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Abstract

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

Muckle-Wells syndrome (MWS) is a dominantly inherited autoinflammatory syndrome. Patients with MWS have a mutation in CIAS1, the gene encoding cryopyrin, a component of the inflammasome that regulates the processing of interleukin-1β (IL-1β). In this report we describe an 8-year-old Japanese girl with MWS who had symptoms of periodic fever, urticarial rash, conjunctivitis, arthropathy, and sensory deafness. Laboratory analysis of the patient's serum showed abnormally high concentrations of C-reactive protein, serum amyloid A, and IL-1β, and she had a heterozygous mutation in the CIAS1 gene, with C-to-T transversion at nucleotide position 778, encoding an arginine-to-tryptophan mutation at position 260 (R260W). Mononuclear cells (MNCs) isolated from the patient secreted large amounts of IL-1β, without stimulation, and were highly sensitive to muramyldipeptide and lipopolysaccharide. After treatment with anakinra, laboratory results normalized, and clinical symptoms, including sensory deafness, disappeared, while MNCs appeared to remain activated. Thus, our case suggests that anakinra possibly affects the cryopyrin inflammasome and markedly improves the clinical and laboratory manifestations of MWS.

Muckle-Wells syndrome (MWS; MIM no. 191900) is an autosomal-dominant autoinflammatory syndrome characterized by chronic recurrent rashes, episodic or periodic fever, arthralgia, progressive sensory deafness, and the potential development of systemic AA amyloidosis (1). MWS is known to be associated with a heterozygous mutation in the cold-induced autoinflammatory syndrome 1 gene (CIAS1) (2). Cryopyrin (also known as NALP3 or PYPAF1), a putative product of CIAS1, is a member of the Nod-like receptor family that is thought to be an intracellular pathogen recognition receptor (3). CIAS1 was initially identified as the causative gene for familial cold-induced autoinflammatory syndrome (FCAS; formerly known as familial cold urticaria) (MIM no. 120100) and MWS, and has also been associated with neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM no. 607115) (2, 4).

Cryopyrin is composed of the N-terminal pyrin domain, the central part of the nucleotide-binding oligomerization domain, and C-terminal leucine-rich repeats. Cryopyrin interacts with ASC through their pyrin domains; then ASC interacts with pro–caspase 1, leading to activation of the cryopyrin inflammasome that processes pro–interleukin-1β (IL-1β) (5, 6). Up-regulation of IL-1β was reported in unstimulated monocytes isolated from a patient with NOMID/CINCA syndrome (7). The very favorable response to a therapeutic trial of a recombinant human IL-1 receptor antagonist, anakinra, in 2 patients with MWS and AA amyloidosis further implicates IL-1β as an important mediator of inflammation in patients with cryopyrin mutations (8).

We herein describe a Japanese girl with MWS and a cryopyrin mutation at R260W that was reported to be associated with sustained and intense chronic inflammation. Treatment with anakinra rapidly and completely improved her clinical symptoms, including sensory deafness, without subsequent secretion of IL-1β or other cytokines.

CASE REPORT

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

The patient, an 8-year-old Japanese girl in whom MWS was diagnosed 1 year previously, was admitted for treatment. She was born at 36 weeks and 5 days' gestation, with a birth weight of 1,528 gm. She had frequently had a urticarial rash on her face beginning 4 months after birth. Her medical records began at age 6 years, when she underwent an examination for periodic fever with persistent urticarial rash, conjunctivitis, and arthralgia. Since then, a biologic inflammatory syndrome had been observed, with the following laboratory values: white blood cell count >10,000/μl, C-reactive protein (CRP) concentration 2–8 mg/dl (normal <0.1 mg/dl), serum amyloid A (SAA) concentration 39–732 μg/ml (normal <8 μg/ml), IL-1β concentration 329 pg/ml, and IgD concentration 27–40 mg/ml (normal <9 mg/ml).

Approximately 18 months after the appearance of periodic fever, when the patient was 8 years old, audiographic examination showed the presence of bilateral sensory high-frequency deafness, which worsened thereafter (Figure 1); nevertheless, she was unaware of the deafness. A bout of conjunctivitis, arthralgia, and urticarial rash persisted, and a fever of 1–2 days' duration occurred every month. The patient had received no treatment with steroids or immunosuppressive or nonsteroidal antiinflammatory agents. Signs of inflammation such as an intense acute-phase response and moderate neutrophilia had been persistent, whereas exhaustive examinations ruled out any diagnosis of autoimmune, infectious, or neoplastic diseases. The other abnormal findings were anemia (hemoglobin concentration 10.9 gm/dl) and an elevated serum IgG level (1,862 mg/dl) attributable to chronic inflammation. The clinical diagnosis of MWS was based on the flares of urticarial rash, conjunctivitis, and arthralgia, without other neurologic symptoms or cold-induced urticaria.

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Figure 1. Audiograms from a Japanese patient with Muckle-Wells syndrome, obtained before and 3 months after treatment with anakinra. Hearing thresholds at the frequency of 0.125–8 kHz were measured with an audiometer. Each response was measured bilaterally when the acoustic sound was delivered to the probed ear.

