Introduction
Schnitzler’s syndrome (SchS) is a chronic disabling autoinflammatory disorder, characterized by a chronic urticarial rash, a monoclonal component (M-protein), arthralgia and other signs and symptoms of systemic inflammation, with the long-term risk of development of a lymphoproliferative disorder [
1‐
3]. The mean age of onset is 51 years, and a positive family history has never been reported. The etiology is unknown, but a pathophysiological clue has been provided by the efficacy of anti-interleukin-1 (IL-1) treatment [
1,
4‐
11], and IL-1β inhibition in particular [
12‐
14]. However, when IL-1 inhibition is discontinued, symptoms will rapidly return after stopping the IL-1 receptor antagonist (IL-1Ra) anakinra, or will gradually return after stopping canakinumab, a monoclonal anti-IL-1β antibody. This implies that the disease process continues upstream of IL-1β [
13]. Also, whereas markers of systemic inflammation all normalize, M-protein concentrations remain unaffected during anakinra and canakinumab treatment [
13].
Previous case reports showed that peripheral blood mononuclear cells (PBMCs) or monocytes from patients with symptomatic SchS produced more IL-1β and IL-6 upon lipopolysaccharide (LPS)-stimulation compared to control PBMCs [
9,
15,
16]. Here, we studied the effect of several toll-like receptor (TLR) ligands on IL-1β, IL-6 and tumor necrosis factor alpha (TNFα) production by PBMCs of eight classical and variant SchS patients, including two variant patients with
NLRP3 mosaicism that were recently described [
17]. Moreover, we performed these experiments, as well as serum cytokine measurements, leukocyte subset analyses and serum free light-chain analyses, on blood samples collected during a symptomatic episode, anakinra treatment, and at several time points during a trial with canakinumab [
13] in order to investigate disease-specific characteristics and the effect of IL-1 on these markers.
We showed that the clinical efficacy of IL-1β inhibition in patients with SchS is associated with suppression of inflammation, and that TLR4 is involved in the enhanced IL-1β production. We also identified MRP8/14 and S100A12 as markers of disease activity in SchS.
Methods
Patients and patient samples
The study was approved by the local medical ethical committee of the Radboud university medical center, as the patients and controls were recruited there. Eight patients with SchS, either classical or variant type, and seventeen healthy controls that were age- and sex-matched as much as possible provided written informed consent. Patients stopped anakinra in order to enter the canakinumab trial and multiple blood samples were collected [
13]. Polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated during anakinra treatment, during disease relapse after discontinuation of anakinra (symptomatic episode), 14 days and 6 months after the first monthly canakinumab injection, and upon disease relapse after discontinuation of canakinumab. At each time point, blood samples from a matched healthy donor control were collected too. B cells and T cells were isolated from blood samples collected during anakinra, canakinumab and during the symptomatic phase. Serum samples were also taken on those occasions, as well as at 3 and 7 days and then monthly after the first canakinumab injection.
PBMC and polymorphonuclear cells (PMNs) processing
PBMCs were isolated from EDTA-blood using Ficoll-paque Plus (GE Healthcare, Eindhoven, The Netherlands) separation, and PMNs were isolated from the pellet by lysing erythrocytes with a hypotonic 155mM NH4Cl, 10 mM KHCO3 lysis buffer. For RNA isolation, 5 million cells of each sample were dissolved in 1 ml Trizol (Invitrogen, Bleiswijk, The Netherlands) and stored until further processing. For protein analysis, 6 million cells were lysed with a lysisbuffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM ethylene glycol tetraacetic acid (EGTA), 10 % glycerol, 1 % Triton X-100, 40 mM β-glycerophosphate, 50 mM sodium fluoride, and 200 mM sodium vanadate, supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany)) and stored at −80 °C until measurement.
PBMC culture
PBMCs isolated at the five indicated time points were also used for in vitro experiments. Cells from patients (N = 8) and controls (N = 17) were stimulated for 24 hours with LPS (TLR4 ligand, 0.1, 1, 10 ng/ml) (Sigma, St Louis, MO, USA;
Escherichia coli serotype 055:B5, purified in our own laboratory), Pam3Cys (TLR2 ligand, 10 μg/ml) (EMC Microcollections, Tubingen, Germany), poly:IC (TLR3 ligand, 5 μg/ml) (InvivoGen, Toulouse, France), or no ligand. For IL-17 assays, cells were stimulated for 7 days with heat-killed (homemade)
Candida albicans (10^6/ml). Supernatants were collected and stored at −80 °C. Cytokine concentrations in serum, supernatants and lysates were measured by means of enzyme-linked immunosorbent assay (ELISA): IL-1β (R&D, DY 201E, Minneapolis, MN, USA), IL-6 (Sanquin, M9316, Nijmegen, The Netherlands), TNF-α (R&D, DY210E), IL-17 (R&D, DY317E), MRP8/14 and S100A12 as previously described [
18,
19].
RNA extraction and quantitative real-time polymerase chain reaction (qPCR)
mRNA was extracted and first-strand cDNA was generated and amplified by means of qPCR as previously described [
20]. Specific qPCR primers were designed with Primer Express 1.0 Software (Applied Biosystems) and purchased from Biolegio (Nijmegen, The Netherlands). By means of the comparative delta-DCt method, relative mRNA expression levels of all examined genes were calculated [
21].
Microarray
A microarray using the Illumina Direct Hybridization Assay (performed by ServiceXs, Leiden, The Netherlands) was performed on purified whole blood RNA samples from two patients with classical SchS and 1 IgG variant case with myeloid-restricted NLRP3 mosaicism. Integrity of the RNA samples was confirmed by Eukaryote Total RNA Nano Bioanalyzer analysis. The microarray data were further analyzed by loading the log expression values into Partek Genomics Suite Software (Version 6.4; Partek, Inc., St Louis, MO, USA). Quantile normalization was performed including all arrays, to correct for large overall expression differences between the arrays. In addition, to adjust for the baseline expression level of each of the individual patients in the different subgroups, a correction for the factor ‘individual’ was performed, as one would do for batch correction, using the batch remove option from the software. On 1 August 2015, the data will be released online in the Gene Expression Omnibus [GSE70019].
Flow cytometric analysis
T-lymphocyte subsets
To detect intracellular expression of the transcription factors Foxp3, RORγt and Tbet in CD3+CD4+ T cells, Ficoll-isolated PBMCs were first labeled with CD3(UCHT1)-ECD and CD4(SFCI12T4D11)-PC7 (Beckman Coulter), and subsequently fixed and permeabilized using Fix/Perm buffer (eBioscience), labeled with FoxP3(PCH101)-FITC (eBioscience), RORγt(AFKJS-9)-APC and T-bet(4B10)-PE (Santa Cruz Biotechnology, Santa Cruz, CA) and measured by five-color flow cytometry (FC500, Beckman Coulter). Data were analyzed using CXP software (Beckman Coulter). Isotype controls were used for gate settings.
B-lymphocyte subsets
Cells from heparinized blood were phenotypically analyzed in a 10-color MoAb conjugate combination using a Navios™ instrument with 10-color PMTs and three solid-state lasers (Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using Kaluza™ software (Beckman Coulter). In order to guarantee that the optics, laser, fluidics and fluorescence intensity were stable during all measurements calibration was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor + Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA, USA). After erythrocyte lysis (BD Pharm-Lyse, BectonDickinson) cells were washed with PBS with 1 % bovine serum albumin before being labeled with fluorochrome-conjugated mAbs. After incubation for 30 minutes at 4 °C in the dark, cells were washed twice to remove unbound antibodies and analyzed. For cell surface staining, the following mAbs were used: IgD-FITC, IgM-PE (both Dako, Denmark) and CD3-ECD, CD4-PECy5.5, CD27-PECy7, CD20-PacB, CD45-KromeOrange, CD56-APC, CD8-APC-Alexa Fluor700 and CD19-APC-Alexa Fluor750 (all Beckman Coulter, Marseille, France). Subsequently, the various lymphocyte subpopulations were analyzed on the flow cytometer using CD45/SSC to gate the lymphocyte population.
M-protein and free light chain analysis
To detect and quantify the presence of an M-protein, agarose gel electrophoresis and immunofixation were performed on the Hydrasys (Sebia, Evry, France) according to the manufacturer’s protocol. Serum free light-chain analysis was performed on a BNII analyzer (Siemens, Marburg, Germany) using Freelite reagents (The Binding Site Ltd, Birmingham, UK) according to the manufacturer’s protocol.
Statistical analysis
Repeated-measures analysis of variance (ANOVA) using SPSS v16.0 (SPSS Inc) was performed on the delta-cycle threshold (dCt) values of the qPCR data corrected for primer efficiency. dCt is the difference between the Ct of the target gene and the reference gene (RPLP0). The ELISA data were analyzed by one-way ANOVA with Bonferroni post hoc testing for the models. Fold changes and p values of the microarray data for the contrast, patients vs. controls, were calculated by conducting multifactorial ANOVA on the factor ‘treatment’.
Discussion
Here, we described the inflammatory response during the symptomatic phase of SchS, during treatment with either anakinra or canakinumab, and during relapse after canakinumab withdrawal. During the symptomatic phase, the circulating concentrations of IL-6 were elevated, as were the protein concentrations of IL-6 and mRNA levels of IL-1β in circulating PBMCs. In this phase there was also augmented LPS-induced production of IL-1β and IL-6. Finally, the MRP8/14 and S100A12 concentrations in serum and S100A12 mRNA levels in PBMCs were elevated. All of these were normalized during treatment with either IL-1Ra or anti-IL-1β antibodies, and both therapies led to a shift of the PBMC transcriptome towards the mRNA signature of healthy controls. Clinical relapse several months following canakinumab withdrawal was not associated with a rise in IL-6 serum concentrations, nor with increased ex vivo cytokine production by PBMCs. Interestingly, irrespective of the treatment condition, PBMCs from SchS patients produced less IL-1β and IL-6 when exposed to Pam3Cys or poly:IC when compared to controls.
Taken together, our data point to an IL-1β-driven disorder, which is in line with the clinical efficacy of IL-1β inhibition and with the few reports on in vitro findings. Recently, we reported two variant SchS patients with
NLRP3 mosaicism in the myeloid lineage, in whom PBMCs produced high constitutive levels of IL-1β and IL-6, which was abolished by in vitro addition of IL-1Ra [
17]. Previously, a few SchS cases were described in which PBMCs or monocytes from symptomatic patients produced more IL-1β and IL-6 upon LPS stimulation compared to control PBMCs [
9,
15,
16], and that this could be reversed by in vivo anakinra treatment [
15]. Spontaneous IL-1β production by PBMCs was present in one more patient [
16], but absent in others [
15].
In our patients with
NLRP3 mosaicism, the hyperproduction of IL-1β is probably due to an overactive NLRP3 inflammasome. In those without this genetic defect the trigger of the enhanced IL-1β production is still unclear. The increased IL-1β concentrations in turn lead to production of IL-6 and an autocrine or paracrine production of more IL-1β. The latter became clear from the striking reduction in spontaneous IL-1β and IL-6 production by PBMCs from the two
NLRP3 mosaic patients when IL-1Ra was added in vitro [
17], the reduced IL1B mRNA levels in circulating PBMCs during in vivo IL-1 inhibition, and the lower LPS-induced IL-1β and IL-6 production in PBMCs sampled during treatment.
The increased downstream production of cytokines leads to an enhanced acute phase response with elevation of C-reactive protein (CRP). The amount of IL-6, which is readily measurable in the circulation, is probably responsible for the fever and other signs of systemic inflammation.
How should we envisage the lack of a rise in serum IL-6 and the still downregulated cytokine production when the patients relapsed several months after canakinumab withdrawal? Probably the best explanation is that the relapse is compartmentalized in its early phase, possibly at the level of the skin, which is continuously exposed to pathogen-associated molecular patterns as well as endogenous ligands of pattern recognition receptors (PRRs). Indeed, PRRs were implicated in the pathophysiology of other inflammatory skin diseases, such as AIM2 (absent in melanoma 2) and dectin-1 in psoriasis [
30‐
32]. The IL-1β positive mast cells we recently identified in SchS skin might not only be involved in the chronic urticaria (de Koning et al., submitted), but also in the induction of systemic inflammation.
As the triggers of the IL-1β production are currently unclear, it is of great interest that we find that TLR4 plays a clear role - and not TLR2, TLR3 and TLR6. It implies that either exogenous TLR4 ligands (such as LPS) or putative endogenous TLR4 ligands (like heat-shock proteins, minimally modified LDL, HMGB1, SAA3, MRP8/14, and S100A12 [
33,
34]) function as triggers for the attacks. Especially, MRP8/14 and S100A12 are interesting in this regard, as serum levels are associated with disease activity in SchS. Several in vitro studies and mouse models of other inflammatory skin diseases have demonstrated a role for TLR4, e.g., nickel-induced allergic contact dermatitis and graft versus host disease [
32]. Intriguingly, expression of both TLR4 and NLRP3 mRNA is extremely low in healthy epidermis, which one might consider a protective strategy preventing continuous stimulation by constituents of the microbiome, for example [
31]. We detected a relatively decreased responsiveness to Pam3Cys (TLR2/6 ligand) and poly:I:C (TLR3 ligand) of the SchS patient PBMCs (sampled both during symptoms and anti-IL-1 treatment) compared to control PBMCs. To our knowledge, such divergent responses to TLR2, TLR3, TLR6 and TLR4 ligands have not been reported in inflammatory diseases before. We speculate that the relative hyporesponsiveness to TLR2 and TLR3 ligands might be a protective mechanism in response to the enhanced proinflammatory response to TLR4 ligands.
Our findings of elevated spontaneous cytokine production are reminiscent of findings in patients with the cryopyrin-associated periodic syndrome CAPS in which systemic inflammation is caused by activating
NLRP3 mutations. CAPS patient PBMCs constitutively produce IL-1β, and treatment with IL-1Ra results in both a dramatic clinical improvement and substantive downregulation of LPS-induced IL-1β secretion by the patients’ cells in vitro [
35]. Typically, the enhanced proinflammatory response to lower LPS concentrations that we observed in SchS is also seen in other autoinflammatory diseases, whereas at the relatively high concentration of 10 ng/ml, the difference is much smaller or absent [
36].
Hence, current and previous findings suggest that an inflammasome is primed in PBMCs in SchS as in CAPS, which explains a substantial IL-1β release in the absence of the second hit that is usually required. Also, the relative hyporesponsiveness to TLR2/6 and TLR3 agonists (in this study) and ATP [
16], and low IL18 mRNA levels in monocytes despite high IL-18 serum levels [
37] suggest the presence of several negative feedback mechanisms.
We previously reported that in the two patients with
NLRP3 mosaicism in the myeloid cell lineage, high constitutive IL-1β and IL-6 production by PBMCs was blocked by in vitro addition of IL-1Ra [
17]. Here we show that this high baseline production was not impaired or was only partially impaired while patients were treated with IL-1 inhibitors. This implies ongoing activation of IL-1β, and may explain why these two patients had the most severe phenotypes and their disease quickly relapsed upon cessation of anti-IL-1 treatment [
13]. TLR2/6 and TLR4 ligands induced the production of IL-1β and IL-6 in these two patients, but a TLR3 ligand did not.
In this study we additionally monitored S100 proteins, B cell and T cell subsets, M-proteins and serum free light-chains. S100A8/A9 (MRP8/14) and S100A12 are secreted after activation of phagocytes via a so-called alternative secretory pathway and cause strong proinflammatory effects on phagocytes and endothelial cells in vitro [
24]. S100A8 and S100A9 and S100A12 have been shown to act as endogenous TLR-4 ligands and binding of S100 proteins to TLR-4 consequently induces NFκB expression via the MyD88-dependent pathway [
33,
34]. Besides their pathophysiological role as DAMPs S100 proteins are regarded as markers of systemic inflammation [
22‐
24] and especially in CAPS, both MRP8/14 and S100A12 levels mirrored disease activity, and were suggested as a sensitive marker even for subclinical disease [
23,
38]. In CAPS patients responding to canakinumab treatment S100A8/A9 serum levels already dropped to the range of normal controls within 8 days [
25].
In our study, S100A12 mRNA levels were significantly higher in circulating PBMCs from symptomatic patients than in PBMCs from treated patients and controls, and serum protein levels of both MRP8/14 and S100A12 correlated with disease activity. In a previous report, serum S100A12 protein levels did not correlate with disease activity in SchS, which might be related to the higher levels found in that study [
5]. In most patients, MRP8/14 levels were higher than in healthy controls, even when they were asymptomatic under anakinra or canakinumab treatment [
24]. Thus, MRP8/14 and S10012 levels are markers of disease activity in SchS. The patient data showed considerable heterogeneity, and in several patients persistent elevated MRP8/14 levels under treatment were found compared to healthy controls, as was previously reported in CAPS patients [
38]. This may indicate subclinical disease activity that is not detected by CRP or IL-6 measurements.
Previously, an increase in transitional B cells, decrease in switched-memory B cells and low levels of peripheral blood plasma cells were reported in a patient with SchS when compared to healthy controls. IL-1Ra treatment had no effect on the patient’s B lymphocytes or the IgM M-protein [
39]. Our analyses on T cell and B cell subsets showed no differences between active disease and anti-IL-1 treatment, neither were there any differences in T cell subsets between patients and controls. We do not know if long-term IL-1 inhibition would affect the T and B cell compartments.
An M-protein is one of the diagnostic hallmarks of Schs. We demonstrated that it is not a marker for disease activity as the serum concentration of the M-protein was not affected by IL-1 inhibition. Moreover, in our two most severely affected patients, only unquantifiable IgG kappa was found. Still, it cannot be excluded that long-term anti-IL-1 treatment could halt a progressive increase in M-protein concentration in view of the B cell activating property of IL-1β (the longest treatment duration of SchS patients on continuous anti-IL-1 treatment is currently 8 years). Moreover, long-term follow up of many SchS patients is needed to determine if IL-1 inhibition can prevent progression to a lymphoproliferative disorder. Indeed, in some patients with smoldering or indolent multiple myeloma, who were at risk of progression to active myeloma, concomitant treatment with IL-1Ra and dexamethasone was found to decrease the myeloma proliferative rate [
40].
The ratio of serum immunoglobulin kappa and lambda light-chain levels is a prognostic factor for multiple myeloma disease progression [
27,
28]. No such correlation was seen in our SchS patient cohort, but in one case, the highest free light-chain level was found prior to starting treatment with IL-1 inhibition. More measurements comparing pre- and post-IL-1 inhibition serum light-chain levels are needed to examine a possible association. We conclude that both the intact M-protein and the free light-chain concentrations are stable biomarkers in SchS patients, and are not affected by disease status or therapeutic intervention.
Competing interests
The clinical trial of canakinumab in SchS during which the samples were collected was an investigator-initiated trial for which HdK and AS received research support from Novartis [
13]. Novartis was not involved in the experiments carried out in this paper, nor with the interpretation of data. The authors declare that they have no competing interests.
Authors’ contributions
HdK conceived of the study, participated in its design and coordination, carried out some PBMC studies, performed analyses of all experiments, and drafted and revised the manuscript. AS conceived of the study, and participated in its design and analyses of all experiments and helped to draft and revise the manuscript. JS conceived of the study, and participated in its design and analyses of all experiments and helped to draft and revise the manuscript. JvdM conceived of the study, and participated in its design and analyses of all experiments and helped to draft the manuscript. MS and JJ carried out the PBMC studies and analyses and revised the manuscript. EV participated in the microarray analyses and revised the manuscript. JFJ the free light-chain studies and revised the manuscript, HK participated in the T cell analysis and revised the manuscript, FP participated in the B cell analyses and revised the manuscript, DH participated in the S100 ELISAs and revised the manuscript, and IJ participated in design of the study and T cell and light-chain analyses and revised the manuscript. All authors read and approved the final manuscript.