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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To examine the synthesis, processing, and secretion of interleukin-1β (IL-1β), as well as the clinical and biologic effects of IL-1 blockade, in patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome and Muckle-Wells syndrome (MWS), in an effort to understand the molecular mechanisms linking mutations of the CIAS1 gene and IL-1β hypersecretion, and the underlying response to IL-1 receptor antagonist (IL-1Ra).

Methods

Six patients with CINCA syndrome or MWS were treated with IL-1Ra and followed up longitudinally. Monocytes obtained from the patients and from 24 healthy donors were activated with lipopolysaccharide (LPS) for 3 hours, and intracellular and secreted IL-1β levels were determined by Western blotting and enzyme-linked immunosorbent assay before and after exposure to exogenous ATP.

Results

LPS-induced IL-1β secretion was markedly increased in monocytes from patients with CIAS1 mutations. However, unlike in healthy subjects, secretion of IL-1β was not induced by exogenous ATP. Treatment with IL-1Ra resulted in a dramatic clinical improvement, which was paralleled by an early and strong down-regulation of LPS-induced IL-1β secretion by the patients' cells in vitro.

Conclusion

Our results showed that the requirements of ATP stimulation for IL-1β release observed in healthy individuals are bypassed in patients bearing CIAS1 mutations. This indicates that cryopyrin is the direct target of ATP and that the mutations release the protein from the requirement of ATP for activation. In addition, the dramatic amelioration induced by IL-1Ra treatment is at least partly due to the strong decrease in IL-1β secretion that follows the first injections of the antagonist. These findings may have implications for other chronic inflammatory conditions characterized by increased IL-1β.

Interleukin-1β (IL-1β) plays a pivotal role in the pathogenesis of autoinflammatory diseases and represents a potential target of therapeutic intervention in both monogenic and multifactorial inflammatory diseases (1). Unlike most cytokines, IL-1β lacks a secretory signal peptide and is externalized by monocytic cells through a nonclassic pathway, arranged in 2 steps (2, 3). First, Toll-like receptor ligands, such as lipopolysaccharide (LPS), induce gene expression and synthesis of the inactive IL-1β precursor (proIL-1β). Monocytes stimulated with LPS alone release only ∼20% of the IL-1β over 24–48 hours (4). A second stimulus, such as exogenous ATP, strongly enhances the proteolytic maturation and secretion of IL-1β (5, 6). ATP-triggered IL-1β secretion is mediated by P2X7 receptors expressed on the surface of monocytes (7) and involves a series of events that have only been partly clarified (8, 9).

A crucial role in IL-1β processing is played by the inflammasome, a multiprotein complex responsible for the activation of caspase 1, which, in turn, converts proIL-1β to the mature, active 17-kd form (10). A key protein of the inflammasome is cryopyrin (also known as NALP3 or cold-induced autoinflammatory syndrome 1 [CIAS-1]) (11, 12). Experimental mouse models have recently revealed that monocytes from knockout mice deficient in cryopyrin cannot activate caspase 1 upon stimulation with LPS and ATP, resulting in a lack of IL-1β secretion (13–15). In contrast, mutations in the CIAS1 gene in humans are associated with diseases characterized by excessive production of IL-1β (11, 16–20). However, the molecular mechanisms connecting mutations in CIAS1 to IL-1β hypersecretion are unclear.

Cryopyrin-associated autoinflammatory diseases include familial cold autoinflammatory syndrome (FCAS; MIM no. #120100), Muckle-Wells syndrome (MWS; MIM no. #191900), and chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM no. #607115), which is also known as neonatal-onset multisystem inflammatory disease (NOMID) (21–23). These conditions represent different phenotypes, from the milder to the most severe, in the context of a clinical continuum (24). FCAS is characterized by episodes of rash, fever, and arthralgia after exposure to cold (18, 21). MWS consists of recurrent episodes of urticarial rash, fever, and abdominal pain. Sensorineural deafness and amyloidosis may represent late complications (22). CINCA syndrome is characterized by a neonatal onset, with urticarial-like skin lesions, fever, arthritis, persistent elevation of acute-phase reactants, and early involvement of the central nervous system (chronic meningitis, central deafness), eyes (uveitis, papilledema), and bones (frontal bossing, epiphyseal and metaphyseal anomalies) (19, 23, 25).

