Homeostatic Role of Interferons Conferred by Inhibition of IL-1-Mediated Inflammation and Tissue Destruction
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免疫学杂志 2005年第13期
Abstract
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-, IFN-, and IFN-, 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- 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--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
The IFNs were first identified on the basis of interference with viral replication and are grouped into type I IFNs (including IFN-, IFN-, and IFN-), type II IFN (IFN-), and IFN-s (1, 2, 3). In addition to mediating antiviral effects, IFNs are important modulators of innate and acquired immune responses (1). IFN- is generally considered to be a proinflammatory factor based on its strong activation of macrophage antimicrobial activity, Ag presentation, and synergy with TNF- and with TLR ligands such as LPS in the activation of macrophage inflammatory functions (4). Anti-inflammatory effects of IFN- 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- comes from the exacerbation of several experimental inflammatory diseases in the absence of IFN- signaling. For example, collagen-induced arthritis was exacerbated in mice lacking IFN- or its receptor (5, 6, 7), and deletion of the IFN- 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- 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
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- or 5 ng/ml (equivalent to 2000 U/ml) of IFN-A for 24 h before stimulation with IL-1. 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- were purchased from Roche Applied Science, PeproTech, and Serotec, respectively. Human IFN-A was obtained from BioSource International. IFN-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 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-B EMSA, 5 μg of nuclear extracts were incubated for 20 min at room temperature with 32P-labeled double-stranded NF-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
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 stimulation, and mRNA levels of cyclooxygenase 2, IL-1, IL-6, IL-8, MIP1 (CCL3), and TNF- were measured using real-time PCR. For all six genes examined, pretreatment with either IFN- or IFN- effectively suppressed IL-1-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- and IFN- 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- 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.
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- or IFN- (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- completely blocked IL-1 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 was also diminished in IFN--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- 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.
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 greatly increased the percentage of macrophages that invaded and crossed a Matrigel membrane in response to CCL2 (Fig. 3A). Preincubation of macrophages with IFN- for 24 h completely abolished IL-1-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 stimulated the infiltration of macrophages into type I collagen gels whereas addition of 5 ng/ml IFN- or IFN- 24 h before IL-1 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-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.
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 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- (Fig. 3D) or IFN-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--treated cells. In human primary macrophages, IL-1 induced degradation of IB and phosphorylation of p38 MAPK, JNK, and ERK in a time-dependent manner (Fig. 4A, lanes 1–4). A 24-h IFN- incubation before IL-1 stimulation abolished IL-1-induced IB degradation and phosphorylation of p38, JNK, and ERK (Fig. 4A, lanes 9–12). However, a 4-h pretreatment with IFN- had only a partial effect (Fig. 4A, lanes 5–8), indicating that IFN--dependent suppression of IL-1 signaling required greater than 4 h to become fully established. We next examined IL-1 activation of NF-B DNA binding activity by EMSA. IL-1 stimulated rapid formation of an NF-B DNA binding complex (Fig. 4B, lanes 1–3). Incubating cells with IFN- alone for 24 h resulted in baseline NF-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--treated cells. Competition with an unlabeled oligonucleotide demonstrated the specificity of DNA binding (Fig. 4B, lanes 7, 8). In contrast to IL-1 signaling, IB 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- (Fig. 4C). Our data show that prolonged IFN- treatment blocks the activation of major signaling pathways downstream of IL-1R. In addition, IFN- does not nonspecifically interfere with signaling of IL-1R/TLR family receptors because LPS responses through TLR4 were intact after IFN pretreatment.
Regulation of IL-1R expression by IFNs in macrophages
The global inhibition of IL-1 signaling by IFN- in macrophages prompted us to search for potential targets of inhibition upstream of the branching point of NF-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- and IFN- dramatically down-regulated basal mRNA expression of IL-1RI in human macrophages (Fig. 5A, top panel). Moreover, IFN- stimulated the expression of IL-1RII, whereas IFN- 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- or IFN- 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- treatment, was maximal at 8 h post IFN- 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- 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- (data not shown), showing specificity of IL-1RI down-regulation. Complete suppression of IL-1RI cell surface expression required overnight incubation with IFN- 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- and subsequent decay in IL-1RI mRNA and protein levels.
The delayed kinetics of IFN--mediated inhibition of IL-1RI expression would predict that coincubation of IFN- with IL-1 would not suppress IL-1 activity (unless IFN- rapidly blocked additional IL-1 signaling components). We tested this notion by incubating macrophages with IFN- either together with IL-1 or before IL-1 stimulation and measuring the secretion of IL-6. Simultaneous addition of IFN- and IL-1 did not substantially alter the pattern of IL-1-induced IL-6 production, while preincubation with IFN- for either 24 or 48 h blocked IL-6 secretion in response to all doses of IL-1 (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- modulates IL-1 signaling in macrophages via a Stat1-dependent mechanism
Stat1 has been implicated as the major mediator of cell activation by IFN-. IFN- 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- was mediated by Stat1-dependent or Stat1-independent pathways. To delineate the requirement for Stat1 in IFN--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- 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- suppressed IL-1-dependent induction of IL-1 expression (Fig. 6A, bottom panel, bars 1 and 2), which recapitulates the phenomenon observed in primary macrophages (Fig. 1A). In contrast, IFN- failed to suppress IL-1-induced IL-1 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- to exert its inhibitory action on IL-1 activity and that, contrary to our prediction, this homeostatic function of IFN- 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- in wild-type macrophages, whereas no such inhibition was observed when Stat1–/– cells were treated with IFN- (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- in wild-type macrophages, and 10 ng/ml IFN- almost completely reduced the surface expression of IL-1RI to isotype control level (Fig. 6C, top panel). However, in Stat1-null macrophages, IFN- 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- was abrogated in the absence of Stat1. Quantitation of the mean fluorescence intensity specific for IL-1RI in Fig. 6C showed that IFN- 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- 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.
Discussion
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- 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- strongly potentiates TLR4 signaling (4) whereas both IFN- and IFN- 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-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- and IFN, 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- suppression of IL-1RI expression, was dependent on Stat1. Stat1 mediates both activating and homeostatic functions of IFN- 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-, may have too many inflammatory effects to be broadly clinically useful, although a limited trial of IFN- and a phase II trial of IFN- 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-B oligonucleotide and Dr. Luk Van Parijs for the lentiviral siRNA vector.
Disclosures
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.).
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@hss.edu
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.
Received for publication February 23, 2005. Accepted for publication April 19, 2005.
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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-, IFN-, and IFN-, 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- 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--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
The IFNs were first identified on the basis of interference with viral replication and are grouped into type I IFNs (including IFN-, IFN-, and IFN-), type II IFN (IFN-), and IFN-s (1, 2, 3). In addition to mediating antiviral effects, IFNs are important modulators of innate and acquired immune responses (1). IFN- is generally considered to be a proinflammatory factor based on its strong activation of macrophage antimicrobial activity, Ag presentation, and synergy with TNF- and with TLR ligands such as LPS in the activation of macrophage inflammatory functions (4). Anti-inflammatory effects of IFN- 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- comes from the exacerbation of several experimental inflammatory diseases in the absence of IFN- signaling. For example, collagen-induced arthritis was exacerbated in mice lacking IFN- or its receptor (5, 6, 7), and deletion of the IFN- 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- 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
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- or 5 ng/ml (equivalent to 2000 U/ml) of IFN-A for 24 h before stimulation with IL-1. 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- were purchased from Roche Applied Science, PeproTech, and Serotec, respectively. Human IFN-A was obtained from BioSource International. IFN-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 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-B EMSA, 5 μg of nuclear extracts were incubated for 20 min at room temperature with 32P-labeled double-stranded NF-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
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 stimulation, and mRNA levels of cyclooxygenase 2, IL-1, IL-6, IL-8, MIP1 (CCL3), and TNF- were measured using real-time PCR. For all six genes examined, pretreatment with either IFN- or IFN- effectively suppressed IL-1-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- and IFN- 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- 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.
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- or IFN- (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- completely blocked IL-1 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 was also diminished in IFN--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- 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.
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 greatly increased the percentage of macrophages that invaded and crossed a Matrigel membrane in response to CCL2 (Fig. 3A). Preincubation of macrophages with IFN- for 24 h completely abolished IL-1-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 stimulated the infiltration of macrophages into type I collagen gels whereas addition of 5 ng/ml IFN- or IFN- 24 h before IL-1 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-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.
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 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- (Fig. 3D) or IFN-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--treated cells. In human primary macrophages, IL-1 induced degradation of IB and phosphorylation of p38 MAPK, JNK, and ERK in a time-dependent manner (Fig. 4A, lanes 1–4). A 24-h IFN- incubation before IL-1 stimulation abolished IL-1-induced IB degradation and phosphorylation of p38, JNK, and ERK (Fig. 4A, lanes 9–12). However, a 4-h pretreatment with IFN- had only a partial effect (Fig. 4A, lanes 5–8), indicating that IFN--dependent suppression of IL-1 signaling required greater than 4 h to become fully established. We next examined IL-1 activation of NF-B DNA binding activity by EMSA. IL-1 stimulated rapid formation of an NF-B DNA binding complex (Fig. 4B, lanes 1–3). Incubating cells with IFN- alone for 24 h resulted in baseline NF-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--treated cells. Competition with an unlabeled oligonucleotide demonstrated the specificity of DNA binding (Fig. 4B, lanes 7, 8). In contrast to IL-1 signaling, IB 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- (Fig. 4C). Our data show that prolonged IFN- treatment blocks the activation of major signaling pathways downstream of IL-1R. In addition, IFN- does not nonspecifically interfere with signaling of IL-1R/TLR family receptors because LPS responses through TLR4 were intact after IFN pretreatment.
Regulation of IL-1R expression by IFNs in macrophages
The global inhibition of IL-1 signaling by IFN- in macrophages prompted us to search for potential targets of inhibition upstream of the branching point of NF-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- and IFN- dramatically down-regulated basal mRNA expression of IL-1RI in human macrophages (Fig. 5A, top panel). Moreover, IFN- stimulated the expression of IL-1RII, whereas IFN- 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- or IFN- 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- treatment, was maximal at 8 h post IFN- 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- 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- (data not shown), showing specificity of IL-1RI down-regulation. Complete suppression of IL-1RI cell surface expression required overnight incubation with IFN- 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- and subsequent decay in IL-1RI mRNA and protein levels.
The delayed kinetics of IFN--mediated inhibition of IL-1RI expression would predict that coincubation of IFN- with IL-1 would not suppress IL-1 activity (unless IFN- rapidly blocked additional IL-1 signaling components). We tested this notion by incubating macrophages with IFN- either together with IL-1 or before IL-1 stimulation and measuring the secretion of IL-6. Simultaneous addition of IFN- and IL-1 did not substantially alter the pattern of IL-1-induced IL-6 production, while preincubation with IFN- for either 24 or 48 h blocked IL-6 secretion in response to all doses of IL-1 (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- modulates IL-1 signaling in macrophages via a Stat1-dependent mechanism
Stat1 has been implicated as the major mediator of cell activation by IFN-. IFN- 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- was mediated by Stat1-dependent or Stat1-independent pathways. To delineate the requirement for Stat1 in IFN--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- 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- suppressed IL-1-dependent induction of IL-1 expression (Fig. 6A, bottom panel, bars 1 and 2), which recapitulates the phenomenon observed in primary macrophages (Fig. 1A). In contrast, IFN- failed to suppress IL-1-induced IL-1 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- to exert its inhibitory action on IL-1 activity and that, contrary to our prediction, this homeostatic function of IFN- 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- in wild-type macrophages, whereas no such inhibition was observed when Stat1–/– cells were treated with IFN- (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- in wild-type macrophages, and 10 ng/ml IFN- almost completely reduced the surface expression of IL-1RI to isotype control level (Fig. 6C, top panel). However, in Stat1-null macrophages, IFN- 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- was abrogated in the absence of Stat1. Quantitation of the mean fluorescence intensity specific for IL-1RI in Fig. 6C showed that IFN- 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- 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.
Discussion
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- 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- strongly potentiates TLR4 signaling (4) whereas both IFN- and IFN- 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-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- and IFN, 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- suppression of IL-1RI expression, was dependent on Stat1. Stat1 mediates both activating and homeostatic functions of IFN- 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-, may have too many inflammatory effects to be broadly clinically useful, although a limited trial of IFN- and a phase II trial of IFN- 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-B oligonucleotide and Dr. Luk Van Parijs for the lentiviral siRNA vector.
Disclosures
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.).
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@hss.edu
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.
Received for publication February 23, 2005. Accepted for publication April 19, 2005.
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