The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, X.
Right arrow Articles by Ivashkiv, L. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, X.
Right arrow Articles by Ivashkiv, L. B.
The Journal of Immunology, 2005, 175: 131-138.
Copyright © 2005 by The American Association of Immunologists

Homeostatic Role of Interferons Conferred by Inhibition of IL-1-Mediated Inflammation and Tissue Destruction

Xiaoyu Hu*, Hao H. Ho*, Olivia Lou{ddagger}, Chisa Hidaka{dagger} and Lionel B. Ivashkiv2,*,{ddagger}

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In addition to their well known immune and proinflammatory activities, IFNs possess homeostatic functions that limit inflammation and tissue destruction in a variety of conditions such as arthritis, osteolysis, and multiple sclerosis. The mechanisms underlying the homeostatic actions of IFNs are not well understood. We report here that both type I and type II IFNs (IFN-{alpha}, IFN-{beta}, and IFN-{gamma}, respectively) suppressed a broad range of proinflammatory and tissue-destructive activities of IL-1, including induction of inflammatory mediators, production of matrix metalloproteinases, macrophage tissue invasion, and cartilage degradation. IFN-{alpha} attenuated IL-1-mediated cell recruitment in vivo. IFNs completely suppressed the activation of IL-1 signal transduction pathways in macrophages. The mechanism of IFN-mediated inhibition of IL-1 action and signaling was modulation of IL-1R expression, which was also observed in vivo. IFN-{gamma}-mediated down-regulation of IL-1R type I expression was dependent on Stat1, a transcription factor typically considered to be a key mediator of macrophage activation by IFNs. These results identify cellular and molecular mechanisms that contribute to the homeostatic role of IFNs in limiting inflammation and associated tissue destruction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The IFNs were first identified on the basis of interference with viral replication and are grouped into type I IFNs (including IFN-{alpha}, IFN-{beta}, and IFN-{tau}), type II IFN (IFN-{gamma}), and IFN-{lambda}s (1, 2, 3). In addition to mediating antiviral effects, IFNs are important modulators of innate and acquired immune responses (1). IFN-{gamma} is generally considered to be a proinflammatory factor based on its strong activation of macrophage antimicrobial activity, Ag presentation, and synergy with TNF-{alpha} and with TLR ligands such as LPS in the activation of macrophage inflammatory functions (4). Anti-inflammatory effects of IFN-{gamma} are less well appreciated but have clearly been described in certain settings in vivo. The most compelling support for the anti-inflammatory action of IFN-{gamma} comes from the exacerbation of several experimental inflammatory diseases in the absence of IFN-{gamma} signaling. For example, collagen-induced arthritis was exacerbated in mice lacking IFN-{gamma} or its receptor (5, 6, 7), and deletion of the IFN-{gamma} gene greatly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE),3 an animal model for multiple sclerosis (MS) (8, 9). Type I IFNs are pleiotropic and can promote immune responses, but they possess anti-inflammatory properties as well. Type I IFNs are used as first line therapeutics for relapsing-remitting MS and are effective at alleviating CNS inflammation (10). In animal studies, administration of type I IFNs markedly reduced inflammation-induced osteolysis (11). It has been proposed that suppression of cell proliferation and promotion of apoptosis contribute to the suppressive effects of IFNs, but the cellular and molecular basis for the homeostatic functions of IFNs is not well understood (4).

IFNs mainly use the Jak-Stat signaling pathway to transmit signals from extracellular ligands to the nucleus. The latent cytoplasmic transcription factor Stat1 is activated by both type I and type II IFNs and plays a key role in mediating their biological effects (1). Stat1 mediates many of the macrophage-activating effects of IFNs (12) and induces expression of many genes involved in microbial recognition and clearance and in Ag presentation (13, 14). On the other hand, Stat1 mediates the antiproliferative and proapoptotic effects of IFNs, suggesting that Stat1 also has the potential to restrain inflammation. A role for Stat1 in limiting inflammation in vivo has been demonstrated in postinfluenza pulmonary inflammation (15), EAE (16), experimental arthritis (17), and bone remodeling (18). Dual and opposing roles for Stat1 conform to a homeostatic paradigm in immune activation in which molecules that promote cell activation and effector function also play a role in limiting the response, such that excessive activation, associated tissue damage, and autoimmunity are avoided.

IL-1 is a multifunctional cytokine produced primarily by monocytes and macrophages in response to numerous endogenous and exogenous stimuli. IL-1 possesses a broad spectrum of bioactivities including induction of cytokines and chemokines, up-regulation of inflammatory mediators, and regulation of the CNS (19). Another prominent function of IL-1 is its ability to promote tissue-invasive and -destructive processes such as cartilage breakdown, bone erosion, and angiogenesis (19). The biological activity of IL-1 is tightly controlled under physiological conditions. IL-1 action can be attenuated at three distinct levels, i.e., suppression of IL-1 production, interference with IL-1 activation of its receptor, and inhibition of IL-1-induced intracellular signal transduction pathways. Glucocorticoids and TGF-{beta} are among the agents that reduce the production of IL-1 (19). At the receptor level, binding of IL-1 to activating IL-1Rs can be suppressed by a competitive antagonist termed IL-1R antagonist (IL-1ra), by soluble IL-1Rs, and by the non-signaling decoy receptor IL-1R type II (IL-1RII) (19). At the level of signal transduction, the intensity of IL-1 signaling can be dampened by single Ig IL-1R-related molecule and ST2, which function by sequestrating receptor-proximal signaling components such as MyD88 and TNF receptor-associated factor 6 (20, 21). The existence of multiple pathways that inhibit IL-1 function at multiple levels signifies the necessity to keep IL-1 activities under control, thereby preventing excessive inflammation.

Consistent with its potent proinflammatory and tissue-destructive nature, IL-1 has been implicated in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), atherosclerosis, and autoinflammatory conditions such as Muckle-Wells syndrome (22, 23, 24). Suppression of IL-1 using rIL-1ra has been effective in the treatment of RA (25, 26), Muckle-Wells syndrome (27, 28), and neonatal-onset multisystem inflammatory disease (29, 30). Thus, IL-1 blockade is a promising approach to treatment of autoimmune and inflammatory diseases. However, therapy with IL-1ra, the only currently available anti-IL-1 therapy, is limited by a short half-life in vivo, the need to achieve high concentrations of IL-1ra at sites of inflammation, and inconvenient route and frequency of administration (31), which hinder the wide use of this drug. Therefore, additional anti-IL-1 therapeutic agents with improved efficacy are desirable for treating RA as well as other inflammatory disorders.

We hypothesized that suppression of the function of inflammatory factors may represent a novel mechanism by which IFNs restrain inflammation and promote homeostasis. Here, we investigated the regulation of IL-1 inflammatory and tissue-destructive effects by IFNs. We report that IFNs suppressed an array of IL-1-mediated effects in vitro and in vivo including induction of cytokines and other inflammatory mediators, production of matrix metalloproteinases (MMPs), cell migration and tissue invasion, and cartilage degradation. The underlying mechanisms involved regulation of IL-1R expression and Stat1-dependent extinction of IL-1R type I (IL-1RI) expression. Our results provide a molecular basis for the anti-inflammatory properties of IFNs and Stat1.


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

Human macrophages were derived from CD14+ blood monocytes cultured with 10 ng/ml M-CSF (PeproTech). CD14+ monocytes were purified from PBMC obtained from disease-free volunteers using anti-CD14 magnetic beads (Miltenyi Biotec) as previously described (32). Purity of monocytes was greater than 97% as verified by FACS. Unless otherwise specified, macrophages were treated with 5 ng/ml (equivalent to 100 U/ml) human IFN-{gamma} or 5 ng/ml (equivalent to 2000 U/ml) of IFN-{alpha}A for 24 h before stimulation with IL-1{beta}. Synovial fibroblasts from osteoarthritis patients were isolated and cultured as described (33). Murine bone marrow-derived macrophages were obtained as described (32) and maintained in medium containing 10 ng/ml M-CSF. Murine-elicited peritoneal cells were harvested from mice 3 days after intraperitoneal injection of 4% thioglycolate. After culturing cells for 4 h, non-adherent cells were removed by washing and adherent cells (>90% CD11b+ macrophages, as confirmed by FACS) were rested in fresh medium overnight before experimentation. Murine splenic macrophages were obtained by harvesting the adherent population from Liberase CI (Roche Applied Science)-digested total splenic cells. C57BL/6J mice were obtained from The Jackson Laboratory. Stat1-deficient animals on a 129S6/SvEv background (34, 35) were obtained from Taconic Farms, and wild-type 129S6/SvEv mice (Taconic Farms) were used as controls for Stat1–/– animals. The experiments using human and murine cells were approved by, respectively, the Hospital for Special Surgery Institutional Review Board and Institutional Animal Care and Use Committee. Human, mouse, and bovine IFN-{gamma} were purchased from Roche Applied Science, PeproTech, and Serotec, respectively. Human IFN-{alpha}A was obtained from BioSource International. IFN-{alpha}A/D, a universal mammalian type I IFN used in bovine and mouse experiments, was purchased from R&D Systems.

Analysis of mRNA level

For real-time PCR, total RNA was extracted using a RNeasy Mini kit (Qiagen), and 1 µg of total RNA was reverse transcribed using a First Strand cDNA synthesis kit (Fermentas). Real-time quantitative PCR was performed using iQ SYBR-Green Supermix and iCycler iQ thermal cycler (Bio-Rad) following the manufacturer’s protocols. Relative expression was normalized for levels of GAPDH. The generation of only the correct size amplification products was confirmed using agarose gel electrophoresis.

ELISA

Culture supernatants were harvested and ELISAs were performed using paired Ab sets, as recommended by the manufacturer (BD Pharmingen).

Invasion assays

Human blood-derived macrophages were placed in the upper chamber of Matrigel (BD Biosciences)-coated Transwell plates (5-µm pore size) and 100 ng/ml CCL2 was added to the bottom chamber. After 4 h, macrophages that had invaded and migrated through the Matrigel layer were counted by FACS using flow cytometry absolute counting standard as an internal control (Bangs Laboratories). For invasion into type I collagen gels, macrophages were seeded on top of the gels and allowed to migrate for 18 h into collagen gels that had been impregnated with 100 ng/ml CCL2. Cells were fixed, stained, and quantitated as previously described (36).

Cartilage proteoglycan release

Articular cartilage explants were harvested with a 5-mm diameter skin biopsy punch from the femoral condyles of young adult steer (>18 mo of age). Explant tissues were incubated with 100 ng/ml IL-1{beta} for 3 days with or without a 24-h IFN pretreatment. The amount of glycosaminoglycan released into the medium from cartilage explants was measured using a dimethyl-methylene blue dye binding assay with a chondroitin sulfate standard (37).

Immunoblotting and EMSA

Whole cell extracts were obtained, and 10 µg of cell lysates were analyzed by immunoblotting as described (32). For NF-{kappa}B EMSA, 5 µg of nuclear extracts were incubated for 20 min at room temperature with 32P-labeled double-stranded NF-{kappa}B binding oligonucleotide, and complexes were resolved on nondenaturing 5% polyacrylamide gels. Unlabeled oligonucleotide was added at 50-fold molar excess relative to 32P-labeled oligonucleotide in competition experiments.

RNA interference and lentiviral transduction

Oligonucleotides encoding several different putative small interfering RNAs (siRNA) that target Stat1 were cloned into the lentivirus-based RNAi vector pLL3.7 (38) that also contains a transcription cassette encoding enhanced GFP driven by a CMV promoter. Constructs that were effective in suppressing Stat1 expression were identified using transient cotransfection of 293T cells with expression plasmids encoding Stat1. The construct that contained the Stat1 siRNA hairpin sequence that was most effective in 293T cells (GCGTAATCTTCAGGATAAT) was used to generate recombinant lentiviral particles as described (38). 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 enhanced GFP expression and was typically >90%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Suppression of IL-1-induced inflammatory mediator production by IFNs

We wished to test whether IFNs can suppress IL-1-induced activation of cells that leads to inflammation and tissue destruction. IL-1 exerts its proinflammatory functions by activating the production of various inflammatory mediators including cytokines, chemokines, and enzymes such as cyclooxygenase 2 (19). To study the impact of IFNs on IL-1-dependent expression of inflammatory genes, we preincubated human primary macrophages with IFNs for 24 h followed by IL-1{beta} stimulation, and mRNA levels of cyclooxygenase 2, IL-1{beta}, IL-6, IL-8, MIP1{alpha} (CCL3), and TNF-{alpha} were measured using real-time PCR. For all six genes examined, pretreatment with either IFN-{gamma} or IFN-{alpha} effectively suppressed IL-1{beta}-induced gene activation (Fig. 1A). To confirm the above findings at the protein level, we performed ELISA to examine the secretion of IL-6 into culture medium by macrophages. Both IFN-{gamma} and IFN-{alpha} pretreatment strongly suppressed IL-1-induced IL-6 production (Fig. 1B). In contrast to inhibition of IL-1 responsiveness by IFNs, treating macrophages with IFN-{gamma} for 24 h enhanced the levels of IL-6 protein production in response to subsequent LPS stimulation, as expected (4) (Fig. 1C). These experiments demonstrate that IFNs can down-regulate the production of inflammatory mediators in response to the endogenous macrophage activator IL-1, and suggest that this action of IFNs may help to limit the extent of inflammation.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. IFNs suppress IL-1 induction of inflammatory mediators. A, Human blood-derived macrophages cultured overnight in the presence or absence of 5 ng/ml IFN-{gamma} or 5 ng/ml IFN-{alpha}A were activated with 100 ng/ml IL-1{beta}, and steady-state mRNA levels were quantitated using real-time PCR. mRNA levels were normalized relative to expression of GAPDH, and results are expressed as mean ± SD of triplicate determinants. These results are representative of five experiments using independent blood donors. B, IL-6 production in human macrophage culture medium analyzed by ELISA. Macrophages were left untreated ({diamondsuit}) or were incubated with IFN-{gamma} ({square}, 0.15 ng/ml; {blacktriangleup}, 5 ng/ml) before an 18-h IL-1{beta} stimulation (top). Cells were untreated ({diamondsuit}) or incubated with IFN-{alpha} ({square}, 0.1 ng/ml; {blacktriangleup}, 1 ng/ml) before an 18-h IL-1{beta} stimulation (bottom). These results are highly reproducible among >10 independent blood donors. C, Control ({diamondsuit}) or IFN-{gamma}-treated ({square}, 0.15 ng/ml; {blacktriangleup}, 5 ng/ml) macrophages were stimulated with the indicated concentrations of LPS. Supernatants were harvested 18 h later and IL-6 protein levels were measured using ELISA.

