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The Journal of Immunology, 2005, 175: 3637-3647.
Copyright © 2005 by The American Association of Immunologists

IFN-{gamma}-Primed Macrophages Exhibit Increased CCR2-Dependent Migration and Altered IFN-{gamma} Responses Mediated by Stat11

Xiaoyu Hu*, Kyung-Hyun Park-Min{dagger}, Hao H. Ho* and Lionel B. Ivashkiv2,*,{dagger}

* Arthritis and Tissue Degeneration Program, Department of Medicine, Hospital for Special Surgery, and {dagger} Graduate Program in Immunology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Priming of macrophages with IFN-{gamma} increases cellular responsiveness to inflammatory stimuli, including IFN-{gamma} itself. We described previously that priming with subactivating concentrations of IFN-{gamma} increased Stat1 expression and resulted in enhanced activation of Stat1 and of a subset of IFN-{gamma}-responsive genes when primed macrophages were restimulated with low doses of IFN-{gamma}. In this study, we determined the effects of IFN-{gamma} priming on the macrophage transcriptome and on transcriptional responses to high saturating concentrations of IFN-{gamma}. At baseline, primed macrophages expressed a small subset of IFN-{gamma}-inducible genes, including CCR2, and exhibited increased migration in response to CCL2. Activation of gene expression by high concentrations of IFN-{gamma} was altered in primed macrophages, such that activation of a subset of IFN-{gamma}-inducible genes was attenuated. A majority of genes in this "less induced" category corresponded to genes that are induced by IFN-{gamma} via Stat1-independent but Stat3-dependent pathways and have been implicated in inflammatory tissue destruction. One mechanism of attenuation of gene expression was down-regulation of Stat3 function by increased levels of Stat1. These results reveal that priming enhances migration to inflammatory chemokines and identify IFN-{gamma}-inducible genes whose expression is attenuated by high levels of Stat1. The increase in Stat1 expression during priming provides a mechanism by which physiological regulation of the relative abundance of Stat1 and Stat3 impacts on gene expression. Our results also suggest that, in addition to inducing hypersensitivity to inflammatory stimuli, IFN priming delivers a homeostatic signal by attenuating IFN-{gamma} induction of certain tissue-destructive genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interferon-{gamma}, the sole type II IFN, possesses a variety of immune regulatory properties that are essential for host defense against infections and tumors (1). IFN-{gamma} acts on a remarkable range of distinct cell populations including immune cells and nonimmune cells. Of these, macrophages are among the most important. IFN-{gamma} activates direct microbicidal functions of macrophages and promotes the Ag processing and presentation capacities of macrophages (2). The significant impact of IFN-{gamma} on macrophage phenotype and function is achieved by a profound alteration of the macrophage transcriptional program in response to IFN-{gamma}. It has been estimated that exposure to IFN-{gamma} results in changes in expression of ~25% of the mouse genome (3). The major IFN-{gamma}-activated signal transduction pathway that leads to transcriptional regulation involves Jak protein tyrosine kinases and Stat transcription factors. As with most cytokines that use the Jak-Stat pathway, two Jaks, Jak1 and Jak2, participate in IFN-{gamma} signaling (1). Stat1 is the major Stat activated by IFN-{gamma} and is essential for many IFN-{gamma} responses (4, 5).

Although Stat1 is a key mediator of IFN-{gamma} signaling, a large number of genes are induced by IFN-{gamma} in Stat1-null cells, and thus IFN-{gamma}-induced but Stat1-independent pathways contribute to IFN-{gamma}-induced gene expression (6, 7, 8). IFN-{gamma}-induced Stat1-independent pathways result in the activation of PI3K, Akt, p38, protein kinase C, I{kappa}B kinases, and Stat3, and have important functions in vitro and in vivo, including promoting cell survival and proliferation (6, 9, 10, 11, 12, 13, 14, 15, 16). Some of the genes induced by IFN-{gamma} in Stat1-null cells are also activated by Stat1, and thus these genes are activated cooperatively by different signaling pathways that act downstream of the IFN-{gamma}R. In contrast, IFN-{gamma} activation of other genes, such as c-myc and c-jun, by Stat1-independent pathways is suppressed by Stat1 (7, 9, 17). Thus, the expression level of a subset of IFN-{gamma}-inducible genes is determined by the relative balance between IFN-{gamma} activation of Stat1-dependent and of Stat1-independent pathways.

IFN-{gamma} signaling is not fixed in amplitude and duration, but is modulated by a number of cross- and autoregulatory mechanisms (18). Cellular responsiveness to IFN-{gamma} is regulated by the microenvironment to which cells are exposed and the activation status of cells. We described previously that priming macrophages with low subactivating concentrations of IFN-{gamma} resulted in enhanced activation of Stat1 when macrophages were restimulated with IFN-{gamma}. Priming did not activate macrophages, but induced a positive feedforward loop by dramatically increasing Stat1 levels without engaging feedback inhibitory mechanisms (19). IFN-{gamma}-primed macrophages did not express elevated amounts of mRNA encoded by many canonical IFN-{gamma}-inducible genes, such as inducible protein 10 (IP-10),3 monokine induced by {gamma} (Mig), and IFN regulatory factor 1 (IRF-1). Instead, primed macrophages were hyperresponsive to restimulation with low concentrations of IFN-{gamma} (0.1–1 U/ml), which induced substantially higher expression of IP-10, Mig, and IRF-1 in primed than in control macrophages (19).