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A genetic examination was carried out following informed consent from the patient and her parents. Genomic DNA derived from the patient's peripheral blood mononuclear cells (MNCs) had a heterozygous mutation in the CIAS1 gene, with C-to-T transversion at nucleotide position 778, encoding an arginine-to-tryptophan mutation at position 260 (R260W). Because inflammation had been persistent and audiograms showed progressive sensory deafness, the patient and her parents consented to a therapeutic trial of anakinra (Amgen, Cambridge, UK). Anakinra (1 mg/kg) was administered by subcutaneous injection once daily. After 2 weeks of treatment with anakinra, laboratory values normalized (CRP level 0.01 mg/dl, SAA concentration 2.5 mg/ml, IL-1β level <20 pg/ml, and IgD level 6.5 mg/dl), along with the disappearance of neutrophilia and languidness. After 3 months of treatment with anakinra, audiographic analysis showed complete recovery from sensory deafness (Figure 1). No bouts of rash, fever, or arthralgia were observed during anakinra treatment. In addition, her body weight gradually increased during anakinra treatment.

Serum cytokine concentrations.

The analysis of patient materials was approved by the Human Research Ethical Committee of Shinshu University. We measured the concentrations of several cytokines in the patient's serum before and after anakinra treatment. The patient's serum was stained using the Cytometric Bead Array system (BD Biosciences, San Jose, CA), and cytokines such as IL-1β, IL-6, IL-8, IL-10, IL-12p70 (IL-12), and tumor necrosis factor α (TNFα) were measured by means of flow cytometric analysis according to the manufacturer's instructions. The lower limit of the assay for all cytokines was 20 pg/ml. Before anakinra treatment, serum levels of IL-1β, IL-6, IL-8, and IL-12 were increased, but serum levels of IL-10 and TNFα were not increased (Table 1). Three days after treatment, the IL-1β level decreased dramatically, and the levels of IL-6, IL-8, and IL-12 became undetectable (Table 1).

Table 1. Cytokine concentrations (pg/ml) in the patient's serum before and after anakinra treatment
 BeforeAfter
Interleukin-1β32972
Interleukin-641≤20
Interleukin-825≤20
Interleukin-10≤20≤20
Interleukin-1257≤20
Tumor necrosis factor α≤20≤20

MNC cytokine secretion in culture supernatant.

Peripheral blood MNCs were separated by Ficoll-gradient centrifugation (GE Healthcare, Piscataway, NJ). Synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine, muramyldipeptide, and lipopolysaccharide (LPS) from Escherichia coli O55:B5, cell culture tested and purified by trichloroacetic acid extraction and gel filtration chromatography, were purchased from Sigma-Aldrich (St. Louis, MO). The cells were cultured in 24-well flat-bottomed plates (BD Biosciences) with a final cell density of 1 × 106/ml in a volume of 1 ml RPMI 1640 including 10% fetal bovine serum, with muramyldipeptide or LPS, or were left untreated for 8 hours at 37°C in a humidified atmosphere with 5% CO2. Concentrations of IL-1β and TNFα in the culture supernatant were measured by enzyme-linked immunosorbent assay, with specific antibodies (BD Biosciences).

Before treatment with anakinra, MNCs isolated from the patient secreted large amounts of IL-1β and exhibited high sensitivity to muramyldipeptide, a candidate activator of cryopyrin inflammasome (9), or LPS compared with MNCs from healthy volunteers (Figure 2). MNCs from the patient secreted a low amount of TNFα and exhibited a low response when incubated with muramyldipeptide or LPS, while MNCs from healthy volunteers exhibited a normal response when incubated with muramyldipeptide or LPS (Figure 2). After 3 weeks of anakinra treatment, the IL-1β secretion pattern of MNCs isolated from the patient was almost as normal as that of healthy volunteers, whereas anakinra treatment did not affect the TNFα secretion pattern of the patient (Figure 2).

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Figure 2. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) secretion by mononuclear cells (MNCs) derived from a Japanese patient with Muckle-Wells syndrome (MWS), before and after anakinra treatment, and MNCs from 3 healthy volunteers. Isolated MNCs were incubated with the indicated amount of muramyldipeptide (MDP) or lipopolysaccharide (LPS) or were left untreated (−) for 8 hours. The concentration of IL-1β or TNFα in the supernatants was measured by enzyme-linked immunosorbent assay. Values are the mean and SD results from triplicate cultures.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

We have described a case of MWS in a Japanese patient treated with the IL-1 receptor antagonist, anakinra. Anakinra treatment dramatically improved the clinical symptoms, especially sensory deafness, as well as the laboratory values for markers of inflammation in her serum (CRP and SAA) and cytokines such as IL-6, IL-8, IL-12, and IL-1β (Table 1). Actually, the elevated level of spontaneous IL-1β secretion and high sensitivity of IL-1β secretion from the patient's MNCs in response to muramyldipeptide or LPS normalized after anakinra treatment.

The association between chronic urticaria, mild arthralgia, progressive sensory deafness, and the spontaneous elevation of CRP concentrations was highly suggestive of MWS. MWS is associated with heterozygous mutations in CIAS1, and the R260W amino acid mutation recognized in our patient has been previously reported (10). CIAS1 mutations were identified as the cause of FCAS, MWS, and NOMID/CINCA syndrome. Disease-specific mutations of CIAS1 are reported to result in constitutive activation, the content of which is thought to affect patient symptoms in each disease (5); thus, in our patient, the diagnosis matched MWS.

Sensory deafness in a patient with MWS is reported to improve following treatment with anakinra (11). Cryopyrin expression is reported to be observed in immune cells and chondrocytes (2, 4). Goldbach-Mansky and colleagues (12) reported that highly sensitive magnetic resonance imaging sequences identify enhancing central nervous system lesions in the cochlea in the majority of patients with NOMID/CINCA syndrome. Those investigators predicted that the striking distribution for cochlear inflammation in NOMID/CINCA syndrome could be caused by local IL-1β production.

Given that amyloidosis has never been observed in the Corti apparatus or the cochlear nerve, amyloid does not directly affect deafness. IL-1β seems to play a role in the pathophysiology of sensory deafness in patients with MWS (11). Consistent with this, the sensory deafness in our patient completely resolved after anakinra treatment, suggesting that a local inflammatory response due to IL-1β secretion may be implicated in the occurrence of deafness rather than a neural development in the Corti apparatus.

In terms of other clinical symptoms, it has been reported that treatments with colchicine, low-dose corticosteroids, azathioprine, mycophenolate mofetil, and infliximab were unsuccessful, and that high-dose corticosteroids and thalidomide were partially effective (8). Anakinra has previously been reported to have a remarkable effect in MWS (13); in our patient, the CRP and SAA levels normalized after 2 weeks of anakinra treatment, and the serum IL-1β level greatly decreased within 3 days of the initiation of treatment.

Consistent with a previous report on patients with the CIAS1 mutation (7), inflammatory cytokines were up-regulated in serum from our patient (Table 1). Monocytes from patients with NOMID/CINCA syndrome and the CIAS1 mutation were reported to produce IL-1β even in the absence of stimuli such as LPS (5–7) or muramyldipeptide, a candidate activator of cryopyrin inflammasome (9). CIAS1 mutations were also reported to cause spontaneous activation of the cryopyrin inflammasome in cells, ultimately leading to increased activation of caspase 1 and processing of IL-1β (5–7). Consistent with these findings, a high concentration of IL-1β was detected in the culture supernatant of our patient's MNCs, even without any prior stimulation, which had high sensitivity in response to muramyldipeptide or LPS (Figure 2).

Notably, a difference in the secretion pattern between IL-1β and TNFα was observed. After anakinra treatment, IL-1β secretion by the patient's MNCs subsided to control levels, whereas the level of TNFα secretion from the patient's MNCs in response to muramyldipeptide or LPS remained low even after anakinra treatment. This phenomenon seems to be explained by the fact that, just as when MNCs are hyperactivated, the characteristics of MNCs do not change after anakinra treatment. TNFα generation is known to be downstream of the transcription factor NF-κB. NF-κB–dependent expression of cyclooxygenase 2 is reported to direct the synthesis of antiinflammatory cyclopentenone prostaglandins in the cells for a negative feedback loop to resolve inflammation (14). The patient's MNCs had constitutively activated cryopyrin with R260W regardless of anakinra treatment that remains to activate NF-κB, which maintain the negative feedback loop for TNFα synthesis paradoxically.

It was previously reported that after treatment of NOMID/CINCA syndrome using anti–IL-6 receptor antibodies, subsequent IL-6 hypersecretion was observed (15); thus, it was thought that blockage of the IL-1β receptor accelerates IL-1β release by a feedback mechanism. In our patient, however, anakinra dramatically improved her symptoms without subsequent hypersecretion of IL-1β. Why does such subsequent hypersecretion of IL-1β not occur with anakinra treatment? Although further experiments are needed, we hypothesize that anakinra not only might block IL-1β receptor itself but also might inhibit the cryopyrin inflammasome, resulting in the disappearance of sensory deafness and other clinical symptoms.

In conclusion, our case report demonstrates that anakinra appeared to be safe and highly effective for alleviating the clinical symptoms of MWS in a Japanese patient, possibly by inhibiting the cryopyrin inflammasome in addition to blocking the IL-1β receptor.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

Dr. Masumoto 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 design. Yamazaki, Matsumoto, Agematsu, Koike.

Acquisition of data. Yamazaki, Masumoto, Agematsu, Sawai, Kobayashi, Shingemura, Yasui.

Analysis and interpretation of data. Yamazaki, Masumoto, Agematsu.

Manuscript preparation. Yamazaki, Masumoto, Agematsu.

Statistical analysis. Yamazaki, Masumoto.

Acknowledgements

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

We thank Tsutomu Oishi (Saitama Children's Medical Center, Saitama, Japan) for DNA sequencing.

REFERENCES

  1. Top of page
  2. Abstract
  3. CASE REPORT
  4. DISCUSSION
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES