Interleukin-1 receptor antagonist autoantibodies in Still’s disease: Mechanistic insights and laboratory testing

Abstract

Neutralizing autoantibodies against interleukin-1 receptor antagonist (IL-1RA) have been identified in patients with Still’s disease, often in association with hyperphosphorylation of the endogenous soluble IL-1RA protein. Although several cytokines and signaling pathways have been implicated in the pathogenesis of Still’s disease, the mechanisms underlying anti-IL-1RA autoantibody production remain unclear. Their detection is challenging due to assay heterogeneity, and therefore, a combination of different detection methods is recommended. This minireview explores potential predisposing factors for IL-1RA autoantibody development and discusses their biological and clinical significance. It also summarizes current diagnostic approaches for their detection, highlighting key challenges, pitfalls, and limitations.

Key Words: Still’s disease; Interleukin-1 receptor antagonist autoantibodies; Pathogenesis; Enzyme-linked immunosorbent; Isoelectric focusing; Western blot; Interleukin-1β signaling reporter cell assays

Core Tip: Still’s disease is a rare, severe systemic inflammatory disorder characterized by elevated levels of neutralizing interleukin-1 receptor antagonist autoantibodies. Their induction is thought to result from a combination of host-related factors, such as human leukocyte antigen class II alleles, and interleukin-1 receptor antagonist intrinsic factors, including hyperphosphorylation. Despite frequent detection of these antibodies using immunoassays and functional cell-based assays, the lack of assay standardization limits their clinical utility. At present, their presence is best regarded as a marker of underlying immune dysregulation rather than a direct pathogenic driver.

INTRODUCTION

Still’s disease is a rare, severe systemic inflammatory syndrome that includes systemic juvenile idiopathic arthritis (sJIA) in children and adult-onset Still’s disease (AoSD) in adults. Both forms share characteristic features, including quotidian spiking fever, evanescent rash, and inflammatory arthritis[1,2]. Phenotypic evidence supporting the autoinflammatory nature of Still’s disease includes elevated levels of several inflammatory biomarkers, including neutrophilic leukocytosis, increased ferritin, C-reactive protein, erythrocyte sedimentation rate, interleukin (IL)-18, and S100 proteins[3,4]. Among these, hyperferritinemia represents a key biological hallmark and is thought to reflect increased levels of IL-18 and soluble IL-2 receptor[5]. Recent findings by Jia et al[6] suggest that ferritin may actively contribute to disease pathogenesis by triggering neutrophil extracellular trap-mediated cytokine storms in AoSD.

Although the recurrent fevers and systemic inflammation observed in Still’s disease closely resemble classic autoinflammatory syndromes driven by innate immune dysregulation, accumulating evidence points to an additional role for adaptive immunity. This is supported by the detection of autoantibodies (AAbs)[7-9] and genetic association with human leukocyte antigen class II (HLA-II) alleles[10,11], implicating contributions from B cells, T cells, and AAb-mediated mechanisms. Recently, Hoffmann et al[7] reported the presence of neutralizing AAbs (Nabs) targeting IL-1 receptor antagonist (IL-1RA) in about 18.9% of individuals with Still’s disease. These AAbs have also been implicated in other conditions, such as vaccine-induced myocarditis[12], multisystem inflammatory syndrome in children (MIS-C)[13], and immunoglobulin G4-related disease (IgG4-RD)[14], suggesting a broader role in inflammatory diseases.

Why only a subset of individuals develops these AAbs remains unclear and appears to be only partly explained by factors such as antibody titer, IgG isotype, and depletion of circulating IL-1RA[7]. The reported prevalence, titers, and biological significance of these AAbs are further confounded by methodological heterogeneity in detection assays. This minireview discusses genetic and environmental factors that may predispose individuals to the development of IL-1RA Nabs and their potential biological and clinical significance in Still’s disease. It also summarizes current laboratory methods for their detection, highlighting key challenges and limitations.

OVERVIEW OF THE IL-1 CYTOKINE FAMILY IN STILL’S DISEASE

The IL-1 cytokine family, which broadly influences Still’s disease, comprises both pro- and anti-inflammatory cytokines that mediate and regulate inflammation. Key proinflammatory members include IL-1α, IL-1β, and IL-18, which promote inflammatory responses[15-17]. In contrast, anti-inflammatory members such as IL-1RA, IL-36RA, and IL-18 binding protein (IL-18BP) act to limit and resolve inflammation[16,17].

