Small circles associated with the IFN-ß and IFN- receptors indicate receptor-associated tyrosine kinases (of the Janus kinase family). Arrows indicate pathways leading to the activation of the indicated transcription factors. Dotted arrows are meant to indicate a lesser degree of activation or accumulation of transcription factors than that indicated by solid arrows. The solid rectangle represents the iNOS gene, and the transcription start point (tsp) is indicated by the arrow located just upstream. The size of the arrowhead associated with the tsp indicates the relative level of expression of iNOS in a given cell population. Smaller filled rectangles upstream of the tsp represent the transcription factor binding sites indicated below each.

Not conveyed in the model depicted in Figure 9 is the fact that each of the transcription factors that bind to the iNOS promoter’s six transcriptional elements apparently must be present simultaneously to achieve activation of this gene. Indications that this is a prerequisite come from four types of observations. The first is similar to that shown in this work, where threshold doses of LPS, despite their ability to activate NF-B (14) and induce IRF-1 (39), do not partially activate the iNOS gene in the absence of an IFN signal. Second, the work of Pace et al. (10) and Alley et al. (19) show that exposure of a population of macrophages to exogenous IFN- or endogenously produced IFN-ß, respectively, in the presence of a threshold dose of LPS increases the number of individual cells in that population that are maximally synthesizing iNOS protein compared with LPS treatment alone. Third, targeted disruption of genes encoding either Stat1 (41) or IRF-1 (9, 16) results in an inability to induce in macrophages, by either LPS plus IFN- or LPS plus IFN-ß, the accumulation of either nitrite or iNOS mRNA. Finally, the octamer-binding transcription factors Oct-1 and Oct-2 are constitutively present in the nucleus of mouse macrophages but are unable to bind the iNOS gene’s octamer site until a proper stimulus is administered (our unpublished observations). Each of these observations is consistent with the hypothesis that the nuclear presence of subsets of those transcription factors necessary for full induction of the iNOS gene does not result in DNA binding or partial activation of the gene in mouse macrophages.

The model outlined in Figure 9 is consistent with that which is known about LPS-induced vs LPS- plus IFN--induced expression of the mouse iNOS gene and provides a framework upon which a more thorough understanding of the regulation of this gene can be achieved. It also underscores the need for two signaling pathways to achieve optimal gene induction. While LPS treatment alone can cause the activation of NF-B and induction of IRF-1, either IFN- or IFN-ß is essential for the activation of transcription factor Stat1, which binds the iNOS enhancer-linked GAS. Stat1 also trans activates the IRF-1 gene, the product of which binds the iNOS enhancer-linked ISRE. This dual stimulus requirement apparently assures that the iNOS gene will be activated only under those circumstances that warrant the synthesis of the potentially toxic NO molecule.

	Acknowledgments

 
We thank Drs. David Morrison, Fang Fan, and Charlotte Zhang for their valuable advice and discussion and Tari Parmely for her excellent technical assistance. We also thank Dr. Chris Schindler for helpful discussions early in the course of this work.

	Footnotes

 
¹ This work was supported in part by Research Grant P01 CA54474 from the National Cancer Institute and the Wilkinson Endowment for Cancer Research (to S.W.R.) and Research Grant AI18797 from the National Institutes of Health (to S.N.V.).

² Address correspondence and reprint requests to Dr. William J. Murphy, Wilkinson Laboratory of the Kansas Cancer Institute, 1008 Wahl Hall West, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7184. E-mail address:

³ Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; NO, nitric oxide; ISRE, IFN-stimulated response element; GAS, IFN--activated site; EMSA, electrophoretic mobility shift assay; anti-P-Tyr, anti-phosphotyrosine; PVDF, polyvinylidene difluoride; IRF-1, IFN regulatory factor-1; CHX, cycloheximide; ISGF-3, IFN-stimulated gene factor-3.

Received for publication February 5, 1998. Accepted for publication July 1, 1998.

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