In each of these diseases, blocking the IL-1 receptors by daily injections of the recombinant human IL-1 receptor antagonist (IL-1Ra) anakinra returns the patients to normalcy, thus indicating the strong IL-1 dependency of the pathogenesis of these syndromes (18, 25–27). Despite these clear clinical results, the in vivo target of anakinra remains to be elucidated.

The present study was aimed at investigating the mode of IL-1 synthesis, processing, and secretion in patients with CIAS1 mutations. Our findings indicate excessive IL-1 secretion by monocytes from these patients. Strikingly, treatment of patients with IL-1Ra resulted in normalization of this in vitro hypersecretion. Both findings have value in elucidating the pathogenesis of CINCA syndrome and MWS and help in understanding the therapeutic effects of treatment with IL-1Ra.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

CINCA syndrome and MWS patients.

Six patients, 5 with CINCA syndrome and 1 with MWS (2 females and 4 males; mean age 13.8 years [range 3.1–33.8 years]), were enrolled in the study. The clinical characteristics of the study patients are shown in Table 1. The CINCA syndrome patients have been described in part elsewhere (28). Disease onset occurred within the first month of life in most of the study patients. The disease course in the CINCA syndrome patients was characterized by daily urticarial rashes, persistent elevation of levels of acute-phase reactants, and neurosensory, ocular, and bone involvement. In the MWS patient, symptoms began at the age of 16 months, with recurrent episodes of fever, urticarial rash, and arthritis/arthralgia, with persistent mild elevations of acute-phase reactant levels, consistent with a clinical diagnosis of MWS. Five of the 6 study patients had a mutation in exon 3 of the CIAS1 gene (Table 1).

Table 1. Clinical characteristics of the patients with CINCA syndrome or MWS*
Patient/age (years)/sexAge at onset of CINCA/MWSFeverRashArthritisHearing lossUveitis/papilledemaFacies/bone dysplasiaDevelopmental delayChronic meningitisCIAS1 mutationTreatment at the time of study
  • *

    The intensity of the clinical manifestations was scored as follows:– = absent, + = present, ++ = severe, and +++ = very severe. Patients C1–C5 have been described in part elsewhere (28). CINCA syndrome = chronic infantile neurologic, cutaneous, articular syndrome; MWS = Muckle-Wells syndrome; NSAID = nonsteroidal antiinflammatory drug.

C1/8.5/M1 week+++++++++++++++++N477KNSAID
C2/5.9/FBirth+++++++D303NNSAID
C3/18.5/M2 weeks+++++++++++++T348MNSAID
C4/33.8/M3 weeks++++++++++++++NegativeNSAID
C5/3.1/M6 months++++++M406INSAID
MWS1/12.8/F16 months++++++E525KNSAID

At baseline, the patients were experiencing daily urticarial rashes (5 of 6 patients), arthritis (4 of 6 patients), and headaches (2 of 6 patients). Elevation of acute-phase reactants and severe leukocytosis were observed in all patients. Hypochromic anemia was present in 4 of the 6 patients. Patients were treated with IL-1Ra (anakinra) at a starting dosage of 1–2 mg/kg/day, delivered subcutaneously (maximum daily dose 100 mg).

Informed consent was obtained from all patients and healthy donors. The study was approved by the Board of Ethics of G. Gaslini Institute.

Cell cultures.

Monocytes from buffy coats obtained from the blood of 24 healthy adult donors matched for sex and ethnicity or from heparinized blood obtained from the study patients were enriched by adherence in RPMI 1640 medium containing 10% fetal bovine serum, and then activated with 1 μg/ml of LPS (all from Sigma-Aldrich, Milan, Italy) for 3 hours at 37°C in RPMI 1640 medium supplemented with 1% Nutridoma-HU (Roche Applied Science, Monza, Italy) as described elsewhere (3, 8, 9). Supernatants were collected and replaced with RPMI 1640/1% Nutridoma-HU in the presence or absence of 1 mM ATP (Sigma-Aldrich) for 15 minutes. After the addition of ATP, supernatants were collected and cells were lysed in 1% Triton X-100 lysis buffer.

Determination of lactate dehydrogenase (LDH) release.

The release of LDH into supernatants as a marker of cell lysis during cocultures of dendritic cells and natural killer cells was determined by standard methods, using an LDH colorimetric assay (Sigma-Aldrich) (3).