IL1-RA is an anti-inflammatory cytokine protein of about 17 kDa composed of two very short 3-10 helices and 12 β-strands[18,19]. Plasma or serum levels of IL-1RA are significantly decreased in anti-IL-1RA seropositive patients with Still’s disease[7]. Structural analyses have identified specific regions of IL-1RA that are critical for antibody recognition. In patients with Still’s disease and MIS-C, residues glycine98-asparagine116 and phenylalanine125-glycine143 located in IL-1RA have been implicated as major epitopes for IL-1RA Nabs[7,13] (Supplementary Table 1). These epitopes partially overlap with residues Met136–Glu152 and Phe100-Gly119 within the IL-1RA/IL-1 type-I receptor (IL-1RI) binding interface, which have been previously associated with Nabs against IL-1RA in the context of IgG4-RD[14] (Supplementary Table 1).

IL-36RA is a receptor antagonist with a molecular mass of about 17 kDa that binds to IL-1R6, thereby inhibiting IL-36-mediated signaling[20]. Anti-IL-36RA AAbs have been reported in approximately 7% of patients with AOSD[7]. These AAbs have also been detected in subgroups of patients with psoriasis without arthritic manifestations and in those with psoriatic arthritis, where they may contribute to disease pathogenesis[21].

Another important IL-1 family cytokine is IL-1β, which is moderately elevated in active Still’s disease[17]. The activity of IL-1β and IL-1α is effectively regulated by decoy receptors and soluble antagonists such as IL-1RA. However, when IL-1RA undergoes hyperphosphorylation, IgG-IL-1RA immune complexes may form, preventing IL-1RA from binding IL-1RI and thereby amplifying IL-1 signaling[22,23]. Similar to IL-1β, IL-18 in Still’s disease serves as a marker of heightened inflammation, with elevated circulating levels observed in patients with active disease[17]. This cytokine is considered pathogenic in macrophage activation syndrome (MAS), a complication that commonly occurs in sJIA and possibly also in AOSD[24,25]. Its biological activity is neutralized by IL-18BP, and to date, no anti-IL-18BP AAbs have been reported in patients with AOSD[7].

IL-1-mediated signaling transduction

The IL-1α/1β signaling pathway transduction can be divided into two branches, as illustrated in Figure 1. Briefly, upon binding of IL-1α or IL-1β to IL-1RI, a receptor complex forms with the IL-1 receptor accessory protein, recruiting the adaptor myeloid differentiation factor 88[26,27]. This initiates phosphorylation of IL-1R-associated kinases and subsequent activation of tumor necrosis factor receptor-associated factor 6. The nuclear factor kappa B (NF-κB) pathway is then triggered through activation of the IκB kinase complex, which phosphorylates IκB, marking it for ubiquitination and proteasomal degradation. The liberated NF-κB heterodimer translocates to the nucleus to induce transcription of pro-inflammatory cytokine genes. In parallel, the mitogen-activated protein kinases (MAPK) cascade is activated via mammalian MAPK kinase kinase, leading to phosphorylation of p38 MAPK[27]. Activated p38 translocates into the nucleus and promotes cytokine production.

Figure 1 Immune response against hyperphosphorylated interleukin-1 receptor antagonist. PP2A: Protein phosphatase 2A; HLA: Human leukocyte antigen; FOXP3: Forkhead box P3; IL-1-RA: Interleukin-1-receptor antagonist; BCR: B-cell receptor; PRR: Pattern recognition receptor; TCR: T-cell receptor; HLA-II: Human leukocyte antigen class II; IL-1RA AutoAbs: Autoantibodies against Interleukin-1 receptor antagonist; IL-1RAP: Interleukin-1 receptor accessory protein; p38 MAPK: P38 mitogen-activated protein kinase; NF-κB: Nuclear factor kappa B; IL-1RI: Interleukin-1 type-I receptor; IRAKs: Interleukin-1 receptor-associated kinases; MyD88: Myeloid differentiation factor 88; AP-1: Activator protein 1; TRAF6: Tumor necrosis factor receptor-associated factor 6.

Approaches for the detection and characterization of anti-IL-1RA AAbs

Laboratory detection and characterization of anti-IL-1RA AAbs in Still’s disease patients are performed using solid-phase immunoassays followed by functional IL-1β activation assays[7].

Solid-phase immunoassays

Detection and quantification of anti-IL-1RA AAbs are performed using in-house indirect enzyme-linked immunosorbent assays (ELISA) with antigen capture (Figure 2). Patient serum or plasma samples are incubated in microtiter wells coated with recombinant progranulin, IL-1RA, IL-18BP, and IL-36RA antigens[28]. Antibody binding is subsequently quantified by measuring optical density at about 490 nm. Notably, lower anti-IL-1RA titers are generally observed in Still’s disease[7] compared with those reported in vaccine-induced myocarditis[12] and MIS-C[13] (Supplementary Table 1). Despite its widespread use, this method has several limitations, including the need for antigen preparation[29], susceptibility to assay variability, lack of standardized cut-offs, and limited reproducibility. In addition, diagnostic specificity is suboptimal, as IL-1RA AAbs have also been detected in several inflammatory and autoimmune conditions[30] and even in some healthy individuals[12]. Accordingly, interpretation should always be guided by local laboratory protocols and integrated with the broader clinical context.

Figure 2 Approaches for the detection and characterization of anti-interleukin-1 receptor accessory autoantibodies. SEAP: Secretion of embryonic alkaline phosphatase; rec hIL-1RA: Recombinant human interleukin-1 receptor antagonist; IL-1RAP: Interleukin-1 receptor accessory protein; IL-1R1: Interleukin-1 receptor type 1; OD: Optical density; HEK: Human embryonic kidney; Anti-FLAG mAb: Anti-FLAG monoclonal antibody; HRP: Horseradish peroxidase; ELISA: Enzyme-linked immunosorbent.

Isoelectric focusing (IEF) combined with alkaline phosphatase treatment allows identification of free hyperphosphorylated IL-1RA at Glycine⁹⁸- Asparagine¹¹⁶ and Phenylalanine¹²⁵- Glycine¹⁴³[7,31]. Western blot analysis using antibodies directed against IL-1RA further confirmed the presence of IL-1RA-IgG immune complexes in the serum of patients with Still’s disease[7]. However, this approach does not provide information about the amount of complexed IL-1RA/anti-IL-1RA AAbs.

Overall, solid-phase immunoassays provide valuable information on the presence of IL-1RA AAbs but cannot differentiate pathogenic from clinically insignificant antibodies. Therefore, functional assays are essential to confirm their pathogenicity.

Functional assays/IL-1β signaling assays

Functional IL-1β signaling reporter cell assays confirm that circulating anti-IL-1RA AutoAbs neutralize the inhibitory function of endogenous IL-1RA. This results in dysinhibition of IL-1β activity, which can be experimentally demonstrated by enhanced activation of NF-κB and MAPK signaling in reporter systems (Figure 2). This functional assay measures the secretion of embryonic alkaline phosphatase by human embryonic kidney IL-1β reporter cells following incubation with patient serum containing anti-IL-1RA antibodies, IL-1β, and recombinant human IL-1RA[7,12,13]. In the presence of clinically relevant anti-IL-1RA AutoAbs, binding to recombinant human IL-1RA impedes its antagonistic function, thereby allowing endogenous IL-1β to engage its receptor on human embryonic kidney cells and trigger secretion of embryonic alkaline phosphatase. The quantitative format of the assay enables measurement of optical density at 655 nm[7]. The IL-1β signaling reporter cell assay, however, is a time-consuming assay, expensive to perform, and requires technical expertise. Prospective studies will be required to determine the relationship between antibody levels and observed dysfunction of IL-1RA bioactivity. Given the potential implications of IL-1 targeting therapies, it is crucial to validate clinical measurements of anti-IL1RA antibodies in more than one center, ensuring analytical parameters such as specificity, predictive value, precision, accuracy, and robustness are rigorously assessed. These laboratory performances should adhere to regulatory guidelines such as those outlined by the International Council for Harmonization[32].

Taken together, a combined triple approach using ELISA, IEF/western blot, and functional assays provides the most comprehensive evaluation of anti-IL-1RA AAbs and enhances understanding of their role in Still’s disease.

HOW DO ANTI-IL-1RA AABS DEVELOP: ROLE OF GENETIC FACTORS AND ENVIRONMENTAL TRIGGERS?

Still’s disease is characterized by increased levels of several AAbs, including IL-1RA AAbs[7], antinuclear antibodies[7,9,33], DEK proto-oncogene AAbs[34], rheumatoid factors, and anticyclic citrullinated peptide antibodies[35]. However, only a subset of patients develops IgG Nabs against hyperphosphorylated IL-1RA, which can impair IL-1RA levels and bioactivity in vitro[7]. Their generation appears to result from an interplay between intrinsic properties of IL-1RA (e.g., phosphorylation) and host-specific determinants (e.g., genetic susceptibility and immune regulation).

Genetic basis of anti-IL-1RA AAb production

The pivotal importance of class-switched, high-affinity IgG1 anti-IL-1RA AAbs in the pathophysiology of Still’s disease implies cooperation between B and CD4⁺ T cells (Supplementary Table 1). Activation of IL-1RA-specific CD4⁺ T lymphocytes necessitates recognition of modified IL-1RA-derived peptides presented by HLA-II molecules to T-cell receptors, thereby implicating HLA-II alleles as key susceptibility factors. This hypothesis is supported by genetic studies across diverse ethnic populations, which have identified specific HLA alleles as risk or protective factors for Still’s disease. Specifically, HLA-DQB1*06: 02, HLA-DRB1*15: 01, and HLA-DQA1*01: 02 are associated with increased susceptibility[36], whereas HLA-DRB*1: 04 and HLA-DRB1*09: 01 appear to confer protection[37,38]. In sJIA, Ombrello et al[11] reported a strong association with HLA-DRB1*11, supporting an allele-specific contribution to disease immunopathogenesis. Anti-IL-1RA AAbs preferentially target two epitopes within IL-1RA (Glycine⁹⁸- Asparagine¹¹⁶ and Phenylalanine¹²⁵-Glycine¹⁴³)[7], although the contribution of HLA-dependent peptide presentation to T cell recognition remains unknown.

Beyond HLA, genetic regulation of immune tolerance also appears relevant. The forkhead box P3 (FOXP3) gene is a key transcription factor required for regulatory T cell (Treg) development and function[39]. Mutations or reduced expression of FOXP3 impair Treg-mediated immune tolerance, leading to the development of AAbs[40,41] and increased susceptibility to several autoimmune diseases[42,43]. In AOSD, FOXP3 expression is reduced in Tregs and inversely correlated with intracellular cytokine levels of interferon gamma, IL-17, and IL-4[44]. Another study found that circulating Treg cells were significantly decreased in AOSD patients[45]. Hence, it is evident that a complete defect in Tregs results in immune system dysfunction in patients with AOSD. However, the role of this defect in the development of IL-1RA AAbs remains unknown.

As previously discussed, the balance between IL-1β and its receptor antagonist IL-1RA plays an important role in the development of Still’s disease. IL-1β and IL-1RA gene polymorphisms are not associated with the development or clinical features of AOSD[46]. However, post-translational modifications of IL-1RA, particularly temporary hyperphosphorylation at threonine 111, may act as an additional immunogenic trigger preceding the loss of peripheral tolerance[12]. Dysregulation of protein phosphatase 2A, a key serine/threonine phosphatase involved in pathways such as extracellular-signal-regulated kinase signaling[47], may promote hyperphosphorylation of autoantigenic proteins, potentially via genetic defects affecting protein phosphatase 2A regulation[48]. Similar patterns of hyperphosphorylation have been observed as a dominant trait in conditions such as monoclonal gammopathy of undetermined significance[49,50], suggesting a potential mechanistic parallel for IL-1RA in Still’s disease.

Environmental factors affecting anti-IL-1RA AAb production

In addition to genetic factors, various environmental triggers are likely to contribute to the development of IL-1RA AAbs. Recent evidence suggests that severe acute respiratory distress syndrome corona virus-2 infection and vaccination may transiently induce AAb response leading to IL-1RA depletion[12,13,30]. Other infectious agents, particularly viruses, may enhance self-antigen immunogenicity through post-translational modifications such as phosphorylation, promoting loss of immune tolerance and potentially contributing to Still’s disease[51-54].

Clinical significance of anti-IL-1RA AAbs in Still’s disease

Anti-IL-1RA AAbs contribute to the depletion of IL-1RA levels and bioactivity in vitro[30]. Nevertheless, their presence has not been associated with disease activity, disability progression, or clinical response to anakinra in Still’s disease[7]. Similarly, no consistent evidence supports an association between these AAbs and clinical presentation or disease severity in patients with MIS-C[13]. By contrast, some studies suggest that these AAbs may predict disease activity in vaccine-associated myocarditis and IgG4-RD[30]. It is therefore likely that the detection of anti-IL-1RA AAbs is best interpreted as a marker of underlying immune dysregulation rather than a direct pathogenic driver. This hypothesis is consistent with the findings of Fijolek et al[55], who reported that antineutrophil cytoplasmic antibody levels were associated with pronounced proinflammatory effects in vitro without a clear in vivo correlate.

The functional consequences of anti-IL-1RA AAbs may also be modulated by additional regulatory factors within the IL-1 pathway, including soluble IL-1RI and soluble or membrane-bound IL-1R2[56]. Further studies are needed to clarify this hypothesis and to define the mechanisms involved.

IL-1-targeted biologics in Still’s disease

IL-1-targeted biologics are therapeutics that interfere with IL-1α/β signaling by either blocking the IL-1 receptor or directly neutralizing IL-1 ligands (Figure 1). Anakinra is a recombinant form of the human soluble IL-1RA that competitively blocks IL-1R1 and has been associated with complete remission in patients with refractory AOSD[57]. Because of its short half-life, it requires once-daily subcutaneous administration.

Canakinumab, a fully human IgG1κ monoclonal antibody (about 150 kDa) that binds and neutralizes IL-1β, has also shown efficacy in Still’s disease[58]; its longer half-life allows for less frequent subcutaneous dosing.

Rilonacept, a soluble decoy receptor fusion protein of about 250 kDa that traps both IL-1α and IL-1β, has demonstrated clinical efficacy in Still’s disease[59]. Although antidrug antibodies have been reported during anakinra therapy in patients with rheumatoid arthritis[60], juvenile idiopathic arthritis[61], and cryopyrin-associated periodic syndromes[62], they are typically transient and do not affect safety or efficacy. In Still’s disease, however, anti–IL-1RA AAbs arise independently of prior anakinra exposure, indicating an endogenous origin[7]. Notably, both anakinra and canakinumab remain effective despite autoantibody-mediated reductions in IL-1RA bioactivity, underscoring the preserved efficacy of IL-1 blockade.

CONCLUSION

The detection of IL-1RA AAbs in a subset of patients with Still’s disease adds valuable insight into the complex immunopathogenesis of this condition. These AAbs are best considered candidate supporting biomarkers that reflect IL-1RA dysregulation in a defined subgroup and therefore require independent validation in larger, prospective cohorts. Their detection may be clinically useful in diagnostically challenging cases, could help biologically stratify patients for IL-1-targeted therapies, and may inform personalized treatment strategies. Multicenter, prospective studies with long-term follow-up are required to establish their true diagnostic and prognostic value and to determine whether AAb status predicts response to IL-1 blockade.

The mechanisms underlying the development of these AAbs remain incompletely understood. Future research should focus on elucidating how HLA polymorphism and the presentation of hyperphosphorylated IL-1RA peptides contribute to AAb generation. These investigations should also explore the interplay between environmental factors, such as viral infections, and HLA class II-associated risk alleles in shaping the IL-1RA autoantibody response.

Currently, laboratory testing for these AAbs is highly dependent on the ELISA protocol employed, underscoring the need for assay standardization to ensure analytical accuracy. Routine detection is not yet implemented in most clinical laboratories, and functional assessments remain confined to specialized, high-complexity research settings. Flow cytometry-based approaches, including phospho-signaling or cytokine readouts, may complement reporter assays. They can demonstrate IL-1β pathway activation by neutralizing antibodies, providing indirect evidence of IL-1RA inhibition in vitro and ex vivo. Although not yet widely available, such approaches have been established for interferon gamma and granulocyte-macrophage colony stimulating factor AAbs by leveraging knowledge of their signaling pathways.

Beyond IL-1-targeted biologics, autoreactive B cells producing pathogenic anti-IL-1RA AAbs may represent a promising therapeutic target in refractory Still’s disease. Although evidence remains limited to case reports and small series, B-cell depletion with anti-CD20 antibodies (e.g., rituximab) has shown potential efficacy in select patients[63,64]. Randomized studies are needed to confirm these observations. Furthermore, tolerance-inducing strategies, such as regulatory T-cell approaches or antigen-specific tolerization, may represent rational next steps and warrant rigorous preclinical and early-phase clinical evaluation to enable personalized therapeutic strategies.