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Breaks Tolerance through Expansion of CD25+ Effector T Cells1
* Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia;
Department of Microbiology and Infectious Diseases, St. Vincent’s Centre for Medical Research, Julia McFarlane Diabetes Research Centre, Melbourne, Australia;
Department of Microbiology, University of Calgary, Canada; and
Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Japan
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Abstract |
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drives proliferation and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25– autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self. |
Introduction |
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is produced by hemopoietic and non-hemopoietic cells in a wide variety of inflammatory responses, with pleiotropic effects including altered cell signaling and migration, cytokine production and fever. Evidence is emerging that a number of arthritic autoimmune diseases are associated with high levels of IL-1 at the inflammatory site, immunogenetic predisposition to IL-1 overproduction associated with autoimmunity, or genetic signatures reflecting IL-1 overactivity (1, 2, 3). Patients with systemic onset juvenile idiopathic arthritis (JIA)3 produce elevated IL-1 but not TNF-
and IL-6 after PBMC activation, and therapy with rIL-1 receptor antagonist (rIL-1Ra) was shown to reverse pre-established arthritis in 6 of 8 patients (2), demonstrating that IL-1 plays a key dysregulatory role and can be successfully corrected using IL-1 blockade. Similar success is being observed in other autoinflammatory diseases associated with mutations in NALP3/cryopyrin and IL-1 overproduction, such as Muckle-Wells and NOMID syndrome and familial Mediterranean fever (4). Exogenous IL-1 has adjuvant effects and also initiates inflammatory arthritis in combination with Ag in several models (5). Moreover, H-2d mice-deficient in IL-1Ra spontaneously develop inflammatory arthritis, and a chromosome 2 haplotype associated with a vigorous IL-1 response to inflammatory stimuli is linked to severity of arthritis in various inbred mouse strains after transfer of arthritic serum (6, 7, 8). By signaling dendritic cells (DC), IL-1 promotes IL-12 and IFN-
production in autoimmunity and exerts direct proapoptotic effects on insulin-producing islet cells of the pancreas in diabetes and on chondrocytes in arthritis (9, 10, 11).
Recently, up to 50% of CD25+ T cells in joint fluids from patients with JIA were found to be CD27–FoxP3– activated effector T cells, compared with around 10% in non-inflamed sites from the same patient (12). In combination with IL-6, IL-1 has been shown to reverse anergy of CD4+CD25+ regulatory T cells (Treg) in vitro (13). Because immune regulation by endogenous CD4+CD25+ Treg holds potentially autoreactive T cells in check (14, 15), and IL-1 is associated with a wide variety of inflammatory events that might trigger autoreactive T cell activation in diseases such as JIA, we tested the hypothesis that IL-1 promotes autoreactive effector T cell function. Using non-autoimmune-prone and autoimmune-prone strains of mice, we show that IL-1 drives proliferation and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of autoreactive CD4+CD25– effectors from suppression.
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Materials and Methods |
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Male B6 mice were obtained from ARC and naive NOD and NOD-SCID from the Walter and Eliza Hall Institute. TCR transgenic strains BDC2.5 (16), and 4.1 NOD (17), were bred, maintained, and backcrossed to the IL-1R1–/– NOD strain at St. Vincent’s Institute. CD45.2 congenic NOD (18) were maintained at Herston Animal Facility Brisbane. IL-1Ra–/– mice on a B6 background were produced as described (6). All mice used were between 6 and 10 wk of age. IL-1R1–/– mice were on the NOD background (19). Recombinant IL-1 was obtained from Biolegend, and rIL-18 and TNF- were purchased from R&D Systems. Anti-IL-1 and anti-IL-18 were obtained from R&D Systems, and anti-TNF- and anti-IL-6 were purchased from Biolegend. CFSE was obtained from Molecular Probes.
Cell purification
CD11c+ cells were purified from spleen by positive immuno-selection using CD11c microbeads and LS columns (Miltenyi Biotec). CD4+CD25+ Treg were purified using a CD4+CD25+ Treg purification kit (Miltenyi Biotec) according to the manufacturer’s protocol with the modifications that CS columns were used for depleting non-T cells, and LS columns were used to positively select CD4+CD25+ cells. To achieve maximum purity, CD25 positive selection was repeated twice. CD4+CD25– T cells were purified from the CD25-depleted fraction using CD4 microbeads and LS columns. Suppression assays were conducted for 72 h with 2 x 104 CD11c+ DC, 1 x 105 CD4+CD25– T cells, and 1 x 105 or varying numbers of CD4+CD25+ Treg and 0.1 to 1 µg/ml anti-CD3 (Biolegend) in a 250 µL final volume in round-bottom wells. Cells were pulsed for the final 18 h of culture with [3H]thymidine (ICN Pharmaceuticals) and counted using a Packard TopCount NXT (Packard Instrument)
CFSE labeling of congenic cells
For detection of CD4+CD25– cell divisions in vitro, CFSE-labeled Thy1.1 CD4+CD25– cells and unlabeled Thy1.2 CD4+CD25+ cells were stimulated with Thy1.2-purified splenic CD11c+ DC and 0.5 µg/ml anti-CD3 for 3 days in the presence or absence of 50 ng/ml IL-1. For detection of CD4+CD25+ cell divisions, CFSE-labeled Thy1.1 CD4+CD25+ cells and unlabeled Thy1.2 CD4+CD25– cells were stimulated with Thy1.2 DC and 0.5 µg/ml anti-CD3 for 3 days in the presence or absence of 50 ng/ml IL-1. FoxP3 expression was detected by intracellular staining following protocols and reagents supplied by the manufacturer (eBiosciences). For detection of IFN-, cells were stimulated for 4 h with 0.5 µg/ml anti-CD3 on the final day of culture in the presence of brefeldin A (Biolegend) and then fixed, permeabilized, and stained with anti-IFN--biotin (Biolegend) followed by SA-PerCP (Biolegend).
Cytokine measurement
IL-1 was measured in supernatants using ELISA kits from Biolegend according to manufacturer’s protocols. IFN- and IL-2 were measured in supernatants using a cytokine bead array following the manufacturer’s protocol (Lincoplex; Linco Research).
Quantitative PCR
RNA was isolated using TriZOL (Invitrogen Life Technologies) and converted to cDNA using Superscript III (Invitrogen Life Technologies) and oligo(dT) (Promega) and random hexamer (Promega). Quantitative PCR was conducted using the following primer sets hypoxanthine phosphoribosyl transferase (HPRT) F-CCTAAGATGAGCGCAAGTTGAA, R-CCACAGGACTAGAACACCTGCTAA; IL-1R1, F-TTATCCTGAGCCCTCGGAATG, R-CCGTGACGTTGCAGATCAGTT; IL-1R2, F-CCTCATGTCTCCTACTTGCAAATCT, R-TCTTTCAGGTCAGGGCACACTA; IL-1Ra, F-CTGGGAAAAGACCCTGCAAG, R-CCAGCAATGAGCTGGTTGTTT; FoxP3, F-CCCAGGAAAGACAGCAACCTT, R-TTCTCACAACCAGGCCACTTG as previously described (20).
Suppression of autoreactivity in vivo
Six-week-old wild-type (wt) NOD mice were injected i.v. with 5 x 105 4.1 CD4+CD25– cells with or without 5 x 105 wt 4.1 CD4+CD25+ or 4.1 IL-1R1–/– CD4+CD25+ T cells. Seven days later, pancreatic draining lymph node (pLN) or spleen CD11c+ DC purified from naive NOD were used to stimulate proliferation of pLN cells harvested from recipient mice in vitro.
Statistical analysis
Student’s two-tailed unpaired t tests were used to compare differences in all experiments.
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Results |
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costimulates IFN- production and reverses suppression by CD4+CD25+ Treg
IL-1 is an important proinflammatory and adjuvant cytokine that influences the development of many autoimmune conditions. Previously, anergy of CD4+CD25+ T cells in vitro could be reversed when cells were stimulated by LPS-activated bone marrow-derived DC, and this was dependent on IL-1 and IL-6 produced by DC (13). However, specific effects of IL-1 on regulatory and effector T cell function in response to Ag presented by DC in the absence of TLR ligand stimulation have not been assessed. We first tested in vitro the hypothesis that IL-1 could break tolerance through inhibition of CD4+CD25+ Treg function, and examined the effect of exogenous IL-1 on the suppressor function of CD4+CD25+ Treg from non-autoimmune-prone B6 mice. Although B6 CD4+CD25+ Treg effectively suppressed proliferation of CD4+CD25– T cells, when stimulated by autologous splenic DC and anti-CD3 (Fig. 1A), rIL-1 costimulated proliferation and IFN- production by CD4+CD25+ T cells alone, and inhibited suppression of CD4+CD25– T cell proliferation (Fig. 1, A and B) or IFN- (Fig. 1C) production by CD4+CD25+ T cells in a dose-dependent manner. Addition of recombinant TNF- had no effect on proliferation (Fig. 1B) and induced minimal IFN- (Fig. 1C). Compared with the activation of CD4+CD25+ T cells alone in the presence of IL-1, proliferation (Fig. 1B) or IFN- production (Fig. 1C) were enhanced when both CD4+CD25– and CD4+CD25+ T cells were present. IL-18 had a similar effect on CD4+CD25– T cell proliferation (Fig. 1B) and IFN- production (Fig. 1C) in the presence of CD4+CD25+ T cells.
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, IL-18, or TNF- induced higher levels of IL-2 production, IL-2 was assayed in supernatants from suppression assays. IL-2 production by CD25+ T cells was equivalent in the presence or absence of each cytokine (Fig. 1D). Furthermore IL-2 production by CD25– T cells was suppressed by Treg even when cultured in the presence of IL-1 or IL-18 and even when T cell proliferation and IFN- production was high (Fig. 1D).
To test whether IFN- was sufficient to reverse suppression by CD25+ T cells, recombinant IFN- was added to suppression assays. However, no differences in suppressive capacity were observed (Fig. 1E). Finally, it was possible that IL-1 exerted its effects through the induction of related proinflammatory cytokines such as IL-6 or TNF- (21, 22). Therefore we added blocking Abs against TNF-, IL-6, or IL-18 in the presence of rIL-1 to similar assays. None of these Abs was able to prevent IL-1-stimulated proliferation or IFN- production (Fig. 1F).
These data indicate that in vitro, IL-1, and IL-18 inhibit suppression by Treg of the proliferative and IFN- responses of CD4+CD25– T cells. Although IL-1 and IL-18 stimulate IFN- production, this cytokine does not itself inhibit suppression by Treg. Furthermore, as IL-2 production is appropriately suppressed by Treg in the presence of IL-1 or IL-18, this cytokine also does not appear to mediate the enhanced T cell proliferative response to IL-1 or IL-18 in the presence of CD4+CD25+ T cells.
IL-1 stimulates CD25+ effector/memory T cell division and resistance to suppression by FoxP3+ Treg
The T cell proliferative response to autologous DC and anti-CD3 was greatly enhanced by IL-1 when both CD4+CD25– and CD4+CD25+ T cells were present. To determine which subset of cells was proliferating in this situation, we labeled either CD4+CD25– or CD4+CD25+ T cells expressing a congenic marker with CFSE, and stimulated them with DC and anti-CD3 in vitro, with or without IL-1. In the absence of CD4+CD25+ cells, CD4+CD25– effector T cells divided extensively, and division was not increased by addition of IL-1 (Fig. 2A), indicating no direct effect of IL-1 on CD4+CD25– cells. Whereas addition of CD4+CD25+ T cells inhibited proliferation of CD4+CD25– T cells, in the presence of IL-1 CD25– T cell proliferation was not inhibited to the same extent (Fig. 2A). In contrast, CD4+CD25+ T cells alone divided weakly, but the proportion of the population entering division increased with IL-1 (Fig. 2B). The data suggest that IL-1 specifically drives CD4+CD25+ T cell proliferation, and CD4+CD25– T cells then resist suppression by Treg.
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75% CD4+CD25+ Treg that express high levels of FoxP3, which confers regulatory function (23, 24, 25) and 25% activated effector/memory CD4+ T cells which are FoxP3 negative (Fig. 2C) (26). Thus, the resistance of CD4+CD25– T cells to suppression in the presence of IL-1 may have resulted either from expansion of CD25+ effector/memory CD4+ T cells, contributing to a cytokine environment favoring resistance to suppression or a direct effect on FoxP3 expression by CD4+CD25+ Treg. To test these possibilities, we examined intracellular FoxP3 expression in CFSE-labeled congenic CD4+CD25+ T cells cultured with DC and CD4+CD25– T cells. At the initiation of culture, 34% of total live cells were FoxP3+ (calculated as follows: 100,000 CD4+CD25+ cells containing 74% FoxP3+ cells were added to 100,000 CD4+CD25– and 20,000 DC). After 72 h, 33% of the total live cells were FoxP3+, but this was reduced to 23% in the presence of IL-1 (Fig. 2D). CFSE-labeling of gated congenic cells showed that both FoxP3– and FoxP3+ populations divided in the presence or absence of IL-1, and FoxP3+ cells retained FoxP3 after division (Fig. 2E). Thus, IL-1 does not directly affect FoxP3 expression by Treg but the FoxP3–CD25+ effector/memory cells expanded more rapidly than Treg. This may be because FoxP3 has been shown to exert repressor activity on T cell cycle progression and cytokine production through NFAT and NF-
B (27). Furthermore, in the presence of IL-1, CD25+ effector/memory cells produced more IFN- (Fig. 2E). Taken together, the data indicate that IL-1 directly affects CD25+ T cells. However, the proliferative response to IL-1 is enhanced when both CD4+CD25– and CD4+CD25+ T cells are present. In the presence of IL-1, CD25+FoxP3+ Treg are diluted through expansion of CD25+ effector/memory T cells with the capacity for cytokine production. Through an unknown mechanism, the suppressive ability of Treg is reduced and CD25– effector T cells expand (Fig. 2A).
IL-1 breaks self-tolerance through a direct effect on CD4+CD25+ effector/memory T cells
Although these data show that IL-1 has direct effects on T cells, IL-1 also stimulates DC maturation through NF-B activation, with associated enhancement of Ag-presenting cell function (28). Furthermore, IL-1 enhances proinflammatory cytokine production by DC, including IL-12, with consequent enhanced IFN- production by CD4+ T cells (29). The importance of this mechanism in autoimmunity was demonstrated when IL-1R1-deficient mice were found to be resistant to induction of myocarditis. Transfer of wt DC pulsed with autoantigen could initiate disease, due to IL-12 production and effective autoantigen presentation in response to IL-1 signaling of transferred DC (11). Because CD86+ mature DC or TLR-ligand-activated DC enhance proliferation of CD4+CD25+ T cells (13, 21, 30) and IL-12 stimulates T cell IFN- production, we assessed whether IL-1 could also signal CD25+ T cell activation indirectly through DC. Therefore, we examined intracellular FoxP3 expression in CFSE-labeled congenic IL-1R–/– CD4+CD25+ T cells cultured with wt DC and CD4+CD25– T cells. In this case, the proportion of FoxP3+ cells was equivalent in the presence or absence of IL-1. Furthermore in the presence of IL-1, dilution of CFSE and IFN- production by CD4+CD25+FoxP3– effector/memory T cells was retarded compared with wt T cells (Fig. 2, E–G). Taken together, the data indicate that IL-1 acts directly on CD25+ effector/memory T cells with resulting dilution of FoxP3+ Treg, rather than through effects on DC. IL-1 similarly enhanced the response of CD25+ T cells alone or CD25+ and CD25– T cells to autologous self-Ag presented in the context of MHC class II, by autologous DC in the absence of anti-CD3 (Fig. 3).
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production in autoimmune-prone strains promotes effector T cell function
The NOD strain is prone to type 1 diabetes and other autoimmune diseases due to a number of immunoregulatory defects, including expansion of autoreactive CD25+ effector T cells with time and reduced function of CD25+ Treg (14). A recent study in NOD found that, despite an increasing number of lymphoid organ CD25+ T cells, the proportion of FoxP3+ CD25+ T cells fell with age, associated with progression of autoimmunity (31). As this in vivo phenomenon resembles the effects of IL-1 on CD25+ T cells described in Fig. 2, we determined whether IL-1 could play a role in the expansion of autoreactive T cells in NOD. IL-1 production by splenocytes was compared in NOD mice and the non-autoimmune-prone B6. Splenocytes isolated from 5 to 7 week old NOD mice produced 10–15 pg/ml IL-1 after incubation in medium for 18 h in contrast to splenocytes from naive B6 mice of the same age, which produced undetectable IL-1 (Fig. 4A), consistent with previous studies showing increased IL-1 production by macrophages in NOD (32). Surprisingly, when signaled through the TCR with anti-CD3, NOD splenocytes produced 3-fold higher levels of IL-1 than B6 splenocytes (Fig. 4B). When CD4+CD25– and CD4+CD25+ T cell populations were purified from naive spleens and stimulated with immobilized anti-CD3, IL-1 secretion was found to be higher in supernatants of CD4+CD25– T cells from NOD compared with B6 (Fig. 4C). To gain insight into the mechanism of IL-1 over-production in NOD, we examined expression levels of molecules important in IL-1 regulation and signaling in splenic T cell populations. The receptor for IL-1, IL-1R1, is competitively antagonized by the non-activating ligand IL-1Ra. In addition, the non-activating soluble decoy receptor IL-1R2 may bind IL-1 thereby preventing signaling (33). Relative mRNA expression of these molecules was examined in freshly isolated CD4+CD25– and CD4+CD25+ T cells from B6 and NOD mice by quantitative PCR. IL-1R1, IL-1R2, and IL-1Ra mRNA were all expressed at higher levels in B6 and NOD CD4+CD25+ than CD4+CD25– T cells (Fig. 4, D–F). Of relevance to the elevated IL-1 produced by NOD T cells, IL-1R2 mRNA expression was decreased in NOD CD4+CD25+ T cells compared with B6 (Fig. 4E), suggesting that NOD T cells have a reduced capacity to modulate IL-1 signaling.
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by NOD T cells, CD4+CD25+ T cells purified from 7-week-old NOD mice failed to suppress CD4+CD25– T cell proliferation or IFN- production in vitro, when stimulated by splenic DC and anti-CD3 (Fig. 5, A–C). This is not surprising, because previous studies have demonstrated that the NOD CD25+ T cell population contains CD4+CD25+CD62low effector T cells and CD4+CD25highCD62high regulatory T cells (34). Because NOD T cells produce IL-1 and IL-1 expands CD25+ effector T cells, we tested whether suppression could be restored by neutralization of IL-1. Addition of anti-IL-1 mAb restored the response of NOD CD25– effector T cells to Treg (Fig. 5, A and B). In contrast, although anti-IL-18 reduced the capacity of CD4+CD25– T cells to proliferate and to produce IFN-, neither anti-IL-18 nor isotype control Ab restored the response of CD4+CD25– cells to Treg (Fig. 5, B and C). The data indicate that increased IL-1 production by NOD myeloid and T cells has important consequences for T cell regulation and IFN- production (35). IFN- plays an important proinflammatory role in autoimmune progression in humans and in mouse models of diabetes (36, 37).
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was added to B6 T cells (Fig. 1A), and suggests that when very high concentrations of CD4+CD25+ T cells are present, some regulation is observed. Furthermore, as CD25+ T cells produce little IL-1, IL-1Ra deficiency does not affect their function. Thus multiple defects in IL-1 signaling may contribute to similar effects on T cells.
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costimulates IFN- and proliferative responses by CD4+CD25+ effector/memory T cells directly through IL-1R1, similarly diluting FoxP3+ Treg (data not shown). The role of IL-1 in dysregulation of NOD CD4+CD25+ T cell function in vitro appears to be essential, as IL-1 neutralization is sufficient to restore suppression.
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on the ability of CD4+CD25+ T cells to regulate self-Ag-specific effector T cells in vivo. CD4+ 4.1 TCR transgenic T cells recognize an unknown islet self-Ag presented by DC in pLN of NOD mice. CD4+CD25– 4.1 T cells were transferred to NOD mice with or without wt or IL-1R1–/– 4.1 CD4+CD25+ T cells. Seven days later, pLN but not spleen DC from naive NOD stimulated equivalent levels of self Ag-specific pLN proliferation from NOD mice receiving 4.1 CD4+CD25– T cells alone or equivalent numbers of 4.1 CD4+CD25– and 4.1 CD4+CD25+ T cells. In contrast, pLN proliferation from mice receiving 4.1 CD4+CD25– and IL-1R1–/– 4.1 CD4+CD25+ T cells was suppressed (Fig. 7D). These data confirm in vivo that IL-1 promotes resistance to self-Ag-specific suppression by CD25+ Treg and that IL-1 reduces the ability of Treg to regulate T cell autoreactivity in vivo.
Our observations in vitro suggested IL-1-mediated dilution of CD4+CD25+FoxP3+ cells in the T cell pool could occur during an immune response, associated with expansion of effector cells. To test this idea in mice over-producing IL-1 in vivo, young NOD mice were primed with keyhole limpet hemocyanin Ag and adjuvant with or without systemic anti-IL-1 mAb. Seven days later, CD4+CD25+ T cells were purified from spleen or draining LN, and FoxP3 mRNA expression was quantitatively compared with naive CD4+CD25+ T cells. FoxP3 mRNA expression was reduced in CD4+CD25+ T cells purified from draining LN of primed mice when compared with naive CD4+CD25+ T cells or splenic CD4+CD25+ T cells of primed animals. However, no reduction was observed when IL-1 was neutralized (Fig. 7E). These results are consistent with a similar dilution of FoxP3+ T cells in the CD4+CD25+ T cell pool in vivo in response to an acute burst of IL-1.
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Discussion |
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affects the interaction of T cells with APC in the immune response, to induce expansion and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, diluting the proportion and thus suppressor function of FoxP3+ Treg within the CD25+ T cell pool (Fig. 8). Furthermore, given that mature DC not only stimulate naive T cells but also promote functional Treg expansion (30, 38), the current and previous data highlight a critical role for IL-1 in priming immunity, through its capacity to acutely tip the balance in favor of effector cell expansion. Although IFN- is also produced by effector/memory cells and can inhibit the generation or activation of Treg when produced by CD8+ tumor Ag-specific T cells (39), our data indicate that IFN- is not required for the enhanced CD25– T cell proliferative response after IL-1 signaling. Furthermore, IL-1-mediated induction of IL-2 does not explain the enhanced CD25– T cell proliferative response. The current data indicate that through an unknown mechanism, the suppressive ability of Treg is reduced and CD25– effector T cells expand. This mechanism could include a cytokine other than IFN- or IL-2, indirect effects of CD25+ effector cells on DC, or indirect effects of IL-1 on CD25+ effector cells through modulation of CD25+ Treg.
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in a genetically predisposed host could be sufficient to initiate a similar self-specific effector cell expansion with inhibition of Treg function and thus trigger autoimmunity. The data strongly suggest that a combination of inappropriate IL-1 production, excessive self-reactivity of effector T cells as a result of thymic selection defects, and declining levels of TGF-1 all contribute to the regulatory dysfunction of NOD mice (31, 40). However, the relatively small delay in the onset of diabetes in IL-1R1–/– NOD suggests that the role of IL-1 in disease development must be seen in the context of a large number of regulatory abnormalities in this model. Importantly however, the current study indicates that resistance of NOD effector/memory T cells to suppression can be restored using anti-IL-1.
It is clear from the current studies that IL-1 specifically regulates CD25+ T cells within the CD4 compartment. However, rather than regulate FoxP3 or Treg directly, IL-1 affects the outcome of DC-T cell interactions through an effect on the small CD25+FoxP3– effector/memory T cell population. IL-1 can now be added to a growing number of cytokines and receptors that costimulate enhanced effector T cell activation and thereby provoke resistance to Treg. These include IL-6, IL-7, IL-15, and glucocorticoid-induced TNFR-L (GITR-L) (12, 21, 41). However, IL-1 is the only mechanism shown to target CD25+FoxP3– T cells. Although it is possible that IL-1 might directly inhibit function of FoxP3+CD25+ T cells in a FoxP3-independent manner, this mechanism is unlikely given the critical role of FoxP3 in Treg function (24, 42). Intriguingly IL-1 is similar in some respects to GITR-L, in which both effector (CD25– in this case) and regulatory populations are expanded in response to costimulation. This dual proliferative response appears to be essential to expand both Ag-specific effectors and Treg in the context of Ag and inflammatory adjuvant. Thus, under normal circumstances, the acute stimulatory effect of IL-1 on effector T cells in inflammation will be rapidly contained by induction of counter-regulatory factors including IL-1Ra, signaled by IL-1 itself (33). Given the inflammatory arthritis that spontaneously occurs in IL-1Ra-deficient BALB/c mice, this counter-regulation appears to be essential to restore self-tolerance after proinflammatory environmental stimuli (6).
Our observations have important implications for understanding the mechanism of autoimmune diseases associated with high levels of IL-1 at the inflammatory site, immunogenetic predisposition to IL-1 overproduction, or genetic signatures reflecting IL-1 overactivity (1, 2). Up to 50% of CD25+ T cells in joint fluids from patients with JIA were CD27–FoxP3– activated effector T cells, compared with around 10% in non-inflamed sites from the same patient (12). Furthermore, rIL-1Ra suppressed arthritis in a high proportion of patients with systemic onset JIA (2). Although CD4+CD25+ populations were not examined in this study, it is tempting to speculate that IL-1 blockade prevents expansion of the CD25+CD27–FoxP3– cells thereby maintaining the FoxP3+ Treg pool—a likely situation given the low proportion of CD27–FoxP3– cells in non-inflamed sites in JIA (12). These clinical studies provide important proof-of-principle to the hypothesis that conditions associated with a break in self-tolerance in which IL-1 plays a key dysregulatory role can be successfully corrected using IL-1 blockade (2, 4). Furthermore, both in vitro and in vivo studies indicate that in rheumatoid arthritis, TNF-—a critical driver of disease pathology—is upstream of IL-1 in the inflammatory cytokine cascade hierarchy (43, 44). Improved suppressor T cell function has been observed in rheumatoid arthritis patients treated with TNF inhibitors likely due to an indirect effect of TNF inhibition on another factor (22), and it will be of interest to test the role of IL-1.
Thus, the current studies provide a novel mechanism for the common association between inflammatory events and autoimmune disease development or flares, in which high levels of IL-1 promote autoantigen-specific effector cells, which prevent regulatory T cells from maintaining tolerance to self-Ag (6, 45). These data illustrate a novel mechanism of progression from "innate to adaptive autoimmunity" (5, 46), in which innate immune system signals impact directly on the ability of T cells to balance tolerance and autoreactivity, particularly in those individuals whose disease-associated MHC class II alleles increase the likelihood of T cell responses to self-peptide (47, 48). Additional effects of IL-1 result from its capacity to enhance IL-12 production by DC and thus IFN- production by Th cells, with stimulation of Ig production (11, 49). To cite two examples, it has been proposed that enterovirus infection is associated with the first appearance of autoantibodies in type 1 diabetes (50). Furthermore, it may be no coincidence that several autoimmune arthritic diseases are associated with EBV, which sets up a life-long infection in the host characterized by a persistent host immune response (51). Of therapeutic relevance, autoantibodies can predict those at risk of disease progression in genetically predisposed individuals in several diseases, including type 1 diabetes and rheumatoid arthritis (52, 53). Suppression of IL-1 before or concomitant with the development of autoantibodies may prevent progression to overt autoimmune disease in such individuals at risk.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by grants from the National Health and Medical Research Council of Australia (to B.O.S. and R.T.), Juvenile Diabetes Research Foundation (to R.T., T.K., and H.T.), and Arthritis Queensland (to R.T.). P.S. is supported by the Canadian Institutes of Health Research and is a Scientist of the Alberta Heritage Foundation for Medical Research. 
2 Address correspondence and reprint requests to Prof. Ranjeny Thomas, Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane, Queensland 4102, Australia. E-mail: rthomas{at}cicr.uq.edu.au
3 Abbreviations used in this paper: JIA, juvenile idiopathic arthritis; Treg, regulatory T cell; GITR-L, glucocorticoid-induced TNFR-L; wt, wild type.
Received for publication December 21, 2005. Accepted for publication March 31, 2006.
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 S. Andre, D. F. Tough, S. Lacroix-Desmazes, S. V. Kaveri, and J. Bayry Surveillance of Antigen-Presenting Cells by CD4+CD25+ Regulatory T Cells in Autoimmunity: Immunopathogenesis and Therapeutic Implications Am. J. Pathol., May 1, 2009; 174(5): 1575 - 1587. [Abstract] [Full Text] [PDF]
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B. B. Ganesh, D. M. Cheatem, J. R. Sheng, C. Vasu, and B. S. Prabhakar GM-CSF-induced CD11c+CD8a--dendritic cells facilitate Foxp3+ and IL-10+ regulatory T cell expansion resulting in suppression of autoimmune thyroiditis Int. Immunol., March 1, 2009; 21(3): 269 - 282. [Abstract] [Full Text] [PDF]
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 S. Karumuthil-Melethil, N. Perez, R. Li, and C. Vasu Induction of Innate Immune Response through TLR2 and Dectin 1 Prevents Type 1 Diabetes J. Immunol., December 15, 2008; 181(12): 8323 - 8334. [Abstract] [Full Text] [PDF]
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 E. Akirav, J. A. Kushner, and K. C. Herold {beta}-Cell Mass and Type 1 Diabetes: Going, Going, Gone? Diabetes, November 1, 2008; 57(11): 2883 - 2888. [Abstract] [Full Text] [PDF]
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 E. C. Lewis, M. Mizrahi, M. Toledano, N. DeFelice, J. L. Wright, A. Churg, L. Shapiro, and C. A. Dinarello {alpha}1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice PNAS, October 21, 2008; 105(42): 16236 - 16241. [Abstract] [Full Text] [PDF]
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 P I Sidiropoulos, G Goulielmos, G K Voloudakis, E Petraki, and D T Boumpas Inflammasomes and rheumatic diseases: evolving concepts Ann Rheum Dis, October 1, 2008; 67(10): 1382 - 1389. [Abstract] [Full Text] [PDF]
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 H. J. P. M. Koenen, R. L. Smeets, P. M. Vink, E. van Rijssen, A. M. H. Boots, and I. Joosten Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells Blood, September 15, 2008; 112(6): 2340 - 2352. [Abstract] [Full Text] [PDF]
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 C. Brinster and E. M. Shevach Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells J. Leukoc. Biol., August 1, 2008; 84(2): 480 - 487. [Abstract] [Full Text] [PDF]
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H. Watanabe, S. Gehrke, E. Contassot, S. Roques, J. Tschopp, P. S. Friedmann, L. E. French, and O. Gaide Danger Signaling through the Inflammasome Acts as a Master Switch between Tolerance and Sensitization J. Immunol., May 1, 2008; 180(9): 5826 - 5832. [Abstract] [Full Text] [PDF]
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 A. Kastbom, D. Verma, P. Eriksson, T. Skogh, G. Wingren, and P. Soderkvist Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (The Swedish TIRA project) Rheumatology, April 1, 2008; 47(4): 415 - 417. [Abstract] [Full Text] [PDF]
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D. A. Rao, K. J. Tracey, and J. S. Pober IL-1{alpha} and IL-1beta Are Endogenous Mediators Linking Cell Injury to the Adaptive Alloimmune Response J. Immunol., November 15, 2007; 179(10): 6536 - 6546. [Abstract] [Full Text] [PDF]
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P. O. Scumpia, M. J. Delano, K. M. Kelly-Scumpia, J. S. Weinstein, J. L. Wynn, R. D. Winfield, C. Xia, C. S. Chung, A. Ayala, M. A. Atkinson, et al. Treatment with GITR agonistic antibody corrects adaptive immune dysfunction in sepsis Blood, November 15, 2007; 110(10): 3673 - 3681. [Abstract] [Full Text] [PDF]
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H. R. Turnquist, G. Raimondi, A. F. Zahorchak, R. T. Fischer, Z. Wang, and A. W. Thomson Rapamycin-Conditioned Dendritic Cells Are Poor Stimulators of Allogeneic CD4+ T Cells, but Enrich for Antigen-Specific Foxp3+ T Regulatory Cells and Promote Organ Transplant Tolerance J. Immunol., June 1, 2007; 178(11): 7018 - 7031. [Abstract] [Full Text] [PDF]
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X. Chen, O. M. Z. Howard, and J. J. Oppenheim Pertussis Toxin by Inducing IL-6 Promotes the Generation of IL-17-Producing CD4 Cells J. Immunol., May 15, 2007; 178(10): 6123 - 6129. [Abstract] [Full Text] [PDF]
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J.-B. Sun, S. Raghavan, A. Sjoling, S. Lundin, and J. Holmgren Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3-CD25- CD4+ Regulatory T Cells J. Immunol., December 1, 2006; 177(11): 7634 - 7644. [Abstract] [Full Text] [PDF]
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