Enzyme-linked immunosorbent assay (ELISA).

IL-1β content in supernatants was determined by ELISA (R&D Systems, Minneapolis, MN).

Western blot analysis.

Cell lysates and trichloroacetic acid–concentrated supernatants were prepared as described previously (8), resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12% gels, and then electrotransferred (4, 8). Filters were probed with 3ZD anti–IL-1β monoclonal antibody (IgG1; Biological Resources Branch, National Cancer Institute, Frederick, MD) or with rabbit anti–caspase 1 antiserum R105 (kind gift of Dr. D. K. Miller, Merck Research Laboratories, Rahway, NJ) followed by the relevant horseradish peroxidase–conjugated secondary antibody (Dako, Glostrup, Denmark) and developed by enhanced chemiluminescence using ECL Plus (Amersham Pharmacia Biotech, Milan, Italy).

Mutational screening of the CIAS1 gene and detection of P2RX7 polymorphisms.

DNA was extracted from peripheral blood samples by standard methods. All coding regions and intronic flanking sequences of the CIAS1 gene were amplified by polymerase chain reaction (PCR) using specific primers (available upon request from the authors) designed with Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Four primer pairs were used to selectively amplify exons 5, 9, 10, 11, and 13 of the P2RX7 gene, as previously described (29).

PCR fragments were purified with enzymatic mix Sap I–Exo III by incubation for 40 minutes at 37°C and for 15 minutes at 80°C, and then analyzed for mutations by direct DNA sequencing using the BigDye Terminator cycle sequencing kit v3.1 and an ABI 3100 automated DNA sequencer (both from Applied Biosystems). DNA analysis of each exon was also performed in 50 adult individuals attending our blood transfusion center.

Statistical analysis.

Comparisons among clinical and laboratory parameters before and after treatment with anakinra were performed using Wilcoxon's matched pairs test for continuous variables and McNemar's chi-square test for categorical variables. All statistical tests were 2-tailed with an alpha level of 5%.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

IL-1β production and secretion by monocytes from CINCA syndrome and MWS patients with CIAS1 gene mutations.

The production and secretion of IL-1β by monocytes from 4 patients with CINCA syndrome (patients C1–C4, Table 1), 1 patient with MWS (patient MWS1, Table 1), and 24 healthy individuals (Figure 1) were compared under resting conditions and after activation with LPS alone or with LPS followed by a brief exposure to exogenous ATP.

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Figure 1. Production, processing, and secretion of interleukin-1β (IL-1β) by monocytes from healthy adult donors (HD). a, Normal monocytes were cultured for 3 hours in the absence (top) and presence (bottom) of 1 μg/ml of lipopolysaccharide (LPS). Aliquots from cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by Western blotting with anti–IL-1β. Migration of the 35-kd and 29-kd proIL-1β molecular forms is indicated. The lower IL-1β bands were not blocked by caspase 1 inhibitors (results not shown) and are probably nonspecific endoproteases that were activated during the preparation of the samples (3). b, Supernatants from normal monocytes cultured for 3 hours with LPS (top) or for 3 hours with LPS followed by 15 minutes with ATP (bottom) were concentrated and analyzed as in a. Shown are 6 representative controls displaying different levels of basal secretion and different responses to ATP: high (donors D and B), low (donors I and O), and intermediate (donors E and F). Migration of the 17-kd mature IL-1β is indicated. Detection of secreted IL-1β was not associated with the release of lactate dehydrogenase (results not shown), which rules out a role of cell lysis in the externalization of the cytokine. c, IL-1β secreted by monocytes from 24 healthy donors during 3 hours of incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation was quantified by enzyme-linked immunosorbent assay. Results are expressed as ng/ml of IL-1β secreted per 106 cells. Values are the mean of at least 3 different experiments.

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In the absence of LPS, adherent control monocytes synthesized little or no proIL-1β (Figure 1a, top, showing 6 representative subjects). Similarly, monocytes from the CINCA syndrome and MWS patients spontaneously produced small or moderate amounts of proIL-1β (Figure 2a, lanes 1–4 and Figure 2c, lane 1). Neither control monocytes (results not shown) nor patient monocytes (Figure 2b, lanes 1, 4, 7, and 10 and Figure 2c, lane 3) secreted detectable IL-1β under these conditions. LPS stimulation drove the synthesis and intracellular accumulation of proIL-1β to a similar extent in monocytes from normal donors (Figure 1a, bottom) and patients (Figure 2a, lanes 5–8 and Figure 2C, lane 2). Notably, the lower IL-1β bands shown in Figure 1a were not blocked by treatment with caspase 1 inhibitors (results not shown) and are probably due to nonspecific endoproteases being activated during the sample preparation (3).

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Figure 2. Production, processing, and secretion of interleukin-1β (IL-1β) by monocytes from 4 patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome and 1 patient with Muckle-Wells syndrome (MWS). Peripheral blood monocytes were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by Western blotting with anti–IL-1β. Migration of the 35-kd and 29-kd proIL-1β molecular forms in cell lysates and the 17-kd mature IL-1β form in supernatants is indicated. a, Aliquots of cell lysates from untreated or lipopolysaccharide (LPS)–treated monocytes from CINCA syndrome patients were analyzed by Western blotting. b, Supernatants from CINCA syndrome patient monocytes were incubated for 3 hours without LPS (lanes 1, 4, 7, and 10), with LPS (lanes 2, 5, 8, and 11), or with LPS followed by 15 minutes with ATP (lanes 3, 6, 9, and 12) and analyzed by Western blotting. Detection of secreted IL-1β was not associated with the release of lactate dehydrogenase (results not shown). c, Aliquots of cell lysates from untreated (lane 1) or LPS-treated (lane 2) monocytes from an MWS patient were analyzed by Western blotting. Supernatants from the MWS patient monocytes were incubated for 3 hours without LPS (lane 3), with LPS (lane 4), or with LPS followed by 15 minutes with ATP (lane 5) and analyzed by Western blotting. d, IL-1β secreted by patient monocytes during 3 hours of incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation was quantified by enzyme-linked immunosorbent assay. Values are the mean of at least 3 different experiments. The mean and SD values in the 24 healthy donors (HD) shown in Figure 1d are provided for comparison. Results are expressed as ng/ml of IL-1β secreted per 106 cells.

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Control monocytes activated with LPS for 3 hours secreted variable amounts of IL-1β, which was detected as a faint 17-kd band (Figure 1b, top). As previously reported (6–9), brief exposure (15 minutes) to extracellular ATP strongly induces the secretion of mature IL-1β (Figure 1b, bottom). Quantification by ELISA (Figure 1c) confirmed the variable secretion of IL-1β during 3 hours of LPS stimulation and the different, but consistent, increases after exposure to ATP in the 24 normal donors. The presence of variable amounts of proIL-1β in supernatants was erratic and was not paralleled by a release of the cytosolic enzyme LDH (results not shown), ruling out a role of cell lysis in the externalization of the cytokine (2, 3).

In contrast, the LPS-induced secretion of mature 17-kd IL-1β by monocytes from the 3 CINCA syndrome patients bearing a CIAS1 mutation who were tested (patients C1–C3, Table 1) was dramatically high (Figure 2b, lanes 2, 5, and 8). Remarkably, the addition of exogenous ATP failed to stimulate further IL-1β secretion in these patients (Figure 2b, lanes 3, 6, and 9). Conversely, monocytes from the single CINCA syndrome patient who lacked CIAS1 mutations (patient C4, Table 1) displayed a pattern of IL-1β secretion similar to that of healthy donors, with low secretion of IL-1β during the 3 hours of stimulation with LPS, which increased following induction with ATP (Figure 2b, lanes 11 and 12). Monocytes from the MWS patient carrying the E525K mutation of the CIAS1 gene (patient MWS1, Table 1) secreted in response to LPS an IL-1β band that was weaker than that observed in the CINCA syndrome patients (Figure 2c, lane 2; compare with Figure 1b, top). However, similar to patients with CIAS1 mutations, ATP was unable to further induce IL-1β secretion (Figure 2c, lane 5).

The results of the Western blot analyses were confirmed and quantified by ELISA (Figure 2d).

Induction of caspase 1 secretion by LPS-stimulated monocytes from CINCA syndrome patients.

ATP-induced IL-1β secretion by monocytes from healthy individuals was accompanied by the conversion of procaspase 1 to active caspase 1, followed by secretion of the enzyme (8, 30, 31). Comparison of caspase 1 production and secretion in healthy individuals and CINCA syndrome patients revealed that both normal and mutated monocytes constitutively produced procaspase 1 (p46), with only a little, if any, increase being detected after LPS stimulation (Figure 3a, lanes 1 and 2). However, while control monocytes did not secrete mature 20-kd caspase 1 unless they were exposed to LPS plus ATP treatment (Figure 3a, top blot, lanes 3–5), in monocytes from patients C1 and C3 (Figure 3a, middle and bottom blots) caspase 1 activation and secretion of abundant p20 active caspase 1 occurred following activation with LPS alone (Figure 3a, lane 4). ATP was unable to drive caspase 1 secretion in cells from CINCA syndrome patients (Figure 3a, lane 5), a finding similar to that for the secretion of IL-1β.

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Figure 3. Production, processing, and secretion of caspase 1 by monocytes from a healthy donor (donor A) and from 2 chronic infantile neurologic, cutaneous, articular (CINCA) syndrome patients with a CIAS1 mutation (patients C1 and C3). Monocytes were obtained a, before or b, 2 days after initiation of treatment with anakinra. Cells were cultured as described in Figures 1 and 2, and cell lysates and supernatants were analyzed for the presence of procaspase 1 (pro-casp-1 p46) or active caspase 1 (casp-1 p20) by Western blotting with anti–caspase 1 antibody. Shown are cell lysates obtained from untreated (lane 1) or lipopolysaccharide (LPS)–treated (lane 2) monocytes and supernatants obtained from monocytes incubated for 3 hours without LPS (lane 3), with LPS (lane 4), or with LPS followed by 15 minutes with ATP (lane 5).

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Lack of relationship between resistance to ATP stimulation in CINCA syndrome or MWS patients and mutations of the gene for the P2X7 receptor.

P2RX7, the gene for the purinergic receptor responsible for the ATP-dependent release of IL-1β (7), is highly polymorphic, and a number of loss-of-function mutations have been characterized (29). The presence of functionally relevant P2RX7 polymorphisms in CINCA syndrome and MWS patients was investigated (Table 2). Patients C1, C2, and C3, whose monocytes secreted little IL-1β following ATP triggering, expressed the wild-type P2RX7, ruling out a role of this receptor in the lack of response to ATP. In contrast, patient C4, who lacked CIAS1 mutations and whose monocytes secreted IL-1β in response to ATP at levels similar to those in healthy individuals, was heterozygous for the 1513 A>C nucleotide mutation; this mutation is associated with a complete loss of function only when 2 copies are present (32, 33). The P2RX7 gene in patient MWS1 carried on both alleles the nucleotide substitution 489 C>T. This variant is present in ∼50% of the population, and although it was proposed to increase the function of P2X7 (34), its functional relevance with respect to IL-1β secretion is uncertain (29).

Table 2. Functionally relevant polymorphisms of P2RX7 in patients with CINCA syndrome or MWS*
PatientP2RX7 genotype
nt 489 C>T (His155Tyr)nt 946 G>A (Arg307Gln)nt 1096 C>G (Thr357Ser)§nt 1513 A>C (Glu496Ala)§nt 1729 T>A (Ile568Asn)
  • *

    CINCA syndrome = chronic infantile neurologic, cutaneous, articular syndrome; MWS = Muckle-Wells syndrome; nt = nucleotide.

  • The effect of His155Tyr substitution is a subject of controversy. Shemon and coworkers (29) reported this mutation as nonfunctional, whereas Cabrini and coworkers (34) proposed that it increases the activity of P2RX7.

  • Arg307Gln and Ile568Asn substitutions have also been reported to confer complete loss of function when 2 copies were present (29).

  • §

    The most common substitutions at amino acid residues 357 and 496 of P2RX7 were demonstrated to induce a complete loss of receptor function when 2 copies were present or in combination with another loss-of-function polymorphism (32).

  • Mutation.

C1C/CG/GC/CA/AT/T
C2C/CG/GC/CA/AT/T
C3C/CG/GC/CA/AT/T
C4C/CG/GC/CA/CT/T
MWS1T/TG/GC/CA/AT/T

Clinical and biologic effects of anakinra treatment in patients with CINCA syndrome or MWS.

The clinical response to anakinra treatment in the study patients is summarized in Figures 4a and b. After the first dose of anakinra was administered, all patients displayed dramatic improvement, with complete resolution of urticarial rash, arthritis, and fever within 1 week from the beginning of the treatment (Figure 4a). A rapid decrease in the levels of acute-phase reactants was also observed in the first weeks of treatment, with complete normalization in the majority of the patients (Figure 4b). Monitoring of the patients during anakinra treatment (mean 12.6 months [range 5–15 months]) revealed that all of them continued to experience complete control of their symptoms of inflammation, with nearly complete amelioration of their general conditions (Figures 4a and b). However, no significant improvement in the visual and/or acoustic impairments has so far been observed in these patients.

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Figure 4. Clinical response to anakinra treatment in 5 patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (patients C1–C5) and 1 patient with Muckle-Wells syndrome (MWS) (patient MW1). Assessments were made at baseline and at 1 week, 4 weeks, and the last followup visit (mean 12.6 months [range 5–15 months]) after treatment with anakinra. a, Number of patients with active arthritis, fever, and cutaneous manifestations before and after treatment with anakinra. = P = 0.04 versus baseline, by nonparametric McNemar's chi-square test. b, C-reactive protein (CRP) and hemoglobin (Hb) values before and after treatment with anakinra. (See also Table 1.) P values are versus baseline, as determined by nonparametric Wilcoxon's matched pairs test. NS = not significant.

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Monocytes were obtained from the patients at different time intervals following the initiation of anakinra therapy and analyzed for IL-1β production and secretion. Whereas LPS-induced IL-1β synthesis remained unchanged in individual patients as compared with the findings before anakinra treatment (results not shown), the amount of IL-1β secreted in response to LPS activation by monocytes from the 3 CINCA syndrome patients with CIAS1 mutations (patients C1–C3) was dramatically decreased (Figure 5a, lanes 2, 5, and 8). Interestingly, the decrease in IL-1β secretion induced by LPS was not accompanied by reestablishment of the responsiveness to ATP, which again failed to induce IL-1β secretion (Figure 5a, lanes 3, 6, and 9). Notably, in patients C1 and C3, secretion of caspase 1 was also strongly reduced after anakinra treatment (Figure 3b), indicating that the secretion of the 2 proteins is tightly associated.

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Figure 5. In vitro secretion of interleukin-1β (IL-1β) by cells from 4 patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (patients C1–C4) and 1 patient with Muckle-Wells syndrome (MWS) (patient MWS1) treated with anakinra. Peripheral blood monocytes were obtained at 2 days (C1 and C3), 1 month (C4 and MWS1), and 4 months (C2) after initiation of anakinra, cultured as described in Figure 2, and analyzed by Western blotting or enzyme-linked immunosorbent assay (ELISA). a, Supernatants from CINCA syndrome patient monocytes were incubated for 3 hours without lipopolysaccharide (LPS) (lanes 1, 4, 7, and 10), with LPS (lanes 2, 5, 8, and 11), or with LPS followed by 15 minutes with ATP (lanes 3, 6, 9, and 12) and analyzed by Western blotting. b, Supernatants from MWS patient monocytes were incubated for 3 hours without LPS (lanes 1), with LPS (lanes 2), or with LPS followed by 15 minutes with ATP (lanes 3) and analyzed by Western blotting. c, IL-1β secreted by patient monocytes during 3 hours of incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation was quantified by ELISA. Values are the mean of at least 3 different experiments. The mean and SD values in the 24 healthy donors (HD) shown in Figure 1c are provided for comparison. Results are expressed as ng/ml of IL-1β secreted per 106 cells.

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In contrast, in the CINCA syndrome patient who lacked a CIAS1 mutation (patient C4) and who displayed a pattern of IL-1β secretion comparable to that in normal individuals before treatment (see Figure 2b), the amount of IL-1β secreted following either LPS activation or ATP stimulation did not change significantly (Figure 5a, lanes 10–12), despite a very good clinical response to anakinra. In addition, cells from patient MWS1, who similarly displayed a good clinical response to anakinra, did not present significant variations in the amount of IL-1β secreted over 3 hours of LPS stimulation and remained insensitive to ATP stimulation (Figure 5b).

Figure 5c shows the quantification of IL-1β present in cell supernatants, as determined by ELISA. Close monitoring of IL-1β secretion by monocytes from CINCA syndrome patients during anakinra treatment showed that, although some fluctuations occurred during the year of therapy, IL-1β secretion was still low after 12 months (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

In this study, we analyzed the synthesis, processing, and secretion of IL-1β in patients with CINCA syndrome and MWS, both before and after treatment with anakinra, and compared the in vitro data with the clinical responses. Our study has generated 2 major new findings. First, monocytes from CINCA syndrome patients following LPS stimulation secrete impressively greater amounts of mature IL-1β despite a cytoplasmic accumulation of the precursor, comparable to that of healthy individuals, but fail to increase IL-1β secretion in response to ATP. These peculiarities are likely to be dependent on the mutated cryopyrin, since they were absent in the single CINCA syndrome patient who lacked known mutations. Interestingly, the MWS patient, who had a CIAS1 mutation and displayed milder clinical manifestations, failed to respond to ATP despite having levels of LPS-induced IL-1β secretion that were comparable to those secreted by normal individuals. The analysis of the sequence coding for the ATP receptor P2X7 revealed that none of the patients expressed nonfunctional P2RX7 variants, which ruled out the possibility that the failure of ATP to induce IL-1β secretion was due to a loss-of-function mutation of the highly polymorphic P2RX7 gene (7, 29).

The second main finding was that the treatment with anakinra in CINCA syndrome or MWS patients, which was associated with a dramatic clinical response, was paralleled by a sharp decrease in LPS-induced IL-1β secretion, but did not restore the ability of the patients' monocytes to secrete IL-1β in response to ATP.

We found that 2 signals are required for inflammasome activation. The dramatic secretion of IL-1β following LPS stimulation of CINCA syndrome monocytes could be dependent upon the induction of proIL-1β synthesis in a cell that, bearing a CIAS1 mutation, displays a constitutively activated inflammasome (11, 12). However, if this were the case, caspase 1, which, unlike IL-1β, is also synthesized by resting monocytes (8), would be processed and secreted even in the absence of LPS stimulation. Our study ruled out this possibility by showing that caspase 1, although normally produced by resting CINCA syndrome monocytes, was processed and secreted only after exposure to LPS.

The different behavior of normal and CIAS1-mutated monocytes (showing low versus high IL-1β and caspase 1 secretion in response to LPS and different responsiveness to ATP) suggests that in normal monocytes, 2 stimuli (LPS and ATP) are required to achieve a rapid activation of the inflammasome, whereas in patients carrying a CIAS1 mutation, LPS alone is sufficient to overactivate procaspase 1 and proIL-1β processing and secretion. In principle, the dramatic increase in IL-1β secretion following LPS stimulation of monocytes from CINCA syndrome patients could account for a “secretory exhaustion” of a cell that is no longer responsive to ATP triggering. However, “secretory exhaustion” cannot account for the lack of response to ATP of MWS monocytes that secreted IL-1β in physiologic amounts following LPS stimulation. Furthermore, the lack of response to ATP persisted in monocytes from CINCA syndrome patients after treatment with anakinra, which dramatically decreased the LPS-induced IL-1β secretion.

Recent data obtained from studies of cryopyrin-deficient mice indicate that cryopyrin is essential for assembly of the inflammasome in response to components of the signaling pathway mediated by Toll-like receptor and ATP (13, 14). Our present findings in humans support the hypothesis that cryopyrin could be the direct target of ATP (most probably mediated by the low intracellular levels of K+ induced by P2X7 receptor triggering [5]), and when mutated, cryopyrin is freed from the requirement of ATP for its activation. LPS would act upstream, possibly inducing the expression and/or activation of a different (known or unknown) inflammasome component.

In the present study, unstimulated monocytes from CINCA syndrome or MWS patients secreted little, if any, IL-1β. This result differs partially from data obtained by other groups of investigators, which showed an increased synthesis (19) and secretion (11, 20, 25) of IL-1β by purified monocytes or mononuclear cells from CINCA syndrome or MWS patients, even in the absence of LPS stimulation. This discrepancy is likely explained by the methods used. The cell adherence time we used in this study before testing proIL-1β content and IL-1β secretion is much shorter than that used by the other groups. In our experience, a longer adherence time also results in the activation of normal monocytes in the absence of LPS, although to different extents in different individuals.

How does anakinra render CIAS1-mutated monocytes refractory to LPS-induced IL-1β secretion? All 6 of our CINCA syndrome or MWS patients experienced complete clinical remission following treatment with anakinra, including the patient who lacked CIAS1 mutations; this supports the results recently reported (25) in a different cohort of patients. In addition, while Goldbach-Mansky et al (25) observed a decrease in IL1B gene expression after 3 months of treatment, we found an early (after 48 hours) and marked down-modulation of LPS-induced IL-1β secretion in all of our patients with CIAS1 mutations, independently of proIL-1β synthesis, that remained substantially unchanged as compared with pretreatment levels in each patient. This observation is consistent with the prompt and dramatic clinical response observed in all CINCA syndrome patients soon after the introduction of anakinra. In contrast, no significant decrease in IL-1β secretion was observed in the CINCA syndrome patient lacking a CIAS1 mutation or in the MWS patient. Whether this is due to a low level of LPS-induced secretion by cells from these 2 patients, which could make the decrease less evident, or whether it is due to other mechanisms remains to be clarified.

The mechanisms related to the dramatic effects of anakinra in patients with autoinflammatory syndromes are still largely unclear. Our study suggests that anakinra could play roles other than a simple competition with free circulating IL-1β for IL-1 receptor type I. In fact, the rapid clinical remission obtained in our patients was associated with a prompt decrease in IL-1β secretion by LPS-stimulated peripheral blood monocytes, which strongly suggests that the monocytes themselves represent a major target of anakinra. This observation is consistent with previous studies highlighting the capacity of IL-1 to induce IL-1 itself (35–37). Anakinra can block this positive feedback, as confirmed by the observation that normal monocytes activated in vitro with LPS in the presence of recombinant IL-1Ra decrease IL-1β secretion (data not shown).

However, the simple occupancy of monocyte IL-1 receptors by anakinra in vivo is unlikely to be responsible for the decreased IL-1β secretion observed in vitro in monocytes from CINCA syndrome patients, since the procedures used to purify and wash the monocytes should greatly reduce the amount of bound recombinant IL-1Ra. If a consistent proportion of LPS-induced IL-1β secretion occurs as a result of IL-1β itself, one could speculate that in monocytes from CINCA syndrome patients treated with anakinra, IL-1 receptor type I is down-regulated, resulting in a reduced secretion of IL-1β. It remains to be elucidated whether interference by anakinra in the vicious circle of IL-1 inducing IL-1 is the major cause of the decreased secretion or whether other mechanisms are involved.

In conclusion, our findings confirm the pivotal role of IL-1β in the pathogenesis of CINCA syndrome or MWS and shed new light on the underlying molecular mechanisms. ATP as a secretory trigger is not operative in monocytes from patients with CINCA syndrome or with MWS who carry mutated cryopyrin genes. This suggests that while in normal monocytes, 2 signals are required to activate the inflammasome, thus tightly controlling IL-1β secretion, in patients with a CIAS1 mutation, a single stimulus, even one that in low amounts (such as danger signals released by injured cells or bacterial products) would be unable to trigger IL-1β secretion in healthy individuals, is sufficient to drive a dramatic inflammatory cascade.

The in vivo encounter with anakinra not only blocks the effects of IL-1 on target cells, but also inhibits the production of IL-1β by monocytes, resulting in an impressive amelioration of the clinical manifestations. Our findings may also have implications for other chronic inflammatory conditions characterized by an increased expression of IL-1β and responsive to IL-1 blockade, such as systemic-onset juvenile idiopathic arthritis and adult Still's disease (38–41).

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Drs. Gattorno and Rubartelli had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Gattorno, Martini, Rubartelli.

Acquisition of data. Gattorno, Tassi, Carta, Delfino, Ferlito, Pelagatti, D'Osualdo, Buoncompagni, Alpigiani, Alessio.

Analysis and interpretation of data. Gattorno, Tassi, Carta, Delfino, Ferlito, Pelagatti, Buoncompagni, Rubartelli.

Manuscript preparation. Gattorno, Martini, Rubartelli.

Statistical analysis. Gattorno.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Prof. M. Ferrarini for critically reading the manuscript. We also thank the Biological Resources Branch of the National Cancer Institute (Frederick, MD) for providing the 3ZD anti–IL-1 monoclonal antibody, Dr. D. K. Miller (Merck Research Laboratories, Rahway, NJ) for the kind gift of the rabbit anti–caspase 1 antiserum R105, and the Blood Centers of Ospedale S. Martino (Genoa, Italy) for providing the buffy coats.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES