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The Journal of Immunology, 2003, 171: 5034-5041.
Copyright © 2003 by The American Association of Immunologists

Reprogramming of IL-10 Activity and Signaling by IFN-{gamma} 1

Carmen Herrero*, Xiaoyu Hu{dagger}, Wai Ping Li*, Stuart Samuels*, M. Nusrat Sharif*, Sergei Kotenko{ddagger} and Lionel B. Ivashkiv2,*,{dagger}

* 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; and {ddagger} Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One important mechanism of cross-regulation by opposing cytokines is inhibition of signal transduction, including inhibition of Janus kinase-STAT signaling by suppressors of cytokine signaling. We investigated whether IFN-{gamma}, a major activator of macrophages, inhibited the activity of IL-10, an important deactivator. Preactivation of macrophages with IFN-{gamma} inhibited two key anti-inflammatory functions of IL-10, the suppression of cytokine production and of MHC class II expression. Gene expression profiling showed that IFN-{gamma} broadly suppressed the ability of IL-10 to induce or repress gene expression. Although IFN-{gamma} induced expression of suppressor of cytokine signaling proteins, IL-10 signal transduction was not suppressed and IL-10 activation of Janus kinases and Stat3 was preserved. Instead, IFN-{gamma} switched the balance of IL-10 STAT activation from Stat3 to Stat1, with concomitant activation of inflammatory gene expression. IL-10 activation of Stat1 required the simultaneous presence of IFN-{gamma}. These results demonstrate that IFN-{gamma} operates a switch that rapidly regulates STAT activation by IL-10 and alters macrophage responses to IL-10. Dynamic regulation of the activation of different STATs by the same cytokine provides a mechanism by which cells can integrate and balance signals delivered by opposing cytokines, and extends our understanding of cross-regulation by opposing cytokines to include reprogramming of signaling and alteration of function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-10 is a potent immunosuppressive and anti-inflammatory cytokine that deactivates macrophages and dendritic cells (1). Mechanisms of IL-10 action include suppression of Ag presentation and of production of cytokines and inflammatory mediators. IL-10 is produced as part of the homeostatic response to infection and inflammation, and plays a critical role in limiting the duration and intensity of immune and inflammatory reactions (1). IL-10 production is tightly regulated, as excessive IL-10 leads to inability to control infectious pathogens, while insufficient IL-10 leads to pathology secondary to tissue injury. IL-10 binds to a heterodimeric receptor consisting of IL-10R1 and IL-10R2 subunits and activates receptor-associated Janus kinase 1 (Jak1)3 and tyrosine kinase 2 (Tyk2) kinases, leading to tyrosine phosphorylation of STAT proteins. IL-10 predominantly activates Stat3 in myeloid cells (2, 3), and Stat3 is required for many anti-inflammatory effects of IL-10 in these cells (4, 5).

The immunosuppressive potency of IL-10 depends upon the timing of IL-10 expression, and IL-10 suppressive activity can become diminished during immune and inflammatory responses. For example, IL-10 effectively suppressed cytokine production when given before, but not when given after, LPS in experimental endotoxemia (6). Similarly, IL-10 activity became diminished during establishment of a chronic infection with the LP-BM5 retrovirus (7). Diminished IL-10 activity during the active phase of an infection can be beneficial to the host, as it will result in enhanced immunity and clearance of pathogens. IL-10 immunosuppressive activity is also deficient after bone marrow or cardiac transplantation, and during the chronic autoimmune and inflammatory diseases rheumatoid arthritis and systemic lupus erythematosus (8, 9, 10, 11, 12). In these diseases, diminished IL-10 activity may be deleterious and may contribute to the inability to control and resolve inflammation. In addition to a loss of anti-inflammatory function, IL-10 can acquire proinflammatory functions during inflammation. When given after LPS, IL-10 induced higher levels of chemokines (6, 13) and IL-10 enhanced graft rejection or graft-vs-host disease when given after transplantation (8, 9). The factors that diminish or alter IL-10 activity during immunity and inflammation have not been identified, and the mechanisms that regulate IL-10 activity are not known.

A key mechanism that inhibits Jak-STAT signaling is induction of suppressors of cytokine signaling (SOCS) proteins, especially SOCS1, SOCS2, SOCS3, and cytokine inducible Src homology 2-containing protein (14). SOCS are rapidly induced in response to multiple cytokines, including cytokines that themselves activate the Jak-STAT pathway. Thus, SOCS proteins play an important role in feedback inhibition and cross-talk and antagonism among different cytokines. SOCS inhibit Jak-STAT signaling by inhibiting Jak catalytic activity, by competing with STATs for receptor docking sites, and by targeting cytokine receptors for degradation by proteosomes (14). One example of cytokine antagonism mediated by SOCS proteins is suppression of IL-4 signaling by IFN-{gamma}-induced SOCS1 (15, 16, 17, 18). We hypothesized that inflammatory cytokines that induce SOCS expression may suppress IL-10 anti-inflammatory activity during immune or inflammatory reactions. We tested the effects of IFN-{gamma}, a major activator of macrophages and of SOCS1 and SOCS3 expression, on the responses of macrophages to IL-10. IFN-{gamma} induced a state of refractoriness to the suppressive effects of IL-10 and dramatically altered the pattern of IL-10-induced gene activation in macrophages. These changes were not associated with suppression of IL-10 signaling, but, rather, a switch in the balance of IL-10 STAT activation from Stat3 to Stat1. These results identify a mechanism by which macrophages can become resistant to IL-10 during inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cell culture

IL-10 was kindly provided by K. Moore (Immunex, Seattle, WA) and recombinant human M-CSF, IL-22, and mAbs against IFN-{gamma} and IL-2 were obtained from R&D Systems (Minneapolis, MN). PBMC were obtained from whole blood from disease-free volunteers by density gradient centrifugation using Ficoll (Life Technologies, Gaithersburg, MD) and monocytes (>97% CD14+) were purified by positive or negative selection using magnetic beads (Miltenyi Biotec, Auburn, CA), as previously described (19). Monocytes and 293T cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (HyClone Laboratories, Logan, UT).

ELISA and flow cytometry

Paired TNF-{alpha} capture and detection Abs were purchased from R&D Systems and used in a sandwich ELISA according to the instructions of the manufacturer. The binding of IL-10 to cell surface IL-10Rs was measured using a Fluorokine kit according to the instructions of the manufacturer (R&D Systems).

Gene expression analysis

cRNA from four independent blood donors was obtained and hybridized to U95Av2 oligonucleotide microarrays according to the instructions of the manufacturer (Affymetrix, Santa Clara, CA). Data were analyzed using Affymetrix Suite 5.0 and Genespring (Silicon Genetics, Palo Alto, CA). The results for 16 genes were verified in the original four donors using real-time PCR, and results for 4 genes were verified in up to five additional independent blood donors. For real-time, quantitative PCR, 1 µg of total RNA was reverse-transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase. Real-time PCR was performed in triplicate using the iCycler iQ thermal cycler and detection system (Bio-Rad, Hercules, CA), and the PCR Core Reagents kit (Applied Biosystems, Foster City, CA), with 500 nM primers; the final Mg2+ concentration was adjusted to 4 mM, as previously described (19). mRNA amounts were normalized relative to GAPDH mRNA. When reverse transcriptase was omitted, the threshold cycle number increased by at least 10, signifying lack of genomic DNA contamination or nonspecific amplification, and the generation of only the correct size amplification products was confirmed using agarose gel electrophoresis.

EMSA and immunoblotting

EMSA were performed with 5 µg of cell extracts and 32P-labeled double stranded high affinity SIS-inducible element (M67) oligonucleotide as previously described (20). For immunoblotting, 10 µg of cell lysates were fractionated on 7.5% polyacrylamide gels using SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). In some experiments, cells were lysed directly in culture wells using SDS-PAGE gel loading buffer with 10% 2-ME and equal volumes of lysates were analyzed by immunoblotting. mAbs against Stat1, Stat3, Jak1, and Tyk2 were obtained from BD Transduction Laboratories (Lexington, KY). Phosphorylation-specific Stat1 (Tyr701), Stat3 (Tyr703), and Tyk2 (Tyr1054/1055) Abs were purchased from Cell Signaling Technology (Beverly, MA). Phosphorylation-specific anti-Jak1 (Tyr1022/1023) was from BioSource International (Camarillo, CA).

Transfections

A plasmid encoding an IL-22R1-IL-10R1 fusion receptor was constructed by amplifying the IL-10R1 cytoplasmic domain using PCR and fusing it with the extracellular and transmembrane domains of IL-22R1 (21). Cloning junctions and the sequence of the IL-10R1 intracellular domain were confirmed by DNA sequencing. 293T cells were transfected in duplicate in six-well plates using Lipofectamine (Life Technologies) and a total of 12 µg of plasmid DNA per well. Cells were cotransfected with plasmids encoding IL-22R1-IL-10R1, either Stat1, Stat3, or an empty control expression vector, and {beta}-galactosidase. Cells were split 18 h after transfection, and then stimulated with cytokines after an additional 24 h of culture. Transfection efficiency was monitored by assaying for {beta}-galactosidase activity. A total of five transfection experiments were performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} impairs IL-10 anti-inflammatory activity

We investigated the effects of IFN-{gamma} on the ability of IL-10 to suppress LPS-induced TNF-{alpha} production. Primary blood monocytes were cultured for 2 days with M-CSF with or without IFN-{gamma}, and then stimulated with LPS in the presence or absence of IL-10. As expected, LPS-induced TNF-{alpha} production in control macrophages was strongly suppressed by IL-10 (Fig. 1A). In IFN-{gamma}-activated macrophages, the effectiveness of IL-10 in suppressing TNF-{alpha} production was diminished (Fig. 1A; a representative experiment of six performed is shown). Similar results were obtained when IL-6 production was analyzed (data not shown). IL-10 suppressed HLA-DR expression in control macrophages and this suppressive effect was diminished in IFN-{gamma}-activated macrophages (Fig. 1B). These results indicate that IFN-{gamma}-activated macrophages are partially refractory to the anti-inflammatory and immunosuppressive effects of IL-10.



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  		FIGURE 1. IFN-{gamma} impairs IL-10 anti-inflammatory activity. Blood monocytes were cultured for 2 days with MCSF (20 ng/ml) with or without IFN-{gamma} (100 U/ml) before adding IL-10. A, IFN-{gamma} suppresses IL-10 inhibition of LPS-induced TNF-{alpha} production. Macrophages were treated with IL-10 for 12 h and then activated with LPS (100 ng/ml). Supernatants were collected 18 h later and TNF-{alpha} levels were measured using ELISA. Total LPS-induced TNF-{alpha} production was 1.1 ng/ml in control cells and 1.3 ng/ml in IFN-{gamma}-activated cells. B, IFN-{gamma} suppresses IL-10 inhibition of HLA-DR expression. Control or IFN-{gamma}-activated macrophages were cultured with IL-10 (100 ng/ml) for 36 h and cell surface HLA-DR was measured using flow cytometry.

 
Altered patterns of IL-10-inducible gene expression in IFN-{gamma}-activated macrophages

IL-10 anti-inflammatory activity requires, at least in part, de novo gene expression (22, 23). We used oligonucleotide microarrays to determine the effects of IFN-{gamma} on IL-10 induction of gene expression. In control macrophages, IL-10 induced statistically significant (p < 0.002) and >3-fold increases in expression of 121 genes after 3 h, and 142 genes after 24 h, of stimulation (Fig. 2A). Strikingly, 93 of 121 (77%) (3-h time point) and 112 of 142 (79%) (24-h time point) genes induced by IL-10 in control cells were no longer induced by IL-10 in IFN-{gamma}-activated macrophages (Fig. 2A). The patterns of gene expression detected by the microarray were verified by real-time PCR, and were confirmed for 16 of 16 genes that were tested; examples of four genes whose expression was no longer induced by IL-10 in IFN-{gamma}-activated macrophages are shown in Fig. 2B. These results indicate that IFN-{gamma} broadly suppressed the ability of IL-10 to induce gene expression; a list of genes whose induction by IL-10 was suppressed in IFN-{gamma}-activated macrophages is shown in Table I. Decreased induction of gene expression by IL-10 in IFN-{gamma}-activated cells was manifested by lower mean fluorescence intensity (Table I), thus indicating diminished magnitude of gene expression. Interestingly, IL-10 activated expression of novel genes in IFN-{gamma}-activated macrophages (Fig. 2A). Genes that were IL-10-inducible in IFN-{gamma}-activated macrophages and more highly expressed (>2-fold) in IFN-{gamma}-activated than in control macrophages after IL-10 stimulation include several genes that subsume a proinflammatory or immune function, including the IL-17R, GM-CSF receptor {beta}, vascular endothelial growth factor, CCR2, MHC class I, {beta}2-microglobulin, and Fc{gamma}RI genes (Table II). This induction of proinflammatory gene expression is consistent with reports of acquisition of proinflammatory activity by IL-10 in vivo (8, 9, 10, 13).



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  		FIGURE 2. IFN-{gamma} alters gene regulation by IL-10. Blood monocytes were cultured for 2 days with MCSF (20 ng/ml) with or without IFN-{gamma} (100 U/ml) and then stimulated with IL-10 (100 ng/ml) for 3 or 24 h. Gene expression was measured using oligonucleotide microarrays as described in experimental procedures. A, Genes induced >3-fold after IL-10 stimulation of control or IFN-{gamma}-activated macrophages. B, Representative real-time PCR results showing relative mRNA levels for four genes that were less induced by IL-10 in IFN-{gamma}-activated macrophages than in control macrophages. C, Genes suppressed >3-fold after IL-10 stimulation of control or IFN-{gamma}-activated macrophages. D, Pattern of expression of genes that were repressed by IL-10 in control macrophages, but not in IFN-{gamma}-activated macrophages.

 

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  		Table I. Genes less induced by IL-10 in IFN-{gamma}-activated macrophages than in control macrophagesa

 

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  		Table II. Genes more highly expressed in IFN-{gamma}-activated macrophages than control macrophages after IL-10 stimulationa

 
In control macrophages, 3 h of IL-10 treatment resulted in lower expression (p < 0.002, >3-fold) of 25 genes (Fig. 2C). IFN-{gamma} almost completely blocked the ability of IL-10 to suppress gene expression (Fig. 2, C and D). The genes that were suppressed by IL-10 in control cells but were no longer suppressed in IFN-{gamma}-activated macrophages included TNF-{alpha}, IL-1, and IL-8. These results are consistent with a loss of IL-10 anti-inflammatory function in IFN-{gamma}-activated macrophages. Taken together, the results show that IFN-{gamma} alters macrophage responses to IL-10 in a global fashion, with a coordinate loss of IL-10-mediated gene regulation and anti-inflammatory function. Altered IL-10-dependent gene regulation may explain some of the IFN-{gamma}-induced changes in IL-10 activity that were observed in Fig. 1.

IFN-{gamma} does not inhibit IL-10 signaling, but couples IL-10 with the activation of Stat1

We reasoned that the suppression of IL-10 function and of IL-10-induced gene expression in IFN-{gamma}-activated macrophages occurred secondary to suppression of IL-10 signal transduction, especially suppression of the activation of Stat3 that is required for IL-10 anti-inflammatory activity (4, 5). Because IFN-{gamma}-activated macrophages express high levels SOCS proteins (Ref. 19 and data not shown), we anticipated that IL-10 signaling would be blocked at a proximal level, resulting in diminished tyrosine phosphorylation of Stat3. We examined IL-10 activation of the Jak-STAT signaling pathway in control and IFN-{gamma}-activated macrophages using immunoblotting and EMSAs to measure, respectively, STAT tyrosine phosphorylation and DNA binding. As expected, IL-10 effectively induced tyrosine phosphorylation of Stat3 in control cells (Fig. 3, top panel), whereas phosphorylation of Stat1 was minimally induced (Fig. 3, second panel, lane 2). Surprisingly, IL-10-induced Stat3 tyrosine phosphorylation was not diminished in IFN-{gamma}-activated macrophages (Fig. 3, top panel, lane 4). In contrast to Stat3, IL-10 activated dramatically higher levels of Stat1 tyrosine phosphorylation in IFN-{gamma}-activated than in control macrophages (Fig. 3, second panel). Increased Stat1 tyrosine phosphorylation was associated with increased Stat1 protein levels that were induced by IFN-{gamma} (Fig. 3, fourth panel). Stat3 levels were modestly increased in this experiment (Fig. 3, third panel), a result that was not consistent among different blood donors. Increased expression and activation of Stat1 by IL-10 in IFN-{gamma}-activated macrophages was reproducibly observed in over 50 independent experiments using different blood donors (data not shown). Signaling by all cytokines that use the Jak-STAT pathway was not nonspecifically sensitized, as there was no increase in STAT activation by GM-CSF or IL-4 in IFN-{gamma}-activated macrophages (data not shown).



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  		FIGURE 3. IFN-{gamma} couples IL-10 with activation of Stat1. Monocytes cultured for 2 days with MCSF (20 ng/ml) with or without IFN-{gamma} (100 U/ml) were stimulated with IL-10 (100 ng/ml) for 10 min and STAT activation was measured by immunoblotting or EMSA with the high affinity SIS-inducible element oligonucleotide.

 
Stat1 and Stat3 can bind to DNA as homodimers or as Stat1:Stat3 heterodimers (24). Consistent with previous reports of activation of Stat3 by IL-10 in myeloid cells (2, 3), IL-10 induced a more slowly migrating DNA binding complex in control macrophages that supershift experiments showed contained Stat3 homodimers and Stat3:Stat1 heterodimers (Fig. 3, bottom panel, and data not shown). In contrast, in IFN-{gamma}-activated macrophages, IL-10 activated a dramatically increased amount of DNA binding activity, with a DNA binding complex of faster mobility that contained Stat1 homodimers and Stat1:Stat3 heterodimers (Fig. 3 and data not shown). These results, taken together, show that IFN-{gamma} did not block IL-10 signaling, but, instead, altered the balance of IL-10-induced STAT activation toward the activation of Stat1, which is typically activated by IFN-{gamma} and mediates its proinflammatory functions (25, 26). Stat1 can function either as an activator or repressor of transcription, and increased activation of Stat1 may contribute to altered IL-10 regulation of gene expression in IFN-{gamma}-activated macrophages that was observed in Fig. 2.

Proximal IL-10 signaling is minimally affected by IFN-{gamma}

We investigated the possibility that stimulation with IFN-{gamma} linked IL-10 with activation of Stat1 by altering IL-10 signaling proximal to activation of STATs, at the level of the IL-10R or associated Jaks. IFN-{gamma} activation of macrophages resulted in a modest increase in cell surface IL-10R expression (Fig. 4A), and levels of tyrosine-phosphorylated Jak1 and Tyk2 were comparable in control and IFN-{gamma}-activated macrophages treated with IL-10 (Fig. 4B). These results are consistent with the comparable IL-10 activation of Stat3 in control and IFN-{gamma}-activated cells (Fig. 3), and indicate that the dramatic increase of Stat1 activation in IFN-{gamma}-activated macrophages cannot be explained on the basis of increased proximal signaling by the IL-10R.



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  		FIGURE 4. IFN-{gamma} does not affect proximal IL-10 signaling. Monocytes were cultured for 2 days with MCSF (20 ng/ml) with or without IFN-{gamma} (100 U/ml). A, Cell surface expression of IL-10 was measured using biotinylated IL-10 and flow cytometry. B, Cells were stimulated for 10 min with IL-10 (100 ng/ml) and activation of Jaks was measured using immunoblotting.

 
Increased Stat1 expression is not sufficient to induce activation of Stat1 by IL-10

We have recently shown that increased macrophage Stat1 expression results in increased IFN-{gamma} Stat1 activation, likely secondary to concentration-dependent increases in the interaction of Stat1 with its docking site on the IFN-{gamma}R (19). To determine whether increased Stat1 expression could also drive increased Stat1 activation by IL-10, we primed macrophages for 2 days with a low concentration of IFN-{gamma} (3 U/ml) that increases Stat1 expression, but does not activate macrophages (19). IFN-{gamma} (3 U/ml) induced a substantial increase in Stat1 levels, comparable to those induced by 100 U/ml IFN-{gamma} (Fig. 5A, middle panel). IL-10 strongly activated Stat1 in macrophages that had been preactivated with 100 U/ml IFN-{gamma} (Fig. 5A, top panel, lane 6), but not in macrophages that had been primed with 3 U/ml IFN-{gamma} (lane 4). Thus, activation of Stat1 by IL-10 was not proportional to increases in Stat1 expression.



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  		FIGURE 5. Increased Stat1 expression does not result in increased Stat1 activation by IL-10. A, Monocytes were cultured for 2 days with either 3 or 100 U/ml of IFN-{gamma}, stimulated for 10 min with IL-10, and Stat1 expression and activation were measured using immunoblotting and EMSA. B, 293T cells were transfected with expression plasmids encoding an IL-22R1-IL-10R1 fusion receptor and Stat3 or Stat1. Transfected cells were stimulated with IL-22 and STAT expression and activation was analyzed using immunoblotting. Positive control = cell extracts from IFN-{gamma}-activated macrophages.

 
To address the effects of elevated Stat1 levels on IL-10 signaling in the absence of any cytokine pretreatment of cells before adding IL-10, we cotransfected 293T cells that do not express IL-10R1 with expression plasmids encoding Stat1 and a fusion receptor containing the IL-22R1 extracellular domain fused with the IL-10R1 cytoplasmic domain. IL-22 ligates IL-22R1 and IL-10R2, and thus addition of IL-22 to cells expressing IL-22R1-IL-10R1 and endogenous IL-10R2 will generate the same signal as IL-10 (21, 27). 293T cells contain low levels of Stat1 and Stat3 relative to macrophages (Fig. 5B), and activation of STATs was not detected after IL-22 stimulation of cells transfected with IL-22R1-IL-10R1 alone (Fig. 5B, lane 3). When Stat3 expression was increased by cotransfecting a Stat3 expression plasmid, IL-22 induced Stat3 tyrosine phosphorylation (Fig. 5B), consistent with the presence of Stat3 docking sites in the IL-10R1 cytoplasmic domain (3). In contrast, comparable overexpression of Stat1 did not result in activation of Stat1 by IL-22 (Fig. 5B). Therefore, we conclude that high Stat1 levels are not sufficient to explain its phosphorylation after IL-10 treatment. These results, taken together with previous evidence that the IL-10R does not contain a Stat1 docking site (3), suggest that activation of Stat1 by IL-10 in IFN-{gamma}-activated macrophages does not occur via direct Stat1-IL-10R interactions.

Activation of Stat1 by IL-10 requires the simultaneous presence of IFN-{gamma}

Previous reports showed that low levels of autocrine IFN-{alpha} increase Stat1 activation by IFN-{gamma} and IL-6 (28, 29, 30). We investigated whether the supernatants of IFN-{gamma}-activated macrophages contained type I IFNs or other soluble factors that promoted Stat1 activation by IL-10. When we removed factors present in culture supernatants by washing IFN-{gamma}-activated cells immediately before adding IL-10, IL-10 no longer activated Stat1 (Fig. 6A). Addition of blocking anti-IFN-{alpha}{beta}R Ab, which completely blocked signaling by this receptor (19), had a minimal effect on Stat1 activation by IL-10 (Fig. 6B). This indicates that activation of Stat1 by IL-10 in our system was not the result of autocrine IFN-{alpha}{beta} production. In contrast, neutralization of IFN-{gamma} in culture supernatants resulted in diminished IL-10 activation of Stat1, while activation of Stat3 was minimally affected (Fig. 6B). These results indicate that IFN-{gamma}, and ongoing ligation of IFN-{gamma}Rs, did not affect the standard IL-10 signaling pathway that activates Stat3, but was required for coupling IL-10 to the activation of Stat1.



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  		FIGURE 6. Activation of Stat1 by IL-10 requires the simultaneous presence of IFN-{gamma}. For A and B, monocytes were cultured for 2 days with MCSF (20 ng/ml) with or without IFN-{gamma} (100 U/ml). A, Cells were harvested after 2 days of culture, washed (where indicated, lane 3), replated, and immediately stimulated with IL-10. STAT activation was measured using EMSA. B, Neutralizing Abs to IFN-{gamma} or IL-2, or a blocking anti-IFN-{alpha}{beta}R Ab were added 15 min or 2 h before adding IL-10. STAT expression and activation were measured using immunoblotting. C, IFN-{gamma} rapidly couples IL-10 to Stat1 activation. Monocytes were treated with IFN-{gamma} for 4 or 6 h, stimulated with IL-10 for 10 min, and STAT expression and activation were measured using immunoblotting.

 
In the previous experiments, we showed that IL-10 signaling became linked to activation of Stat1 in macrophages that expressed high levels of Stat1 protein after 2 days of activation with IFN-{gamma} (Fig. 3). We tested whether shorter incubations with IFN-{gamma} that did not increase Stat1 expression were sufficient to alter IL-10 signaling. Four hours after addition of IFN-{gamma}, IFN-{gamma}-induced Stat1 tyrosine phosphorylation had decayed to background levels, and Stat1 levels were not increased (Fig. 6C). IL-10 strongly activated Stat1 when added 4 h after IFN-{gamma}, before any increase in Stat1 levels (Fig. 6C). IL-10 also strongly activated Stat1 when added 6 h after IFN-{gamma}, when there was minimal change in Stat1 levels (Fig. 6C). This shows that elevated Stat1 expression was not necessary for IL-10 activation of Stat1, and further supports the argument that IL-10 activation of Stat1 does not occur solely as a consequence of high Stat1 expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-10 is an important homeostatic cytokine that acts to limit the duration and intensity of immune and inflammatory responses and prevent excessive tissue damage. In vivo, IL-10 can be expressed within the first few hours of immune and inflammatory reactions (1, 4), and the potential risk of excessive IL-10 activity early during an immune response is ineffective clearance of infectious pathogens. Emerging data suggest that IL-10 immunosuppressive and anti-inflammatory activity is modulated during the course of an immune or inflammatory response, and this modulation plays a role in fine-tuning the balance between effective clearance of pathogens and excessive inflammation (6, 7, 8, 9, 10, 11, 12, 13, 31). We now identify IFN-{gamma} as an important factor that alters macrophage responses to IL-10 and dampens IL-10 immunosuppressive and anti-inflammatory activity. In the presence of IFN-{gamma}, IL-10 was less effective at suppressing cytokine and chemokine production, and in down-regulating MHC class II expression. Thus, during the active phase of an immune or inflammatory response characterized by high levels of IFN-{gamma} production, IL-10 activity would be restrained, such that pathogens could be effectively cleared. This type of block in IL-10 activity has been observed in vivo under conditions where IFN-{gamma} is expressed (6, 11, 31). The suppression of IL-10 activity in IFN-{gamma}-activated macrophages was partial, but we have found that IFN-{gamma} also sensitizes macrophages such that IL-10 signaling can be nearly completely blocked after Fc{gamma}R ligation by immune complexes (L. Ivashkiv, unpublished data). Thus, different inflammatory factors may work together with IFN-{gamma} to regulate IL-10 signaling during the course of an immune response. After pathogens are cleared and the immune response begins to wane and IFN-{gamma} levels drop, IL-10 that is already present would become more active in deactivating cells and resolving inflammation. Regulation of IL-10 activity by IFN-{gamma} thus provides an additional level of flexibility in regulating macrophage activation beyond that provided by regulation of IL-10 expression alone.

IFN-{gamma} redirected IL-10 signaling from activation of Stat3, which is anti-inflammatory in macrophages, to activation of Stat1, which is proinflammatory. Stat3 and Stat1 oppose each other in the regulation of several important cellular functions, including proliferation and inflammation (32, 33, 34). Opposing STATs are typically activated by different cytokines, but recent evidence shows that individual cytokines can activate STATs that oppose each other’s functions (35, 36). For example, Stat3 suppresses Stat1 activation by IL-6, with concomitant suppression of activation of Stat1 target genes (35), and Stat1 and Stat4, both activated by IFN-{alpha}, oppose each other during viral infection in vivo (36). In these previously described cases, Stat3 suppressed IL-6-induced tyrosine phosphorylation of Stat1, and Stat1 suppressed IFN-{alpha}-induced tyrosine phosphorylation of Stat4. The underlying mechanisms by which tyrosine phosphorylation was inhibited have not been resolved. In contrast, in our experiments, Stat3 function was suppressed, possibly secondary to heterodimerization with high levels of activated Stat1, but levels of Stat3 tyrosine phosphorylation were preserved. The intact activation of Jak1 and Stat3 by IL-10 in IFN-{gamma}-activated macrophages is consistent with previous reports showing that IL-10 signaling can be relatively resistant to inhibition by SOCS proteins in a cell type-dependent manner (37, 38). Our results showing that IFN-{gamma} regulates the balance of Stat1 and Stat3 activation by IL-10, together with the results from Biron and O’Shea and colleagues (36) demonstrating differential activation of Stat1 and Stat4 by IFN-{alpha}{beta} over the time course of a viral infection in vivo, support the notion that dynamic regulation of the activation of different STATs by cytokines plays an important role in determining the activity of pleiotropic cytokines.

The coupling of IL-10 to activation of Stat1 was dependent upon the simultaneous presence of IFN-{gamma}. As the IL-10R does not contain Stat1 docking sites (3), it is possible that IFN-{gamma} mediates an interaction between the IL-10R and either the IFN-{gamma}R or another receptor that contains a Stat1 docking site. Addition of IL-10 then would lead to transphosphorylation of this receptor and Stat1 activation. This notion is supported by previous reports showing transphosphorylation and activation of heterologous receptors by ligands that do not directly bind that receptor (30, 37, 39, 40, 41, 42), interactions between receptor subunits in the type II receptor family that includes IFN-{alpha}, IFN-{gamma}, and IL-10 (27, 28), and activation of Stat1 by an IL-10R mutated in known STAT docking sites (37). To date, we have not been able to document an association of IL-10R and IFN-{gamma}R subunits (C. Herrero, unpublished data), but these experiments have been limited by available reagents and the low expression of these receptors in primary macrophages in which IFN-{gamma} regulates IL-10 activation of Stat1. Another possible explanation of our findings is that IFN-{gamma} signaling is down-regulated in cells cultured in the presence of IFN-{gamma}, and IL-10 reverses this down-regulation, thereby restoring the magnitude of the signal generated by IFN-{gamma} present in culture supernatants. We were unable to detect increased tyrosine phosphorylation of the IFN-{gamma}R after IL-10 stimulation of IFN-{gamma}-activated macrophages, or increased Stat1 tyrosine phosphorylation after incubation with the protein tyrosine phosphatase inhibitor orthovanadate (C. Herrero and X. Hu, unpublished data). Thus, IL-10 did not restore activation of the IFN-{gamma}R to a level that could be detected by phosphotyrosine immunoblotting, and did not work by inhibiting a phosphatase that constitutively dephosphorylated Stat1. However, it remains possible that IL-10 restored low level IFN-{gamma}R phosphorylation that was not detected, or that IL-10 restored IFN-{gamma} signaling by reversing other mechanisms that down-regulate IFN-{gamma} signaling in these cells.

Antagonism among opposing cytokines is often explained on the basis of signal transduction cross-talk leading to a block in cytokine activity. Examples include IFN-{gamma}-induced suppression of the anti-inflammatory cytokines IL-4 and TGF-{beta} by induction of, respectively, the inhibitory proteins SOCS1 (15, 16, 17) and Sma- and Mad-related protein 7 (43). In these examples, IFN-{gamma} solely suppressed IL-4 and TGF-{beta} signaling and function. In contrast, IFN-{gamma} not only suppressed the anti-inflammatory effects of IL-10, but redirected IL-10 signaling to activate the proinflammatory Stat1 protein. Thus, IFN-{gamma} co-opts IL-10 to signal more like IFN-{gamma} itself, and allows IL-10 to activate Stat1 at a time when IFN-{gamma} activation of Stat1 has been down-regulated by feedback inhibition (19). Activation of Stat1 may mediate some of the proinflammatory functions of IL-10 that have been described during inflammation in vivo (6, 8, 9, 10, 13). A switch in cytokine activity that is induced by an antagonistic cytokine adds an additional level of complexity to cytokine cross-regulation that goes beyond simple inhibition of signaling. It is interesting to propose that reprogramming of cytokine activity may play a role in cytokine cross-regulation in other systems that are regulated by opposing cytokines, such as T cell differentiation (44).


   Acknowledgments
 
We thank Kevin Moore for helpful discussions and kindly providing IL-10, Anne Davidson for helpful suggestions, and Peggy Crow and Zhimei Du for critical review of the manuscript.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health and Kirkland Center for Systemic Lupus Erythematosus Research (to L.B.I.) and National Institutes of Health Grant AI51139 (to S.K.). 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: Jak, Janus kinase; Tyk, tyrosine kinase; SOCS, suppressor of cytokine signaling. Back

Received for publication May 15, 2003. Accepted for publication September 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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