Systemic-onset juvenile idiopathic arthritis (JIA), an autoimmune disease characterized by arthritis and systemic features, such as spiking fever, skin rash, generalized lymphadenopathy, hepatosplenomegaly, and serositis (1), was first described by the British pediatrician George F. Still in 1897 (2). The pathogenesis of systemic-onset JIA differs from that of other types of JIA in several respects, such as the lack of association with HLA type (3) and the absence of autoantibodies or autoreactive T cells (1). In fact, systemic-onset JIA has similarities to autoinflammatory diseases, as exemplified by a central role of the innate immune system and by the cytokines involved (e.g., interleukin-1 [IL-1], IL-6, and IL-18) (4). These cytokines are thought to be responsible for at least part of the clinical symptoms of the disease. Moreover, the blocking of both IL-1 and IL-6 has been shown to be efficacious in the treatment of systemic-onset JIA (5–7).
Besides the unrestrained production of cytokines, a role of natural killer (NK) cells in the pathogenesis of systemic-onset JIA has been suggested (8, 9). A key molecule in the granule-mediated exocytosis pathway in both NK cells and cytotoxic T lymphocytes (CTLs) is perforin. In familial hemophagocytic lymphohistiocytosis (FHL), a rare genetic disease, mutations are found in genes encoding perforin and related proteins that are involved in direct cell-mediated killing or cellular processes in NK cells (10, 11). The clinical features of FHL bear a marked resemblance to a common complication of systemic-onset JIA, namely, the macrophage activation syndrome (MAS) (1, 4). This resemblance between FHL and MAS is underscored by the observation of decreased NK cell function as well as decreased levels of perforin in CTLs and NK cells isolated from patients with systemic-onset JIA (8, 12, 13). It is still unclear what drives the cytokine production in relation to the observed NK cell dysfunction in systemic-onset JIA.
Recently, we showed that elevated levels of IL-18 in the plasma of patients with systemic-onset JIA correlated with disease activity (14). When IL-18 is bound to IL-18 receptor α (IL-18Rα), the ligand-binding chain of the receptor, IL-18Rβ is then recruited to form a high-affinity heterotrimeric complex with IL-18Rα and IL-18. Complex formation results in phosphorylation of the IL-18R β-chain, leading to recruitment of intracellular adaptor molecules (myeloid differentiation factor 88, interleukin-1 receptor–associated kinase, and tumor necrosis factor receptor−associated factor 6), and resulting in activation of ERK, JNK, and p38 MAP kinases (15–17). When NK cells are stimulated with IL-18, this cytokine can also potently induce interferon-γ (IFNγ) and increase NK cell activity.
We questioned why, despite increased circulating levels of IL-18, patients with systemic-onset JIA have decreased levels of perforin in CTLs and impaired NK cell function. We assumed that a defect in the IL-18/NK cell axis might contribute to the immunologic abnormalities found in systemic-onset JIA and set out to explore the functionality of NK cells in relation to IL-18. We found that although the increased levels of IL-18 in the plasma of patients with systemic-onset JIA is functional, NK cells in these patients show impaired IL-18–mediated activation due to defective phosphorylation of the IL-18R β-chain. This was not observed in patients with polyarticular JIA or in healthy control subjects. This finding has major implications for understanding and eventually treating systemic-onset JIA.
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Systemic-onset JIA is an autoinflammatory-like disease with characteristic clinical features resembling those of hereditary autoinflammatory disorders (1, 29). Unlike the classic monogenetic autoinflammatory diseases, systemic-onset JIA is far more common and is expected to have a more complex multifactorial pathogenesis. So far, abnormalities have been found in 2 broad areas: increased cytokine-inducing capacity and dysfunction of NK cells. We show herein that these 2 areas are linked with regard to IL-18 and NK cell dysfunction, since CD3–CD16+CD56dim NK cells from patients with systemic-onset JIA were found to be refractory to IL-18 stimulation because of defective IL-18 receptor signal transduction.
Clearly, this defect prohibited the activation of NK cells by IL-18 in patients with systemic-onset JIA. The IL-18 receptor complex consists of an α-chain that is responsible for binding extracellular IL-18 and a nonbinding signal-transducing β-chain, both of which belong to the IL-1 receptor family (15). When IL-18 binds to the IL-18R α-chain, the IL-18R β-chain is recruited, inducing intracellular signaling pathways that are shared with IL-1R (15, 16).
In T and B cells, activation of this signaling pathway requires additional costimulation with IL-12 to yield cell activation (28). We have established the existence of an NK cell population that has the capacity to respond to IL-18 to induce IFNγ without costimulation with IL-12 and can thus activate the innate immune system. Patients with systemic-onset JIA have increased plasma levels of IL-18, but the levels of other potent NK cell stimulators, such as IL-2, IL-12, and IL-15, are in the normal range (14). When plasma samples from patients with systemic-onset JIA were added to NK cells from either healthy controls or patients with polyarticular JIA, NK cell function was augmented, indicating the presence of stimulatory molecules. Clinical symptoms arising during the pathogenesis of systemic-onset JIA can be explained by the high levels of IL-18, as reported in studies of humans as well as mice (30–33), but the question remains, what causes the induction of extremely high IL-18 levels in the plasma of patients with systemic-onset JIA?
IL-18, a member of the IL-1 cytokine superfamily, is stored in inflammasomes as precursor protein and, upon activation, is cleaved by caspase 1 in a manner similar to that of IL-1β and IL-33 to yield active cytokine (34–36). This cytokine is one of the most effective at regulating NK cell activity (37, 38). IL-1 has been suggested to play a role in the pathogenesis of systemic-onset JIA, and indeed, treatment with an IL-1 receptor antagonist has been reported to be clinically beneficial (6). Since the plasma levels of IL-1 and the numbers of gene transcripts of the IL-1 family are normal in systemic-onset JIA (6, 14, 39), it is not expected that IL-18 levels would be increased through hyperactivity of caspase 1. This hypothesis is underscored by the fact that no changes in the levels of IL-18 or pro–IL-1β were observed in a cohort of systemic-onset JIA patients before and after treatment with anti–IL-1 receptor antagonist (5).
Until now, no mutations have been found in cytokines, cytokine receptors, or downstream signal transduction molecules of the IL-1 family. Polymorphisms in the IL-18 promoter are related to increased levels of IL-18 in systemic-onset JIA patients, but even without these polymorphisms, IL-18 levels are still well above normal in these patients (40). Consistent with a recent gene expression profiling study (41) showing an overall down-regulation of the NK cell receptor profile in patients with systemic-onset JIA, we found a decreased expression profile of NK cell receptors on the CD3–CD16+CD56dim NK cell subset in systemic-onset JIA patients, while approximately one-third of these patients lacked the CD3–CD16+CD56bright NK cell subset in the circulating lymphocyte population. One explanation for these differentially activated NK cell populations could be that in systemic-onset JIA patients, there is expansion of one or more specific NK cell populations, as seen in other arthritic diseases, such as rheumatoid arthritis, spondylarthritis, and enthesitis-related arthritis (42). The low expression profile of NK cell receptors and reduced perforin content therefore contribute to diminished NK cell function and a different activation state of NK cells in systemic-onset JIA.
T-bet is a key transcription factor that regulates IFNγ gene expression in NK cells following activation by either JNK, ERK-1/2, or p38 MAP kinases (34, 43, 44). In addition, activation of the perforin lytic signaling pathway requires ERK-1/2 signaling (45, 46). We found increased basal activity of pERK in systemic-onset JIA patients; however, we were unable to find a causal link between IL-18 and activation of this MAP kinase. The high degree of spontaneous phosphorylation of ERK-1/2 seen in unstimulated cells from systemic-onset JIA patients suggests a different activation state of NK cells in systemic-onset JIA, which is possibly linked to the perforin pathway. Previous studies have also shown a direct link between phosphorylated ERK-1/2 and several genes that encode dysregulated IL-1R/Toll-like receptor–mediated inflammation, as well as NK cell receptors, in systemic-onset JIA (39, 41).
In conclusion, we have shown that the nonfunctional IL-18/NK cell axis is due to defective IL-18 receptor function in patients with systemic-onset JIA. Further research will be required to determine whether this defect represents a reversible tolerogenic effect due to the high levels of IL-18 or whether it represents a possible genetic defect either in the IL-18 receptor itself or in essential signaling molecules downstream of the IL-18 receptor. We believe that this novel finding is of importance for the understanding of the complex immune pathogenic mechanisms at play in systemic-onset JIA and may lead to new avenues for therapeutic intervention.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. de Jager had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. De Jager, Vastert, Kuis, Coffer, Prakken.
Acquisition of data. De Jager, Vastert, Beekman, Wulffraat.
Analysis and interpretation of data. De Jager, Beekman, Wulffraat, Kuis, Coffer, Prakken.