In this study, we used gene expression profiling to gain insight into mechanisms that contribute to the primed macrophage phenotype and to analyze the influence of IFN-{gamma} priming on IFN-{gamma}-induced transcriptional responses. The analysis of transcriptional changes during priming may help to illuminate the process by which macrophages adapt to alterations in the cytokine microenvironment that occur early during innate immune responses as infections are established. We were interested in discovering novel patterns of IFN-{gamma}-dependent gene regulation rather than confirming and extending previous results that low concentrations of IFN-{gamma} activate higher expression of well-known IFN-{gamma}-inducible genes in primed than in control macrophages. Therefore, instead of using low concentrations of IFN-{gamma}, we restimulated control and primed macrophages with high saturating concentrations of IFN-{gamma}. This experimental design enabled us to test the hypotheses that maximal activation of Stat1 would result in expression of novel genes, and would attenuate the expression of genes that are negatively regulated by Stat1, such as the genes that are superinduced in Stat1-deficient cells (6, 7, 9, 17). We found that IFN-{gamma} priming activated a very limited number of genes relative to the genes activated by full activating concentrations of IFN-{gamma}. Priming increased macrophage CCR2 expression and chemotactic capacity. IFN-{gamma} transcriptional responses were altered in primed macrophages, with the most prominent finding being attenuated induction of a subset of IFN-{gamma}-inducible genes that correspond to genes that are induced by Stat1-independent but Stat3-dependent pathways. Our results suggest that the counterbalancing action between Stat1 and Stat3 is regulated under physiological settings such as IFN priming of macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

PBMCs were obtained from whole blood from disease-free volunteers by density gradient centrifugation using Ficoll (Invitrogen Life Technologies). CD14+ monocytes were purified from fresh PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec), as recommended by the manufacturer. Purity of monocytes was >97% as verified by FACS. Human control macrophages were derived from CD14+ blood monocytes cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FBS (HyClone) in the presence of 10 ng/ml human macrophage-CSF (M-CSF) (PeproTech). IFN-{gamma}-primed macrophages were obtained by culturing CD14+ monocytes with both M-CSF and low concentrations of IFN-{gamma} (3 U/ml) for 48 h. Murine bone marrow-derived macrophages were obtained as described (20) and maintained in DMEM supplemented with 20% FBS containing 10 ng/ml murine M-CSF (PeproTech). Stat1-deficient animals (4, 5) on a 129S6/SvEv background were from Taconic.

Microarray analysis

Total RNA was isolated from human macrophages using an RNeasy mini kit (Qiagen). Isolated RNA from four individual blood donors was evenly pooled and three independent pools of RNA that represented a total of 12 donors were reversed transcribed using Superscript II (Invitrogen Life Technologies). cDNA was transcribed in vitro with BioArray HighYield RNA Transcript Labeling kit (Enzo Life Sciences) to obtain biotin-labeled cRNA. cRNA was fragmented and hybridized to U95Av2 oligonucleotide microarrays (Affymetrix) according to the instructions of the manufacturer. Primary images were obtained using Gene-Array Scanner (Affymetrix) and analyzed using Microarray Analysis Suite 5.0 (Affymetrix). Images were scaled to an average hybridization intensity of 250. Further data analysis was performed using GeneSpring software (Silicon Genetics). All readings on each chip were normalized to the mean value of that chip. Each gene was compared between two treatment groups. Only genes with present calls according to Microarray Analysis Suite 5.0 in designated conditions were included in the analysis. For example, a gene considered to be up-regulated in a certain treatment group relative to the control group must be "present" in such treatment condition in all three independent replicates. Fold induction or suppression was calculated with the average signal intensity from three replicates. In addition, individual data points from three replicates were used to calculate p values based on a Welch t test with the Benjamini-Hochberg correction for multiple test comparisons. When p ≤ 0.05, the expression of a gene was considered significantly different between two treatment groups. The complete data set was deposited in the Gene Expression Omnibus public database and can be accessed at <www.ncbi.nlm.nih.gov/projects/geo/> with accession no. GSE1925.

Real-time quantitative RT-PCR (qPCR)

For real-time PCR, total RNA was extracted using an RNeasy mini kit and 1 µg of total RNA was reverse transcribed using a First Strand cDNA Synthesis kit (Fermentas). qPCR was performed using iQ SYBR Green Supermix and iCycler iQ thermal cycler (Bio-Rad) following the manufacturer’s protocols. Triplicate reactions were run for each sample, and expression of a tested gene was normalized relative to levels of GAPDH. The generation of only the correct size amplification products was confirmed using agarose gel electrophoresis.

Flow cytometry

A total of 2 x 105 human macrophages was stained using PE-conjugated monoclonal anti-human CCR2 Ab (R&D Systems) or isotype-matched control Ab. Samples were read on FACScan (BD Biosciences), and data were analyzed using CellQuest software.

Migration assay

A total of 5 x 105 control or IFN-{gamma}-primed macrophages was placed in the upper chambers of Transwell plates (5-µm pore size) and CCL2-containing medium was added to the bottom chambers. After 4 h of incubation at 37°C, macrophages that had migrated through the Transwell membrane into the bottom chambers were collected and counted by FACS using Flow Cytometry Absolute Counting Standard as an internal control (Bangs Laboratories).

Lentiviral gene transduction and RNA interference (RNAi)

A lentivirus-based vector expressing the human Stat1 or Stat1{beta} cDNA driven by a human phospho-glycerol kinase promoter was used to generate recombinant lentiviral particles as described (21). A construct that contained a transcription cassette encoding enhanced GFP (eGFP) driven by the human phospho-glycerol kinase promoter was used to generate control viral particles for Stat1 overexpression experiments. For Stat3 RNAi, oligonucleotides encoding several different short duplex putative interfering RNAs that target human Stat3 were cloned into the lentivirus-based RNAi vector pLL3.7, which also contains a transcription cassette encoding eGFP driven by a CMV promoter (21). Constructs that were effective in suppressing Stat3 expression were identified using transient cotransfection of HEK 293T cells with expression plasmids encoding Stat3. The construct that was most effective in HEK 293T cells (containing the sequence 5'-AGTCAGGTTGCTGGTCAAA-3') was used to generate recombinant lentiviral particles. Lentiviral particles encoding interfering RNA against red fluorescence protein DSRed2 were used as control for Stat3 RNAi experiments. THP-1 cells were incubated overnight with recombinant lentiviral particles at a ratio of 1:50 in the presence of 4 µg/ml polybrene. The efficiency of transduction was evaluated using flow cytometry and fluorescence microscopy to monitor eGFP expression and was typically >90%.

Immunoblotting and EMSA

Whole-cell and nuclear extracts were obtained, and protein levels were quantitated using the Bradford assay (Bio-Rad), as previously described (20). For immunoblotting, 5 µg of whole-cell lysates were fractionated on 7.5% polyacrylamide gels using SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore), and incubated with specific Abs, and ECL was used for detection. mAbs against Stat1 and Stat3 were obtained from BD Transduction Laboratories. Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology. For EMSA, 5 µg of nuclear extracts were incubated for 15 min at room temperature with 0.5 ng of 32P-labeled double-stranded high-affinity SIS-inducible element oligonucleotide in 15 µl of a binding reaction containing 40 mM NaCl and 2 µg of poly(dI:dC) (Pharmacia), and complexes were resolved on nondenaturing 4.5% polyacrylamide gels. For Stat3 supershift assay, nuclear extracts were incubated with anti-Stat3 Ab on ice for 1 h before the addition of labeled oligonucleotide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Up-regulation and suppression of gene expression by IFN-{gamma}

To investigate how priming with subactivating concentrations of IFN-{gamma} affects gene expression upon restimulation with IFN-{gamma}, we conducted gene expression profiling analysis on control and IFN-{gamma}-primed primary human macrophages. In the context of this paper, "priming" refers to treating macrophages with low doses of IFN-{gamma} without phenotypically activating them, and "stimulation" refers to treating cells with saturating doses of IFN-{gamma} to achieve full activation. In our experimental system, priming of macrophages with low doses of IFN-{gamma} for 2 days consistently yielded augmented Stat1 activation upon IFN-{gamma} restimulation with minimal cell loss. To prepare samples for microarray analysis, control macrophages or macrophages primed for 2 days with 3 U/ml (150 pg/ml) IFN-{gamma} were stimulated with an activating dose of IFN-{gamma} (100 U/ml) for either 3 or 24 h. Thus, there were six treatment conditions, i.e., control unstimulated cells, control cells stimulated with 100 U/ml IFN-{gamma} for 3 or 24 h, primed cells that were not restimulated with IFN-{gamma}, and primed cells stimulated with 100 U/ml IFN-{gamma} for 3 or 24 h. Expression levels in each condition were assessed using data from three independent donor pools as described in Materials and Methods. As an initial step toward understanding the impact of priming on gene expression, we determined the effects of IFN-{gamma} on gene expression in control macrophages. The expression of ~12,000 genes was monitored, and data were analyzed using stringent criteria that restricted the identification of regulated genes to those that were reproducibly induced or suppressed by IFN-{gamma} in three independent pools (p ≤ 0.05 by Welch t test with Benjamini-Hochberg correction for multiple test comparisons). First, activation of gene expression in control macrophages after 3 and 24 h of stimulation with IFN-{gamma} was examined. Induction of gene expression was confirmatory of previous studies (Table I and data not shown). As previously reported (19), the expression of Stat1 was activated by IFN-{gamma} stimulation (Table I). One noteworthy point is that more genes were induced at 24 h than at 3 h, and examples of genes that were induced by IFN-{gamma} with delayed kinetics are shown in Table I. IFN-{gamma} exerts its biological functions not only by inducing but also by inhibiting gene expression. We next examined the suppressive effects of IFN-{gamma} on gene expression. Stimulation of control macrophages with 100 U/ml IFN-{gamma} for 3 h did not result in the suppression of any genes that reached statistical significance as described in Materials and Methods. In contrast, a large number of genes were significantly down-regulated after 24 h of IFN-{gamma} treatment (Table II). Overall, our results extend analysis of IFN-{gamma} regulation of gene expression to primary human macrophages and to later time points.


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  		Table I. Examples of genes that are induced by IFN-{gamma} in primary human macrophages

 

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  		Table II. Examples of genes that are suppressed by IFN-{gamma} at 24 h (>10-fold)a

 
Gene expression pattern and phenotype of IFN-{gamma}-primed macrophages

We showed previously that macrophages primed with 3 U/ml IFN-{gamma} did not exhibit an activated phenotype or increased expression of canonical IFN-{gamma}-inducible genes (19), which is consistent with the subactivating concentrations of IFN-{gamma} used for priming. To gain insight into the primed macrophage phenotype and potential mechanisms that sensitize these cells to extracellular ligands, we determined the gene expression profile of primed macrophages. In contrast to the profound alteration of gene expression in macrophages activated by high concentrations of IFN-{gamma}, primed macrophages exhibited only limited changes in gene expression. Fifteen genes were up-regulated and 20 genes were down-regulated >3-fold by priming (Table III). The pattern of gene expression in primed macrophages at the mRNA level (Table III) did not yield clues about why these cells are hyperresponsive to stimulation with IFNs, cytokines, and TLR activators. However, there was a striking ~10-fold increase in mRNA encoding the chemokine receptor CCR2 in primed macrophages, which was confirmed by qPCR in two additional donors (Fig. 1A), and at the protein level by flow cytometry (Fig. 1B). Moreover, primed macrophages exhibited increased migration in response to the CCR2 ligand, CCL2 (MCP-1) (Fig. 1C). Thus, priming enhanced the capacity of macrophages to migrate into inflammatory sites where chemokines are produced. Another interesting gene whose expression was consistently elevated in primed macrophages was the proteosome regulator PA28{beta} (Table III and Fig. 1A). Genes down-regulated by IFN-{gamma} priming included a subset of canonical type I IFN-inducible, IFN-stimulated gene factor 3-dependent genes, such as myxovirus resistance 1 (mx1) and several oligoadenylate synthetase (oas) genes (Table III). Suppressed expression of two of these genes was confirmed using real-time PCR (data not shown). Consonant with the low-level constitutive type I IFN signaling that has been described (22, 23), these genes were consistently expressed at baseline in control macrophages, and IFN-{gamma} treatment suppressed this baseline expression, possibly by promoting formation of Stat1:Stat1 homodimers relative to IFN-stimulated gene factor 3, by sequestration of Stat2, or by indirect effects. A caveat concerning changes in gene expression observed after priming (and after restimulation; see below) is that secondary effects will take place during the priming period, and thus observed changes in gene expression may not be directly regulated by Stat1.


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  		Table III. Genes with expression that differs >3-fold in primed macrophages compared with control macrophages

 


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  		FIGURE 1. Priming with low doses of IFN-{gamma} alters macrophage gene expression. A, Human macrophages were primed with 3 U/ml IFN-{gamma} for 2 days, and steady-state mRNA levels of CCR2 and PA28{beta} were quantitated using real-time PCR in two independent experiments. Results are expressed as fold increase in primed cells relative to control cells ({square}) and compared with the fold increase calculated using average signal intensities derived from microarray analysis ({blacksquare}). B, Flow-cytometric analysis of cell surface expression of CCR2 on human macrophages. Upper panel, Control macrophages. Lower panel, IFN-{gamma}-primed macrophages. Thin lines denote staining with isotype-matched control Ab. Thick lines represent specific fluorescence for CCR2. C, Migration assay was performed as described in Materials and Methods. CCL2 was used as the chemoattractant. Results are expressed as mean ± SD of triplicate cultures. Data shown are representative of three independent experiments.

 
IFN-{gamma} activation of IFN-{gamma}-inducible Stat1-independent genes is attenuated in primed macrophages

Next, we analyzed the impact of priming on IFN-{gamma}-mediated gene regulation in response to restimulation of primed cells with high saturating concentrations of IFN-{gamma}. The patterns of gene expression in control and primed macrophages after 3 and 24 h of stimulation with IFN-{gamma} (100 U/ml) were compared. A gene was considered differentially regulated by IFN-{gamma} in control and primed cells if the following two criteria were both fulfilled: 1) the fold induction or suppression upon IFN-{gamma} stimulation was >2-fold in a statistically significant manner (p < 0.05; Benjamini-Hochberg correction) in one group but not in the other group; 2) expression levels after IFN-{gamma} treatment were >2-fold different between control and primed macrophages. The latter criterion was included to ensure that absolute levels of mRNA transcripts differed between two conditions, and thus to exclude genes where differences in basal gene expression levels between unstimulated control and unstimulated primed cells could confound results. Genes whose induction by IFN-{gamma} differed between control and primed macrophages fell into four groups based upon the pattern of regulation (Table IV). The first group, termed "newly induced," consists of three genes that were induced by IFN-{gamma} in primed, but not in control, macrophages. Of note, genes previously reported to be more strongly induced by low concentrations of IFN-{gamma} in primed macrophages (19) were not identified in this group, because these genes, such as IP-10, Mig, and IRF-1, were strongly induced in control cells by the high concentration of IFN-{gamma} (100 U/ml) used in the current experiments. Thus, although IFN-{gamma} induction of known IFN-{gamma}/Stat1 target genes was enhanced in primed macrophages (19), priming did not result in the activation of a large number of novel genes in response to IFN-{gamma} restimulation (Table IV).


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  		Table IV. Genes that are differentially regulated by IFN-{gamma} between control and primed cellsa

 
A second group of genes, termed "less induced," consisted of genes whose induction by IFN-{gamma} was attenuated in primed relative to control macrophages (Table IV). The pattern of expression of four of these genes, IL-1{beta}, IL-6, cyclooxygenase 2 (Cox-2), and VCAM-1, was independently confirmed by real-time PCR in macrophages from an additional three to five blood donors (Fig. 2A). Next, we wished to determine whether these less induced genes shared any common characteristics in terms of their induction by IFN-{gamma}. Notably, four genes in this less induced group, including early growth response 2 (EGR2), IL-1{beta}, matrix metalloproteinase 1 (MMP1), and Fas, were previously reported to be activated by IFN-{gamma} in Stat1-null cells, and thus can be activated by IFN-{gamma}-induced but Stat1-independent pathways (6, 7, 9). We investigated the requirement for Stat1 for activation of the other eight genes in the less induced category using bone marrow-derived macrophages from Stat1-deficient mice. As expected (6, 7), up-regulation of IL-1{beta} gene expression by IFN-{gamma} was observed in Stat1-deficient macrophages (Fig. 2B, upper panel). Moreover, in macrophages lacking Stat1, IFN-{gamma} activated expression of IL-6, Bcl2-related protein A1 (Bcl2A1), CCL5, and VCAM-1 (Fig. 2B, middle and lower panels, and data not shown). Thus, among 12 genes whose induction by IFN-{gamma} was attenuated in primed macrophages (Table IV), at least 8 genes were induced by IFN-{gamma} independently of Stat1. These results suggest that strong activation of Stat1 in primed macrophages antagonizes IFN-{gamma}-induced gene activation that is mediated by Stat1-independent pathways.



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  		FIGURE 2. IFN-{gamma} priming selectively suppresses the induction of a subset of IFN-{gamma}-activated genes. A, Control or primed human macrophages were activated with 100 U/ml IFN-{gamma} for 3 h (for analysis of IL-1{beta}, IL-6, and Cox-2) or 24 h (for analysis of VCAM-1). Steady-state mRNA levels were determined by real-time PCR, and fold induction by IFN-{gamma} relative to basal expression in control and IFN-{gamma}-primed cells is shown. Each line represents an individual experiment performed with an independent blood donor. B, Stat1-independent induction of IFN-{gamma}-responsive genes. Murine macrophages from Stat1-deficient mice were cultured with 10 ng/ml IFN-{gamma} for the indicated time periods, and mRNA levels of IL-1{beta}, IL-6, and Bcl2A1 were analyzed using real-time PCR and normalized relative to expression of GAPDH. Results are expressed as mean ± SD of triplicate determinants.  

 
In addition to IFN-{gamma} up-regulated genes, the expression pattern of IFN-{gamma} down-regulated genes was also modulated by priming. As described above, 3 h of IFN-{gamma} treatment did not lead to significant suppression of gene expression in control macrophages. In contrast, IFN-{gamma} significantly suppressed the expression of nine genes after 3 h of IFN-{gamma} treatment of primed cells (Table IV), suggesting that IFN-{gamma} more effectively suppresses gene expression in primed than in control macrophages.

Increased Stat1 levels mediate attenuated gene induction in primed macrophages

We next investigated the mechanism by which expression of genes whose induction by IFN-{gamma} did not require Stat1 was attenuated in primed macrophages. Suppressor of cytokine signaling 1 (SOCS1) is an important physiological regulator of IFN-{gamma} signaling (24). We wished to determine whether SOCS1 may play a role in priming-mediated inhibition of Stat1-independent genes. As assessed by real-time PCR, the expression of SOCS1 mRNA was transiently induced by a priming dose of IFN-{gamma}, returned to basal levels at 4 h, and remained low during the rest of the 2-day priming period (Fig. 3A, middle panel). In addition, SOCS1 activation by IFN-{gamma} did not significantly differ between control and primed macrophages (Fig. 3A, lower panel). Because priming did not alter basal or IFN-{gamma}-induced SOCS1 expression, SOCS1 is unlikely to mediate the suppression of Stat1-independent genes in primed macrophages. In contrast, IFN-{gamma} priming resulted in sustained elevation of Stat1 expression (Ref.19 and Fig. 3A, upper panel), and we wished to determine whether increased Stat1 expression could contribute to the reduced induction of select IFN-{gamma} target genes in primed cells. Human monocytic THP-1 cells were transduced to overexpress full-length Stat1 or Stat1{beta}, an alternatively spliced isoform that lacks the transcriptional activation domain (25, 26). Overexpression of either Stat1 or Stat1{beta} led to enhanced Stat1 tyrosine phosphorylation upon IFN-{gamma} stimulation (Fig. 3B, upper panel). Moreover, increased expression of Stat1 resulted in increased activation of Mig expression, whereas Stat1{beta} suppressed IFN-{gamma} induction of Mig (Fig. 3C), consistent with the previously reported dominant-negative function of this transcriptionally inactive variant (27). In contrast to Mig, elevated expression of Stat1 suppressed IFN-{gamma} induction of genes belonging to the less induced group whose induction was Stat1 independent (Table IV and Fig. 2), namely CD63, EGR2, Fas, and VCAM-1 (Fig. 3D, bars 5–8). Interestingly, elevated expression of Stat1{beta} inhibited the activation of these genes as efficiently as did full-length Stat1 (Fig. 3E). These results indicate that Stat1 is a negative regulator of a subset of IFN-{gamma}-activated genes and that this inhibition does not require the transcriptional activation domain of Stat1. Elevated Stat1 or Stat1{beta} expression did not result in increased down-regulation of the "newly suppressed" genes in Table IV (data not shown), suggesting that Stat1 did not serve as a direct transcriptional repressor of these genes.



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  		FIGURE 3. Stat1 overexpression suppresses IFN-{gamma} activation of genes induced by Stat1-independent pathways. A, Upper and middle panels, Human primary macrophages were treated with 3 U/ml IFN-{gamma} as indicated. Stat1 and SOCS1 mRNA levels were quantitated using real-time PCR. Lower panel shows the average signal intensities of SOCS1 derived from microarray analysis. B, THP-1 monocytic cells transduced with eGFP, Stat1, or Stat1{beta}-encoding lentiviral particles were activated with 100 U/ml IFN-{gamma} for 15 min. Whole-cell lysates were subjected to immunoblotting with anti-tyrosine phosphorylated Stat1 (pY-Stat1), anti-Stat1, and anti-Stat3 Abs. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN-{gamma} for 3 h and mRNA levels of Mig were quantitated using real-time PCR. D, THP-1 cells transduced with eGFP or Stat1-encoding lentiviral particles were stimulated with 100 U/ml IFN-{gamma} for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR. E, THP-1 cells were stimulated with 100 U/ml IFN-{gamma} for 24 h, and mRNA levels were determined using real-time PCR.

 
Elevated Stat1 expression suppresses IFN-{gamma} induction of Stat3-dependent genes

IFN-{gamma} weakly activates Stat3 and genetic ablation of Stat1 results in stronger IFN-{gamma} activation of Stat3, along with activation of certain Stat3-dependent genes, in murine embryonic fibroblasts (9). We wished to test whether the converse situation applied in our experiments, namely that elevated Stat1 expression suppressed IFN-{gamma} activation of certain genes by suppressing Stat3 activation. First, we tested the Stat3 dependence of genes that were suppressed by high levels of Stat1 in macrophages. We used lentiviral-mediated RNAi (21) to down-regulate Stat3 expression in THP-1 cells (Fig. 4A, top panel). As an expected consequence of strongly diminished Stat3 expression, the activation of SOCS3, a Stat3-dependent gene (28), by IFN-{gamma} was abrogated (Fig. 4B). Notably, IFN-{gamma} activation of the CD63, EGR2, Fas, and VCAM-1 genes that was observed in control cells was almost completely abrogated in cells expressing low levels of Stat3 (Fig. 4C).



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  		FIGURE 4. Stat3 mediates IFN-{gamma} activation of certain Stat1-independent genes. A, THP-1 cells were transduced with lentiviral particles encoding DSred2 small interfering RNA (siRNA) or Stat3 siRNA. Cell lysates from lentiviral-transduced THP-1 cells were subjected to immunoblotting. B, Lentiviral-transduced THP-1 cells were stimulated with 100 U/ml IFN-{gamma} for 3 h, and mRNA levels of SOCS3 were quantitated using real-time PCR. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN-{gamma} for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR.

 
These results showed that the genes suppressed by high levels of Stat1 were Stat3 dependent, and suggested that high Stat1 expression may block Stat3 activation or function. Therefore, we tested the effects of high Stat1 expression on Stat3 activation by IFN-{gamma}. As expected (19), increased Stat1 protein expression resulted in increased Stat1 tyrosine phosphorylation (Fig. 5A, panels 1 and 2). In contrast, Stat3 tyrosine phosphorylation was comparable in IFN-{gamma}-treated cells that expressed either low or high Stat1 levels (Fig. 5A, panels 3 and 4). Thus, increased Stat1 expression did not alter activation of Stat3 by the IFN-{gamma}R. Next, we wished to test whether increased Stat1 expression altered Stat3 translocation to the nucleus. The weak activation of Stat3 by IFN-{gamma}, basal nuclear localization of a fraction of Stat3 molecules, and nuclear translocation of only a fraction of activated STATs precluded reliable measurement of Stat3 nuclear translocation using immunoblotting (data not shown). However, IFN-{gamma} induction of Stat3 nuclear DNA-binding activity was detected using a supershift assay, and was diminished in IFN-{gamma}-primed cells (Fig. 5B). Diminished nuclear Stat3 activity in cells expressing high Stat1 levels would explain the diminished IFN-{gamma} activation of Stat3-dependent genes that was observed (Figs. 3 and 4) and predicts that sufficiently high Stat1 expression would inhibit expression of a canonical Stat3-dependent gene such as SOCS3 (28). Indeed, overexpression of Stat1 or Stat1{beta} in THP-1 cells abrogated the induction of SOCS3 expression (Fig. 5C). Overall, the results show that increased Stat1 expression antagonized Stat3 function and suppressed activation of Stat3-dependent genes.



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  		FIGURE 5. Regulation of Stat3 activation in primed macrophages expressing high Stat1 levels. A, Primary human macrophages were primed with 3 U/ml IFN-{gamma} for 2 days and restimulated with 10 U/ml IFN-{gamma} for the indicated time periods. Whole-cell extracts were subjected to immunoblotting. B, Control or IFN-{gamma}-primed primary macrophages were stimulated with 10 U/ml IFN-{gamma} for 15 min. Nuclear extracts were subjected to EMSA with high-affinity SIS-inducible element oligonucleotide probe. Stat3 was specifically supershifted using anti-Stat3 Abs as previously described (44 ). C, THP-1 cells transduced with eGFP, Stat1, or Stat1{beta}-encoding lentiviral particles were stimulated with 100 U/ml IFN-{gamma} for the indicated time periods, and mRNA expression of SOCS3 was measured by real-time PCR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Priming is a well-described phenomenon (29) in which pre-exposure of cells to low subactivating concentrations of IFNs sensitizes cells to produce enhanced responses to extracellular inflammatory stimuli, including IFNs themselves, TNF, and TLR activators. In this study, we used microarray analysis to gain insights into mechanisms by which priming alters macrophage responses, with a focus on IFN-{gamma} responses. One significant finding was that priming enhances migration of macrophages to the inflammatory chemokine CCL2/MCP-1. Thus, priming not only enhances inflammatory responses (29) but also promotes recruitment into inflammatory sites. Another finding was that priming alters macrophage transcriptional responses to subsequent restimulation with IFN-{gamma}, such that activation of a subset of IFN-{gamma}-inducible genes is suppressed by a mechanism that is dependent on increased Stat1 expression. Many of these genes are Stat3 dependent, and thus physiological regulation of the relative abundance of Stat1 and Stat3 during priming results in the attenuation of Stat3-dependent responses.

Priming of macrophages with subactivating concentrations of IFN-{gamma}, without restimulation, had a limited impact on the macrophage transcriptional program. This result confirmed on a global level that primed macrophages indeed do not exhibit an activated macrophage phenotype, at least at the mRNA level. However, increased expression of the PA28{beta} proteasome regulator that alters proteosome function and substrate specificity (30) suggests that control and primed macrophages may differ more substantially at the level of protein expression than mRNA expression. Indeed, the dramatic increase in Stat1 expression that occurs during priming is mediated in part by stability and accumulation of Stat1 protein (19), and increases in Stat1 mRNA in primed macrophages were more modest. Priming did not induce expression of receptors or signaling components that mediate inflammatory responses, such as cytokine receptors or TLRs, at least at the mRNA level. However, IFN-{gamma} suppressed expression of the Tyro3 and Mer protein tyrosine kinases that are important negative regulators of macrophage activation (31). An important component of IFN-{gamma}-induced macrophage activation may be the lowering of activation thresholds by suppressing expression of inhibitory receptors like Tyro3 family members.

One gene whose expression was elevated at both mRNA and protein levels in primed macrophages was CCR2, a chemokine receptor that plays a key role in recruitment of monocytes to inflammatory sites. Recently, it has been proposed that blood monocytes consist of two distinct subpopulations, and CCR2 is one of the surface markers that differentiate these two subsets (32, 33). One subset of monocytes expresses CX3CR1 and preferentially migrates into noninflamed tissues to perform homeostatic functions. Another subset expresses high CCR2 levels and preferentially migrates into inflammatory sites, presumably to participate in host defense. Our results suggest that exposure to low systemic concentrations of IFN-{gamma} will increase the CCR2high monocyte pool and will help mobilize these cells for migration to sites of infection and inflammation.

Cytokines simultaneously activate several signaling pathways that can synergize or oppose each other. As discussed in the signal orchestration model proposed by Hirano and coworkers (34), the outcome of cytokine signaling is determined by integration of the output of these signaling pathways and thus by the balance between signaling pathways that oppose each other’s activity. A large body of work (1) has demonstrated that Stat1 is a key and predominant mediator of IFN-{gamma} function. However, emerging evidence supports an important role for IFN-{gamma}-activated but Stat1-independent pathways in mediating IFN-{gamma} regulation of gene expression and cell survival/proliferation (6, 7, 9, 17). Of particular interest, in the absence of Stat1, IFN-{gamma} strongly activates Stat3 and thereby drives the transcription of select IFN-{gamma}-inducible genes in mouse embryonic fibroblasts (9). Thus, Stat3 activation seems to contribute to the induction of so-called "Stat1-independent" genes.

The previous studies showing Stat1-independent IFN-{gamma} responses were performed using Stat1-deficient cells, and their conclusions are subject to the caveat that Stat1-independent pathways that become apparent in Stat1-deficient cells may not occur in physiological settings in cells that express Stat1. Our findings extend this work by demonstrating that physiological increases in Stat1 expression that occur during macrophage priming negatively regulate IFN-{gamma}-induced Stat3-dependent pathways (and possibly other Stat1-independent pathways as well). These results support the notion advanced by Stark and colleague (9) that the relative abundance of Stat1 and Stat3, and their cross-regulation, is a key determinant of cellular responses to cytokines. Our results also provide an explanation for the discrepancy between the IFN-{gamma} induction of Stat3-dependent genes such as IL-1 and IL-6 in unprimed human monocyte-derived macrophages, but not in murine bone marrow-derived macrophages (Fig. 2 and Ref.6). This is because IFN-{gamma} activation of Stat3 was substantially weaker in murine bone marrow-derived macrophages than in human macrophages (X. Hu, unpublished data), and thus the IFN-{gamma} Stat3-dependent response was suppressed by Stat1 in murine cells even in the absence of priming. In contrast, in human macrophages, IFN-{gamma} more strongly activated Stat3 and knockdown of Stat1 using RNAi did not result in an increased Stat3-dependent response (X. Hu, unpublished data). Thus, basal levels of Stat1 were insufficient to suppress Stat3-dependent IFN-{gamma} responses in human macrophages, and an increase in Stat1 expression, such as occurs during priming, was required (Fig. 3). The relative abundance of Stat1 and Stat3 varies according to cell type and activation conditions, and Stat1 expression is elevated during immune responses to viruses (35) and in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (36, 37). It is likely that regulation of Stat1 expression plays a role in determining cytokine responses in a variety of physiological and pathophysiological settings.

It has been long appreciated that Stat1 and Stat3 have opposing biological functions, and evidence is emerging that these two transcription factors antagonize each other’s action in cytokine signal transduction pathways (9, 28, 38, 39). Our results further support the notion that Stat1 suppresses the activation of gene expression mediated by Stat3-dependent pathways (schematically illustrated in Fig. 6). There are several potential mechanisms for the counterbalancing effects between Stat1 and Stat3. For IFN-{gamma} signaling in mouse embryonic fibroblasts, it was shown that Stat1 and Stat3 compete for the same receptor docking site, resulting in enhanced Stat3 activation in the absence of Stat1 (9). In the context of IL-6R signaling, Stat1 and Stat3 cross-regulate each other’s activation via induction of inhibitory molecules, including SOCS3 (28, 38, 40, 41, 42). Tyrosine phosphorylation of STATs is regulated in both of these examples (9, 28, 38). In contrast, in our system Stat3 tyrosine phosphorylation was not affected and Stat3 function was blocked by the transcriptionally inactive Stat1{beta} (Fig. 5, A and C). Therefore, our results cannot be explained by docking site competition or Stat1-dependent induction of SOCS. Instead, our results suggest a mechanism whereby excess Stat1 sequesters Stat3 (Fig. 5B) and thereby blocks its action.



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  		FIGURE 6. Hypothetical model for regulation of IFN-{gamma} transcriptional responses by priming. The thickness of arrow lines and the font size of signaling molecules represent the intensity of signal transduction. In control human macrophages, IFN-{gamma} induces the expression of both Stat1- and Stat3-dependent genes, with minimal repression of Stat3 function by basal Stat1 levels. In IFN-{gamma}-primed cells, high Stat1 levels suppress Stat3-mediated gene activation without altering Stat3 tyrosine phosphorylation.

 
The IFN-{gamma}-inducible genes whose activation was suppressed in primed macrophages included genes that encode well-known inflammatory mediators such as IL-1, Cox-2, IL-6, MMP1, CCL5, and VCAM-1 (Table IV and Fig. 2). Interestingly, IL-1, COX-2, and MMP1 play a prominent role in mediating tissue destruction that can occur during inflammation and is deleterious for the host. IFNs are well known to posses homeostatic as well as inflammatory functions (43), and our results suggest that priming activates homeostatic mechanisms, such that primed macrophages are strongly activated at inflammatory sites but do not cause excessive tissue destruction. In conclusion, our results suggest that priming with IFNs does more than induce cells that are hyperresponsive to inflammatory stimuli (29), but also modulates cell migration, homeostatic mechanisms, and possibly additional macrophage phenotypes.


   Acknowledgments
 
We are grateful to Weill Medical College Microarray Core Facility for technical assistance. We thank Dr. James E. Darnell for the Stat1{beta} expression plasmid, Yang Hu for helpful discussions, and Drs. Paul K. Paik and Ioannis Tassiulas for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (to L.B.I.) and a Hospital for Special Surgery Pilot and Feasibility Grant (to X.H.). Back

2 Address correspondence and reprint requests to Dr. Lionel B. Ivashkiv, Department of Medicine, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address: ivashkivl{at}hss.edu Back

3 Abbreviations used in this paper: IP-10, inducible protein 10; Mig, monokine induced by {gamma}; IRF-1, IFN regulatory factor 1; M-CSF, macrophage-CSF; qPCR, quantitative real-time PCR; RNAi, RNA interference; eGFP, enhanced GFP; Mx1, myxovirus resistance 1; OAS, oligoadenylate synthetase; Cox-2, cyclooxygenase 2; EGR2, early growth response 2; MMP1, matrix metalloproteinase 1; Bcl2A1, Bcl2-related protein A1; SOCS, suppressor of cytokine signaling. Back

Received for publication December 8, 2004. Accepted for publication July 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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