 
Inhibition of IL-1-induced MMP expression by IFNs

Besides inflammation, IL-1 promotes tissue destruction by inducing the production of MMPs that degrade extracellular matrix (39). MMP-3 (stromelysin-1) is abundantly expressed in RA tissues and has been implicated in RA pathogenesis (40). In primary human macrophages, MMP-3 mRNA expression was induced by IL-1 in a time-dependent fashion, and this activation of MMP-3 was completely blocked in cells pretreated with IFN-{gamma} or IFN-{alpha} (Fig. 2A). MMP-9 (gelatinase B) is a type IV collagenase that has been strongly associated with tissue invasion (39). Treating human macrophages with IFN-{gamma} completely blocked IL-1{beta} induction of MMP-9 mRNA expression (Fig. 2B, top panel). In addition to the change at the mRNA level, the accumulation of MMP-9 protein in macrophage culture supernatants in response to IL-1{beta} was also diminished in IFN-{gamma}-pretreated cells (Fig. 2B, bottom panel). Synovial fibroblasts have been strongly implicated in MMP-mediated tissue destruction and are considered to be a major pathogenic cell type in RA. Therefore, we examined the impact of IFNs on IL-1-induced activation of MMP expression in synovial fibroblasts. IFN-{gamma} pretreatment strongly suppressed IL-1-activated MMP-1 (interstitial collagenase) expression in synovial fibroblasts (Fig. 2C). These results demonstrate that IFNs suppress IL-1-induced MMP expression in cell types important in inflammation and tissue degeneration.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. IL-1-induced expression of MMPs is inhibited by IFNs. A, Human primary macrophages were stimulated with IL-1{beta} (100 ng/ml) for indicated time periods with or without IFN preincubation, and steady-state MMP-3 mRNA levels were determined using real-time PCR. B, Human control or IFN-{gamma}-pretreated macrophages were stimulated with IL-1{beta} for 3 h for analysis of MMP-9 mRNA using real-time PCR (top panel) or for 48 h for analysis of MMP-9 protein in culture supernatants using immunoblotting (bottom panel). C, Human synovial fibroblasts were preincubated with 5 ng/ml IFN-{gamma} and activated with 10 ng/ml IL-1{beta}. mRNA levels of MMP-1 were assessed by real-time PCR.

 
Inhibition of IL-1-mediated cell invasion and tissue destruction by IFNs

Invasion and destruction of tissues by macrophages contribute to the pathogenesis of several inflammatory diseases, including RA (41), MS (10), and atherosclerosis (42). Since MMPs mediate matrix remodeling and tissue destruction (39), we hypothesized that IFN-mediated inhibition of IL-1-activated MMP expression in macrophages may have functional significance regarding macrophage tissue invasion. We evaluated the tissue invasive capacity of human primary macrophages using the well-established Matrigel invasion assay. Addition of IL-1{beta} greatly increased the percentage of macrophages that invaded and crossed a Matrigel membrane in response to CCL2 (Fig. 3A). Preincubation of macrophages with IFN-{gamma} for 24 h completely abolished IL-1{beta}-triggered migration across Matrigel membranes (Fig. 3A). We also evaluated the invasion of macrophages into type I collagen gels as a measure of macrophage invasion and destruction of connective tissues. IL-1{beta} stimulated the infiltration of macrophages into type I collagen gels whereas addition of 5 ng/ml IFN-{gamma} or IFN-{alpha} 24 h before IL-1{beta} treatment greatly reduced the invasive properties of these cells (Fig. 3B). These results demonstrate that IFNs are capable of suppressing IL-1-induced macrophage invasion of extracellular matrices that are representative of basement membranes and connective tissue. To further test the physiological relevance of IFN-mediated inhibition of cell invasion, we analyzed the impact of IFN administration on IL-1-induced cell recruitment in vivo. Intraperitoneal IL-1 injection resulted in a substantial peritoneal exudate whereas administration of IFN-{alpha}A/D, a universal mammalian type I IFN, before IL-1 injection prevented IL-1-mediated cell recruitment (Fig. 3C). These results demonstrate important functional consequences of suppression of IL-1 responses by IFNs and suggest that this inhibitory activity may contribute to the beneficial effects of IFNs in suppressing tissue damage.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. IFNs inhibit IL-1-induced tissue invasion and destruction. A, Macrophage invasion through model basement membrane. Human blood-derived macrophages were left untreated ({diamondsuit}) or were treated with IFN-{gamma} ({blacksquare}), stimulated with the indicated doses of IL-1{beta}, and assessed for their ability to cross Matrigel membranes. Results are presented as mean ± SD of duplicate cultures. B, Macrophage infiltration into type I collagen gels. Control macrophages ({diamondsuit}) and IFN-{gamma} ({blacksquare}) or IFN-{alpha} ({triangleup})-cultured macrophages were activated with IL-1{beta} and seeded on top of CCL2-impregnated type I collagen gels. The data are expressed as the ratio of cells that penetrated into collagen gels to cells that remained on top of collagen gels. Results are presented as mean ± SD, n = 4. C, Eight-week-old C57BL/6J mice were injected i.p. with PBS or 0.5 µg of IFN-{alpha}A/D. Twenty-four hours after the initial injection, mice were injected with PBS or 0.5 µg of IL-1{beta}. Peritoneal cells were harvested and counted 6 h later. Average peritoneal cell numbers from each treatment group are shown, and each dot represents a single mouse. The unpaired Student’s t test was used to claculate the p value. D and E, Bovine articular cartilage explants were pretreated with either bovine IFN-{gamma} (D) or IFN-{alpha}A/D (E) followed by IL-1{beta} stimulation. Explant media were tested for proteoglycan release by dimethyl-methylene blue dye binding assay from each of four replicate cultures. Results are expressed as amounts of proteoglycan released per milligram of cartilage. Histograms are representative of results from four independent experiments.

 
Another activity of IL-1 with particular relevance to the pathogenesis of RA is its ability to induce cartilage degradation (43). We examined the roles of IL-1 and IFNs on cartilage breakdown in cultured bovine articular cartilage explants. IL-1{beta} promoted the degradation of cartilage as measured by the release of proteoglycan into culture supernatants (Fig. 3, D and E). Pretreating the cartilage tissue for 24 h with either bovine IFN-{gamma} (Fig. 3D) or IFN-{alpha}A/D (Fig. 3E) prevented the increase in proteoglycan release induced by IL-1. Our data show that IL-1-mediated cartilage damage is suppressed by IFNs.

Inhibition of IL-1 signal transduction pathways by IFNs

In macrophages, we had observed that IFNs strongly and globally suppressed a number of IL-1-activated genes and cellular functions. This global inhibition of IL-1 responsiveness in macrophages raised the possibility that instead of targeting a particular promoter and antagonizing the transcription of any single IL-1-dependent gene, IFNs likely interfered with upstream IL-1 signal transduction machinery. We tested this hypothesis by comparing the activation of IL-1 signaling pathways in control and IFN-{gamma}-treated cells. In human primary macrophages, IL-1{beta} induced degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK, JNK, and ERK in a time-dependent manner (Fig. 4A, lanes 1–4). A 24-h IFN-{gamma} incubation before IL-1 stimulation abolished IL-1-induced I{kappa}B{alpha} degradation and phosphorylation of p38, JNK, and ERK (Fig. 4A, lanes 9–12). However, a 4-h pretreatment with IFN-{gamma} had only a partial effect (Fig. 4A, lanes 5–8), indicating that IFN-{gamma}-dependent suppression of IL-1 signaling required greater than 4 h to become fully established. We next examined IL-1 activation of NF-{kappa}B DNA binding activity by EMSA. IL-1 stimulated rapid formation of an NF-{kappa}B DNA binding complex (Fig. 4B, lanes 1–3). Incubating cells with IFN-{gamma} alone for 24 h resulted in baseline NF-{kappa}B activity that was not further enhanced by IL-1 stimulation (Fig. 4B, lanes 4–6), indicating a lack of IL-1 responsiveness in IFN-{gamma}-treated cells. Competition with an unlabeled oligonucleotide demonstrated the specificity of DNA binding (Fig. 4B, lanes 7, 8). In contrast to IL-1 signaling, I{kappa}B{alpha} degradation and phosphorylation of p38, JNK, and ERK stimulated by LPS, which shares many signaling components with IL-1 (44), were preserved or enhanced in cells that had been pretreated with IFN-{gamma} (Fig. 4C). Our data show that prolonged IFN-{gamma} treatment blocks the activation of major signaling pathways downstream of IL-1R. In addition, IFN-{gamma} does not nonspecifically interfere with signaling of IL-1R/TLR family receptors because LPS responses through TLR4 were intact after IFN pretreatment.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. IFN-{gamma} inhibits IL-1 but not LPS-activated signal transduction. A, Following either short term (4 h) or long term (24 h) preincubation with IFN-{gamma}, human macrophages were activated with 100 ng/ml IL-1{beta}. Cell extracts were analyzed using immunoblotting. Results representative of five independent experiments are shown. B, Control or IFN-{gamma}-cultured macrophages were stimulated with IL-1{beta}, and nuclear extracts were subjected to EMSA using a specific NF-{kappa}B binding oligonucleotide. The same extracts were assayed for specificity protein 1 (Sp1) levels by immunoblotting. C, Macrophages were treated with IFN-{gamma} for 24 h and subsequently stimulated with 1 ng/ml LPS. Cell extracts were analyzed using immunoblotting.

 
Regulation of IL-1R expression by IFNs in macrophages

The global inhibition of IL-1 signaling by IFN-{gamma} in macrophages prompted us to search for potential targets of inhibition upstream of the branching point of NF-{kappa}B and MAPK pathways. First, we investigated whether the expression of IL-1R was modulated by IFNs. The functional IL-1R is comprised of two subunits, IL-1RI and IL-1R accessory protein (IL-1R-AcP). IL-1RII also binds IL-1 but does not transmit a signal, thus functioning as a decoy receptor that suppresses IL-1 signaling in a dominant fashion (19). 24 h of treatment with IFN-{gamma} and IFN-{alpha} dramatically down-regulated basal mRNA expression of IL-1RI in human macrophages (Fig. 5A, top panel). Moreover, IFN-{alpha} stimulated the expression of IL-1RII, whereas IFN-{gamma} had no such effect (Fig. 5A, middle panel). Both IFNs induced expression of IFN regulatory factor 1 (IRF-1) as expected (13, 14) (Fig. 5A, bottom panel). Exposure to IFN-{gamma} or IFN-{alpha} did not alter expression of IL-1R-AcP or induce significant levels of IL-1ra (data not shown), which could otherwise explain the IFNs’ inhibitory functions. A kinetic experiment revealed that suppression of IL-1RI RNA expression was apparent after 4 h of IFN-{gamma} treatment, was maximal at 8 h post IFN-{gamma} treatment, and was sustained over a course of two days (Fig. 5B). Next, we performed FACS analysis to evaluate the cell surface expression of IL-1RI. As expected (19), the expression of IL-1RI was low on primary cells. Nevertheless, we could still appreciate a dim yet consistent staining of IL-1RI relative to isotype control in untreated cells (Fig. 5C, upper panel). In cells treated with IFN-{gamma} for 24 h, IL-1RI-specific fluorescence above the isotype control level was no longer detected (Fig. 5C, lower panel). In contrast, the surface level of CD11b was not reduced by IFN-{gamma} (data not shown), showing specificity of IL-1RI down-regulation. Complete suppression of IL-1RI cell surface expression required overnight incubation with IFN-{gamma} and correlated with a block in all IL-1 activities that were tested. The kinetics of IL-1RI expression are consistent with the delayed kinetics of IFN-mediated signaling inhibition (Fig. 4A). The delayed kinetics of decreased IL-1RI expression are likely secondary to suppression of IL-1RI expression by IFN-{gamma} and subsequent decay in IL-1RI mRNA and protein levels.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. IFNs modulate IL-1R expression. A, Human primary macrophages were incubated with 5 ng/ml IFN-{gamma} or IFN-{alpha} for 24 h, and steady-state mRNA levels of IL-1RI, IL-1RII, and IRF-1 were determined by real-time PCR. B, Human macrophages were treated with IFN-{gamma}, and cells were harvested at the indicated time points and mRNA expression of IL-1RI was measured by real-time PCR. C, Flow cytometric analysis of cell surface expression of IL-1RI on murine peritoneal macrophages obtained from C57BL/6J mice. Top panel, control macrophages; bottom panel, IFN-{gamma}-treated macrophages. Dashed lines denote staining with isotype-matched control antibody. Thick lines represent specific fluorescence for IL-1RI. D, Human macrophages were stimulated with IL-1{beta} alone ({diamondsuit}), treated simultaneously with IFN-{gamma} and IL-1{beta} ({square}), or pretreated with IFN-{gamma} before activation ({blacktriangleup}, 24-h IFN-{gamma}-pretreatment; x, 48-h IFN-{gamma} pretreatment). IL-6 in culture supernatants was measured using ELISA. E, Regulation of IL-1RI expression in vivo. Splenic cells from C57BL/6J mice were harvested 24 h after intraperitoneal injection of PBS or poly(I:C) (100 µg/per animal). IL-1RI and oligoadenylate synthetase mRNA expression in splenic macrophages was quantitated using real-time PCR. Bars indicate mean ± SD. Results are based on seven mice in each group and the p value is calculated by the unpaired Student’s t test.

 
The delayed kinetics of IFN-{gamma}-mediated inhibition of IL-1RI expression would predict that coincubation of IFN-{gamma} with IL-1 would not suppress IL-1 activity (unless IFN-{gamma} rapidly blocked additional IL-1 signaling components). We tested this notion by incubating macrophages with IFN-{gamma} either together with IL-1{beta} or before IL-1 stimulation and measuring the secretion of IL-6. Simultaneous addition of IFN-{gamma} and IL-1{beta} did not substantially alter the pattern of IL-1{beta}-induced IL-6 production, while preincubation with IFN-{gamma} for either 24 or 48 h blocked IL-6 secretion in response to all doses of IL-1{beta} (Fig. 5D). Next we investigated whether the modulation of IL-1R expression that we observed in vitro could occur in vivo. Injection of mice with poly(I:C), a mimic of viral dsRNA that is a physiological inducer of type I IFN production (44), down-regulated the expression of IL-1RI mRNA in splenic macrophages in a statistically significant manner (Fig. 5E, top panel). As expected, poly(I:C) administration induced mRNA expression of oligoadenylate synthetase-like 9, a canonical type I IFN target gene (Fig. 5E, bottom panel). These data suggest that IL-1R expression can be suppressed in vivo during immune/inflammatory processes where IFNs are abundantly expressed.

IFN-{gamma} modulates IL-1 signaling in macrophages via a Stat1-dependent mechanism

Stat1 has been implicated as the major mediator of cell activation by IFN-{gamma}. IFN-{gamma} also activates Stat1-independent pathways (45), including the anti-inflammatory factor Stat3 (46), and it has been proposed that these Stat1-independent pathways may mediate suppressive actions of IFNs (47). Therefore, it was important to resolve whether suppression of IL-1 responses by IFN-{gamma} was mediated by Stat1-dependent or Stat1-independent pathways. To delineate the requirement for Stat1 in IFN-{gamma}-mediated inhibition of IL-1-activated gene expression, we first used lentiviral-mediated RNA interference (38) to knock down Stat1 expression in THP-1 human monocytic cells. Immunoblotting analysis revealed effective suppression of constitutive Stat1 protein expression in THP-1 cells stably transduced with lentiviral particles that encode a short siRNA that targets Stat1 mRNA for degradation (Fig. 6A, top panel). As an expected consequence of diminished Stat1 expression, the activation of expression of inducible protein 10 (IP-10), a Stat1-dependent gene (13, 14), by IFN-{gamma} was suppressed in cells transduced to express Stat1 siRNA relative to cells transduced with control lentiviral vectors (Fig. 6A, middle panel). Pretreatment of control THP-1 cells with IFN-{gamma} suppressed IL-1{beta}-dependent induction of IL-1{beta} expression (Fig. 6A, bottom panel, bars 1 and 2), which recapitulates the phenomenon observed in primary macrophages (Fig. 1A). In contrast, IFN-{gamma} failed to suppress IL-1{beta}-induced IL-1{beta} expression in THP-1 cells in which Stat1 expression had been knocked down using siRNA (Fig. 6A, bottom panel, bars 3 and 4). These results demonstrate that Stat1 is required for IFN-{gamma} to exert its inhibitory action on IL-1 activity and that, contrary to our prediction, this homeostatic function of IFN-{gamma} does not involve Stat1-independent pathways. To further assess the role of Stat1 in the regulation of IL-1 responses, we used murine macrophages derived from wild-type or Stat1-null mice (34, 35). IL-1RI mRNA levels were suppressed by IFN-{gamma} in wild-type macrophages, whereas no such inhibition was observed when Stat1–/– cells were treated with IFN-{gamma} (Fig. 6B, top panel). The induction of IRF-1 was Stat1-dependent as described (13, 14) (Fig. 6B, bottom panel). Furthermore, flow cytometric analysis revealed a dose-dependent down-regulation of cell surface IL-1RI by IFN-{gamma} in wild-type macrophages, and 10 ng/ml IFN-{gamma} almost completely reduced the surface expression of IL-1RI to isotype control level (Fig. 6C, top panel). However, in Stat1-null macrophages, IFN-{gamma} treatment did not alter IL-1RI-specfic fluorescence relative to isotype control staining (Fig. 6C, bottom panel), indicating that suppression of IL-1RI by IFN-{gamma} was abrogated in the absence of Stat1. Quantitation of the mean fluorescence intensity specific for IL-1RI in Fig. 6C showed that IFN-{gamma} inhibited IL-1RI expression by 80% in wild-type macrophages, whereas no inhibition was observed in Stat1-deficient macrophages (Fig. 6D). In summary, both RNA interference and gene knockout data demonstrate that IFN-{gamma} inhibition of IL-1 action is dependent on Stat1. Additional signaling pathways activated by IFNs may contribute to inhibition of IL-1 signaling, and activation of Stat1 may not be sufficient to inhibit IL-1 signaling. Thus, IL-1 signaling would not necessarily be suppressed by all cytokines that activate Stat1, because these cytokines may not activate the additional necessary signaling pathways that are activated by IFNs, or because they activate signaling pathways that modulate the effects of Stat1 and prevent Stat1-dependent suppression of IL-1 signaling.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 6. Regulation of IL-1-inducible gene expression and IL-1RI by IFN-{gamma} is dependent on Stat1. A, THP-1 cells were transduced with a lentiviral vector encoding Stat1 siRNA or a control vector. Cell lysates from non-transduced or lentiviral-transduced THP-1 cells were subjected to immunoblotting (top panel). Expression of IP-10, an IFN-{gamma}-inducible gene, was analyzed in cells treated with IFN-{gamma} for 24 h (middle panel). Cells transduced with control or Stat1 siRNA vectors were preincubated with 5 ng/ml IFN-{gamma} and subsequently activated with 10 ng/ml IL-1{beta} for 3 h. mRNA levels of IL-1{beta} were determined using real-time PCR. The results are presented as -fold induction by IL-1{beta} over baseline (bottom panel). B, Murine macrophages from wild-type or Stat1-deficient mice were cultured with 10 ng/ml IFN-{gamma} for the indicated time periods, and mRNA levels of IL-1RI and IRF-1 were analyzed using real-time PCR. C, Murine peritoneal macrophages from wild-type or Stat1-deficient animals were treated with IFN-{gamma} for 24 h. Cell surface levels of IL-1RI in control (purple shaded area) and IFN-{gamma}-cultured cells (green lines, 1 ng/ml IFN-{gamma}; pink lines, 10 ng/ml IFN-{gamma}) were analyzed using flow cytometry. Blue dashed lines represent isotype control staining. D, The IL-1RI-specific mean fluorescence intensity in C shown as percentage of inhibition relative to untreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A role for IFNs and Stat1 in attenuating inflammation and associated tissue damage in vivo in MS, EAE, experimental arthritis, osteolysis, and certain viral infections has been well established (5, 6, 7, 8, 9, 10, 11, 15, 16, 17, 18, 48), but underlying cellular and molecular mechanisms are not well understood. In this report, we describe a specific molecular mechanism underlying the suppressive effects of IFNs, namely regulation of IL-1R expression that results in lack of responsiveness of macrophages to IL-1, a potent inflammatory cytokine strongly implicated in tissue destruction (19). In addition, we identified suppression of macrophage migration, tissue invasion, and protease and inflammatory mediator production as cellular mechanisms of IFN- and Stat1-mediated suppression of inflammation. Inhibition of IL-1 responses likely contributes to the previously described suppressive effects of IFN-{gamma} and Stat1 on IL-1-dependent diseases, such as arthritis and osteolysis (5, 6, 7, 11, 17, 18, 48). In addition, suppression of cell invasion and MMP expression may contribute to the beneficial effects of type I IFNs in the treatment of MS.

The IL-1R and TLR4, which mediates LPS responses, activate similar signaling pathways with many shared components. Interestingly, IL-1R and TLR4 activities are differentially regulated by IFNs. IFN-{gamma} strongly potentiates TLR4 signaling (4) whereas both IFN-{alpha} and IFN-{gamma} reduced cellular responsiveness to IL-1 as reported here. It is logical that IFNs potentiate inflammation when pathogenic microbes that activate TLRs are present, but limit the extent of tissue damage and inflammation in response to endogenous cytokines, as excessive activity of cytokines such as IL-1 can be deleterious to the host if left uncontrolled. IFN-mediated suppression of IL-1RI expression was observed in macrophages, osteoclasts, and T cells (Fig. 5) (X. Hu, unpublished data) and was associated with almost complete suppression of all IL-1 responses in macrophages that were tested. Thus, IFNs and Stat1 will most effectively suppress IL-1-dependent inflammation in processes and diseases in which these cells play a prominent role, such as arthritis and osteolysis. This notion is supported by a substantial body of literature documenting suppression of arthritis and osteolysis by IFNs and Stat1 (5, 6, 7, 11, 17, 18, 48). IL-1 promotes osteoclast-mediated bone resorption (49). Therefore, IFN and Stat1 anti-bone resorptive activity may be mediated by suppression of IL-1 responses in addition to previously described mechanisms involving suppression of receptor activator of NF-{kappa}B-dependent osteoclastogenesis (11, 48).

As delineated in the signal integration model (50), the outcome of cytokine signaling is often determined by the balance between opposing signaling pathways triggered by the same cytokine. Stat1 has been established as a key mediator of IFN activation of cells and an indispensable component of IFN-dependent innate defense mechanisms against viral infections (34, 35). However, IFN-{gamma} and IFN{alpha}, {beta} also activate Stat1-independent signaling pathways, such as Stat3 and PI3K (46, 51), which have suppressive effects on macrophage activation (52, 53) and would be predicted to mediate the homeostatic functions of IFNs. Thus, it is noteworthy that the homeostatic function that we analyzed, IFN-{gamma} suppression of IL-1RI expression, was dependent on Stat1. Stat1 mediates both activating and homeostatic functions of IFN-{gamma} in macrophages, and our findings highlight and extend our appreciation of the pleiotropic and suppressive activities of Stat1. The exact contribution of Stat1 to disease pathogenesis will be determined by the balance between Stat1 pro- and anti-inflammatory activities in the cell types important for pathogenesis, and by the effects of IFNs and Stat1 on other factors that drive disease pathogenesis. For example, although the expression of Stat1 and several Stat1 target genes was elevated during RA synovitis (32, 54), it is not known whether this overexpression of Stat1 is pathogenic or protective (47). Indeed, Stat1 is protective in one animal model of RA (17) and may be at least partially protective in human RA, in which IL-1 and macrophages are important in pathogenesis (40).

Blockade of IL-1 action using IL-1ra has been effective in the treatment of RA, especially in suppression of joint damage, and appears promising in the treatment of autoinflammatory disorders such as Muckle-Wells syndrome (25, 26, 27, 28). The efficacy of IL-1ra therapy is limited by pharmacodynamic and practical issues, and complete and global suppression of IL-1 in multiple cell types in vivo is potentially limited by associated immunosuppression (55). Our results suggest that a therapeutic approach directed at extinguishing IL-1RI expression in hemopoietic cells will be effective in diseases mediated by these cells and by IL-1, especially in suppressing inflammation-associated tissue damage. IFNs, especially IFN-{gamma}, may have too many inflammatory effects to be broadly clinically useful, although a limited trial of IFN-{gamma} and a phase II trial of IFN-{beta} in RA either appeared beneficial or did not result in disease flares (56, 57); the effects of IFNs on joint damage in RA have yet to be determined. Instead of using IFNs as therapeutic agents, we suggest that further characterization of pathways downstream of IFNs that suppress IL-1RI expression would aid in designing new therapeutic approaches for inflammatory disorders.


   Acknowledgments
 
We thank Dr. Theresa Lu and Kyung-Hyun Park-Min for reviewing the manuscript. We are grateful to Dr. Shiquan Liu for the NF-{kappa}B oligonucleotide and Dr. Luk Van Parijs for the lentiviral siRNA vector.


   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: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; IL-1ra, IL-1R antagonist; IL-1RII, IL-1R type II; RA, rheumatoid arthritis; IL-1RI, IL-1R type I; MMP, matrix metalloproteinase; siRNA, small interfering RNA; IL-1R-AcP, IL-1R accessory protein; IRF-1, interferon regulatory factor 1; IP-10, inducible protein 10; Sp1, specificity protein 1. Back

Received for publication February 23, 2005. Accepted for publication April 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264.[Medline]
  2. Sheppard, P., W. Kindsvogel, W. Xu, K. Henderson, S. Schlutsmeyer, T. E. Whitmore, R. Kuestner, U. Garrigues, C. Birks, J. Roraback, et al 2003. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat. Immunol. 4: 63-68.[Medline]
  3. Kotenko, S. V., G. Gallagher, V. V. Baurin, A. Lewis-Antes, M. Shen, N. K. Shah, J. A. Langer, F. Sheikh, H. Dickensheets, R. P. Donnelly. 2003. IFN-{lambda}s mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4: 69-77.[Medline]
  4. Schroder, K., P. J. Hertzog, T. Ravasi, D. A. Hume. 2004. Interferon-{gamma}: an overview of signals, mechanisms and functions. J. Leukocyte Biol. 75: 163-189.[Abstract/Free Full Text]
  5. Manoury-Schwartz, B., G. Chiocchia, N. Bessis, O. Abehsira-Amar, F. Batteux, S. Muller, S. Huang, M. C. Boissier, C. Fournier. 1997. High susceptibility to collagen-induced arthritis in mice lacking IFN-{gamma} receptors. J. Immunol. 158: 5501-5506.[Abstract]
  6. Vermeire, K., H. Heremans, M. Vandeputte, S. Huang, A. Billiau, P. Matthys. 1997. Accelerated collagen-induced arthritis in IFN-{gamma} receptor-deficient mice. J. Immunol. 158: 5507-5513.[Abstract]
  7. Guedez, Y. B., K. B. Whittington, J. L. Clayton, L. A. Joosten, F. A. van de Loo, W. B. van den Berg, E. F. Rosloniec. 2001. Genetic ablation of interferon-{gamma} up-regulates interleukin-1{beta} expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain. Arthritis Rheum. 44: 2413-2424.[Medline]
  8. Krakowski, M., T. Owens. 1996. Interferon-{gamma} confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26: 1641-1646.[Medline]
  9. Willenborg, D. O., S. Fordham, C. C. Bernard, W. B. Cowden, I. A. Ramshaw. 1996. IFN-{gamma} plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157: 3223-3227.[Abstract]
  10. Zamvil, S. S., L. Steinman. 2003. Diverse targets for intervention during inflammatory and neurodegenerative phases of multiple sclerosis. Neuron 38: 685-688.[Medline]
  11. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, H. Oda, K. Nakamura, N. Ida, et al 2002. RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-{beta}. Nature 416: 744-749.[Medline]
  12. Ivashkiv, L. B.. 2000. Jak-STAT signaling pathways in cells of the immune system. Rev. Immunogenet. 2: 220-230.[Medline]
  13. Ramana, C. V., M. P. Gil, Y. Han, R. M. Ransohoff, R. D. Schreiber, G. R. Stark. 2001. Stat1-independent regulation of gene expression in response to IFN-{gamma}. Proc. Natl. Acad. Sci. USA 98: 6674-6679.[Abstract/Free Full Text]
  14. Gil, M. P., E. Bohn, A. K. O’Guin, C. V. Ramana, B. Levine, G. R. Stark, H. W. Virgin, R. D. Schreiber. 2001. Biologic consequences of Stat1-independent IFN signaling. Proc. Natl. Acad. Sci. USA 98: 6680-6685.[Abstract/Free Full Text]
  15. Durbin, J. E., A. Fernandez-Sesma, C. K. Lee, T. D. Rao, A. B. Frey, T. M. Moran, S. Vukmanovic, A. Garcia-Sastre, D. E. Levy. 2000. Type I IFN modulates innate and specific antiviral immunity. J. Immunol. 164: 4220-4228.[Abstract/Free Full Text]
  16. Nishibori, T., Y. Tanabe, L. Su, M. David. 2004. Impaired development of CD4+CD25+ regulatory T cells in the absence of STAT1: increased susceptibility to autoimmune disease. J. Exp. Med. 199: 25-34.[Abstract/Free Full Text]
  17. de Hooge, A. S., F. A. van de Loo, M. I. Koenders, M. B. Bennink, O. J. Arntz, T. Kolbe, W. B. van den Berg. 2004. Local activation of STAT-1 and STAT-3 in the inflamed synovium during zymosan-induced arthritis: exacerbation of joint inflammation in STAT-1 gene-knockout mice. Arthritis Rheum. 50: 2014-2023.[Medline]
  18. Kim, S., T. Koga, M. Isobe, B. E. Kern, T. Yokochi, Y. E. Chin, G. Karsenty, T. Taniguchi, H. Takayanagi. 2003. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. Genes Dev. 17: 1979-1991.[Abstract/Free Full Text]
  19. Dinarello, C. A.. 1996. Biologic basis for interleukin-1 in disease. Blood 87: 2095-2147.[Abstract/Free Full Text]
  20. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. McKenzie, L. A. O’Neill, F. Y. Liew. 2004. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol 5: 373-379.[Medline]
  21. Wald, D., J. Qin, Z. Zhao, Y. Qian, M. Naramura, L. Tian, J. Towne, J. E. Sims, G. R. Stark, X. Li. 2003. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4: 920-927.[Medline]
  22. Dayer, J. M.. 2003. The pivotal role of interleukin-1 in the clinical manifestations of rheumatoid arthritis. Rheumatology (Oxford) 42:(Suppl 2): ii3-ii10.
  23. Kirii, H., T. Niwa, Y. Yamada, H. Wada, K. Saito, Y. Iwakura, M. Asano, H. Moriwaki, M. Seishima. 2003. Lack of interleukin-1{beta} decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23: 656-660.[Abstract/Free Full Text]
  24. Agostini, L., F. Martinon, K. Burns, M. F. McDermott, P. N. Hawkins, J. Tschopp. 2004. NALP3 forms an IL-1{beta}-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20: 319-325.[Medline]
  25. Bresnihan, B., J. M. Alvaro-Gracia, M. Cobby, M. Doherty, Z. Domljan, P. Emery, G. Nuki, K. Pavelka, R. Rau, B. Rozman, et al 1998. Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum. 41: 2196-2204.[Medline]
  26. Cohen, S., E. Hurd, J. Cush, M. Schiff, M. E. Weinblatt, L. W. Moreland, J. Kremer, M. B. Bear, W. J. Rich, D. McCabe. 2002. Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 46: 614-624.[Medline]
  27. Hawkins, P. N., H. J. Lachmann, M. F. McDermott. 2003. Interleukin-1-receptor antagonist in the Muckle-Wells syndrome. N. Engl. J. Med. 348: 2583-2584.[Free Full Text]
  28. Hawkins, P. N., H. J. Lachmann, E. Aganna, M. F. McDermott. 2004. Spectrum of clinical features in Muckle-Wells syndrome and response to anakinra. Arthritis Rheum. 50: 607-612.[Medline]
  29. Hawkins, P. N., A. Bybee, E. Aganna, M. F. McDermott. 2004. Response to anakinra in a de novo case of neonatal-onset multisystem inflammatory disease. Arthritis Rheum. 50: 2708-2709.[Medline]
  30. Hoffman, H. M., D. D. Patel. 2004. Genomic-based therapy: targeting interleukin-1 for autoinflammatory diseases. Arthritis Rheum. 50: 345-349.[Medline]
  31. Fleischmann, R. M.. 2003. Addressing the safety of anakinra in patients with rheumatoid arthritis. Rheumatology (Oxford) 42:(Suppl 2): ii29-ii35.
  32. Hu, X., C. Herrero, W. P. Li, T. T. Antoniv, E. Falck-Pedersen, A. E. Koch, J. M. Woods, G. K. Haines, L. B. Ivashkiv. 2002. Sensitization of IFN-{gamma} Jak-STAT signaling during macrophage activation. Nat. Immunol. 3: 859-865.[Medline]
  33. Deon, D., S. Ahmed, K. Tai, N. Scaletta, C. Herrero, I. H. Lee, A. Krause, L. B. Ivashkiv. 2001. Cross-talk between IL-1 and IL-6 signaling pathways in rheumatoid arthritis synovial fibroblasts. J. Immunol. 167: 5395-5403.[Abstract/Free Full Text]
  34. Durbin, J. E., R. Hackenmiller, M. C. Simon, D. E. Levy. 1996. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 84: 443-450.[Medline]
  35. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431-442.[Medline]
  36. Schenkel, A. R., Z. Mamdouh, X. Chen, R. M. Liebman, W. A. Muller. 2002. CD99 plays a major role in the migration of monocytes through endothelial junctions. Nat. Immunol. 3: 143-150.[Medline]
  37. Farndale, R. W., C. A. Sayers, A. J. Barrett. 1982. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res. 9: 247-248.[Medline]
  38. Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, D. L. Rooney, M. M. Ihrig, M. T. McManus, F. B. Gertler, et al 2003. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33: 401-406.[Medline]
  39. Brinckerhoff, C. E., L. M. Matrisian. 2002. Matrix metalloproteinases: a tail of a frog that became a prince. Nat. Rev. Mol. Cell Biol. 3: 207-214.[Medline]
  40. Firestein, G. S.. 2003. Rheumatoid synovitis and pannus. M. C. Hichberg, and A. J. Silman, and J. S. Smolen, and M. E. Weinblatt, and M. H. Weisman, eds. In Rheumatology Vol. 1: 2855-2864 Mosby, London. .
  41. Arend, W. P.. 2001. Cytokines and cellular interactions in inflammatory synovitis. J. Clin. Invest. 107: 1081-1082.[Medline]
  42. Ridley, A. J., M. A. Schwartz, K. Burridge, R. A. Firtel, M. H. Ginsberg, G. Borisy, J. T. Parsons, A. R. Horwitz. 2003. Cell migration: integrating signals from front to back. Science 302: 1704-1709.[Abstract/Free Full Text]
  43. Abramson, S. B., A. Amin. 2002. Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage. Rheumatology (Oxford) 41: 972-980.
  44. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511.[Medline]
  45. Ramana, C. V., M. P. Gil, R. D. Schreiber, G. R. Stark. 2002. Stat1-dependent and -independent pathways in IFN-{gamma}-dependent signaling. Trends Immunol. 23: 96-101.[Medline]
  46. Qing, Y., G. R. Stark. 2004. Alternative activation of STAT1 and STAT3 in response to interferon-{gamma}. J. Biol. Chem. 279: 41679-41685.[Abstract/Free Full Text]
  47. Ivashkiv, L. B., X. Hu. 2003. The JAK/STAT pathway in rheumatoid arthritis: pathogenic or protective?. Arthritis Rheum. 48: 2092-2096.[Medline]
  48. Takayanagi, H., K. Ogasawara, S. Hida, T. Chiba, S. Murata, K. Sato, A. Takaoka, T. Yokochi, H. Oda, K. Tanaka, et al 2000. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-{gamma}. Nature 408: 600-605.[Medline]
  49. Teitelbaum, S. L.. 2000. Bone resorption by osteoclasts. Science 289: 1504-1508.[Abstract/Free Full Text]
  50. Kamimura, D., K. Ishihara, T. Hirano. 2003. IL-6 signal transduction and its physiological roles: the signal orchestration model. Rev. Physiol. Biochem. Pharmacol. 149: 1-38.[Medline]
  51. Choudhury, G. G.. 2004. A linear signal transduction pathway involving phosphatidylinositol 3-kinase, protein kinase Cepsilon, and MAPK in mesangial cells regulates interferon-{gamma}-induced STAT1{alpha} transcriptional activation. J. Biol. Chem. 279: 27399-27409.[Abstract/Free Full Text]
  52. Fukao, T., S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol. 24: 358-363.[Medline]
  53. Takeda, K., B. E. Clausen, T. Kaisho, T. Tsujimura, N. Terada, I. Forster, S. Akira. 1999. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10: 39-49.[Medline]
  54. van der Pouw Kraan, T. C., F. A. van Gaalen, P. V. Kasperkovitz, N. L. Verbeet, T. J. Smeets, M. C. Kraan, M. Fero, P. P. Tak, T. W. Huizinga, E. Pieterman, et al 2003. Rheumatoid arthritis is a heterogeneous disease: evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues. Arthritis Rheum. 48: 2132-2145.[Medline]
  55. Irikura, V. M., E. Hirsch, D. Hirsh. 1999. Effects of interleukin-1 receptor antagonist overexpression on infection by Listeria monocytogenes. Infect. Immun. 67: 1901-1909.[Abstract/Free Full Text]
  56. Genovese, M. C., E. F. Chakravarty, E. Krishnan, L. W. Moreland. 2004. A randomized, controlled trial of interferon-{beta}-1a (Avonex®) in patients with rheumatoid arthritis: a pilot study. [ISRCTN03626626]. Arthritis Res. Ther. 6: R73.[Medline]
  57. Machold, K. P., K. Neumann, J. S. Smolen. 1992. Recombinant human interferon {gamma} in the treatment of rheumatoid arthritis: double blind placebo controlled study. Ann. Rheum. Dis. 51: 1039-1043.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 NeurologyHome page
G. Birnbaum, B. Cree, I. Altafullah, M. Zinser, and A. T. Reder
Combining beta interferon and atorvastatin may increase disease activity in multiple sclerosis
Neurology, October 28, 2008; 71(18): 1390 - 1395.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Hu, X. Hu, L. Boumsell, and L. B. Ivashkiv
IFN-{gamma} and STAT1 Arrest Monocyte Migration and Modulate RAC/CDC42 Pathways
J. Immunol., June 15, 2008; 180(12): 8057 - 8065.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
X. Hu, J. Chen, L. Wang, and L. B. Ivashkiv
Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways in macrophage activation
J. Leukoc. Biol., August 1, 2007; 82(2): 237 - 243.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
R Ahmad, H Y Qureshi, M El Mabrouk, J Sylvester, M Ahmad, and M Zafarullah
Inhibition of interleukin 1-induced matrix metalloproteinase 13 expression in human chondrocytes by interferon {gamma}
Ann Rheum Dis, June 1, 2007; 66(6): 782 - 789.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
H. Kelchtermans, S. Struyf, B. De Klerck, T. Mitera, M. Alen, L. Geboes, M. Van Balen, C. Dillen, W. Put, C. Gysemans, et al.
Protective role of IFN-{gamma} in collagen-induced arthritis conferred by inhibition of mycobacteria-induced granulocyte chemotactic protein-2 production
J. Leukoc. Biol., April 1, 2007; 81(4): 1044 - 1053.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. Hausmann, A. Pagenstecher, K. Baur, K. Richter, H.-J. Rziha, and P. Staeheli
CD8 T Cells Require Gamma Interferon To Clear Borna Disease Virus from the Brain and Prevent Immune System-Mediated Neuronal Damage
J. Virol., November 1, 2005; 79(21): 13509 - 13518.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, X.
Right arrow Articles by Ivashkiv, L. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, X.
Right arrow Articles by Ivashkiv, L. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS