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  2. Abstract

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome is a severe inflammatory disease that recently was associated with mutations in CIAS1. It was hypothesized that these mutations may lead to enhanced inflammatory responses. Herein, we provide evidence that inflammation in the CINCA syndrome is characterized by enhanced interleukin-1β (IL-1β) and IL-18 release upon stimulation of blood cells and show that this release is caspase 1 dependent.

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome is an inflammatory disease characterized by persistent skin rash and chronic aseptic meningitis, with extensive polymorphonuclear cell infiltration at the sites of inflammation. Patients with the CINCA syndrome experience recurrent fever and joint symptoms and have dysmorphic features, such as a saddle nose and a prominent forehead, that give them their characteristic appearance. Recently, it was reported that patients with the CINCA syndrome have mutations in CIAS1 (1), a gene previously associated with 2 other inflammatory syndromes, i.e., Muckle-Wells syndrome (MWS) and familial cold autoinflammatory syndrome (FCAS; formerly known as familial cold urticaria) (2, 3).

CIAS1, which also is designated PYPAF1 and NALP3, is expressed in polymorphonuclear cells, monocytes, chondrocytes, and activated T cells (1, 4). Cryopyrin, the CIAS1 product, contains a pyrin domain, a nucleotide-binding site of the NACHT family, and a leucine-rich repeat. All of the mutations found to date are point mutations located in exon 3 (i.e., in or around the NACHT domain). Patients with CINCA syndrome are heterozygous, which indicates a dominant effect of the altered gene product. Other investigators have hypothesized that the CIAS1 mutations lead to aberrant function of the cryopyrin protein, resulting in disturbed regulation of the inflammatory process, but no experimental data have been presented to support this hypothesis.

In transfection experiments, cryopyrin was shown to be an activator of caspase 1 (5), a member of the proinflammatory caspase family. Caspase 1 cleaves pro–interleukin-1β (proIL-1β) into biologically active IL-1β, which activates, among other proteins, NF-κB. Like IL-1β, IL-18 also is cleaved by caspase 1 to become biologically active (6). IL-1β is known to inhibit the apoptosis of polymorphonuclear cells (7) and, for instance, chondrocytes (8). Therefore, a comprehensive hypothesis explaining the pathophysiologic events in the CINCA syndrome would involve a hyperactive state of caspase 1 attributable to enhanced activation by mutated cryopyrin in cells expressing CIAS1, leading to superfluous release of IL-1β and IL-18 and an influx of polymorphonuclear cells to a site of inflammation. The similarly affected apoptotic process may then explain the persistence of the polymorphonuclear cell infiltrates and the dysmorphic features observed in patients with the CINCA syndrome. We found support for this hypothesis in our studies of lipopolysaccharide (LPS)–induced release of IL-1β and IL-18 by peripheral blood cells in a patient with the CINCA syndrome.


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  2. Abstract

The patient, a 10-year-old girl, was diagnosed as having CINCA syndrome when she was 3 years old. At birth, a maculopapular rash was present. Gradually, arthritis, chronic aseptic meningitis, and uveitis developed. At the time of the diagnosis of CINCA syndrome, the child was experiencing recurrent febrile episodes that lasted up to 2 weeks; the characteristic facial features of the syndrome developed later. Since then, she was treated with prednisone, 5 mg daily, and during this treatment the number and duration of febrile episodes had gone down. Sequence analysis of genomic DNA revealed that the patient had a G907A mutation in CIAS1 leading to a D303N amino acid alteration, and thus in the NACHT domain, a neutral residue was replaced with a highly conserved acidic residue. Both parents were homozygous for the wild-type sequence, indicating that the mutation in their daughter had appeared de novo.

To assess the activity of caspase 1 in peripheral blood cells from this patient, we stimulated ex vivo 5-fold diluted whole blood with increasing amounts of LPS, as described by van der Linden et al (9) and, after 18 hours, measured IL-1β and IL-18 release using commercially available enzyme-linked immunosorbent assays (ELISAs). The ELISA kits used for the detection of these cytokines are specific for the cleaved/secreted IL-1β and IL-18 and do not cross-react with the pro form of these cytokines. (According to Sanquin Research/CLB [Amsterdam, The Netherlands], the supplier of the IL-1β ELISA, there is no cross-reaction at all with proIL-1β, and according to BioSource International [Camarillo, CA], the supplier of the IL-18 ELISA, cross-reaction with proIL-18 is <1%).

Even without stimulation, blood cells from the patient produced detectable amounts of IL-1β, a finding we did not observe in blood cells obtained from 9 healthy controls. The addition of increasing amounts of LPS resulted in a 5-fold higher release of IL-1β in the blood cells of the patient compared with controls (Figure 1A). In the 9 healthy control samples, IL-1β production in response to stimulation with 10 ng/ml of LPS ranged from 195 to 1,613 pg/ml (mean ± SD 679 ± 426 pg/ml). Similarly, tumor necrosis factor α (TNFα), another potent proinflammatory stimulus that induces responses that overlap with those of LPS, failed to induce IL-1β release in controls but resulted in very high production of IL-1β in the patient's blood cells (Figure 1C). This response was determined not to be attributable to LPS contamination of our TNFα preparation, because a boiled TNFα sample did not induce cytokine production in whole blood obtained from controls (data not shown).

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Figure 1. Production of interleukin-1β (IL-1β) and IL-18. Samples of whole blood obtained from the patient, her mother, and a healthy control were stimulated with increasing amounts of lipopolysaccharide (LPS) (A and B) or with tumor necrosis factor α (TNFα) (C). Eighteen hours later, IL-1β (A and C) and IL-18 (B) were measured in the supernatant. Shown are representative results from 4 independent experiments (A), 3 experiments (C), and 2 experiments (B). ND = not detectable.

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The significant differences observed between the patient and controls occurred despite the fact that the patient was receiving prednisone; glucocorticoids likely dampen IL-1β production. Similar to IL-1β, IL-18 release also was dramatically increased in the patient as compared with her mother, even without LPS stimulation (Figure 1B). The synthesis of TNFα and IL-6, two other cytokines that are under the control of NF-κB, was 3–10-fold lower in the patient than in her mother (Figure 2) and was clearly outside of the range measured in healthy controls; this presumably was attributable to the prednisone treatment. IL-10 production, which is not under NF-κB control, was only somewhat lower in the patient and was only just outside of the range measured in healthy controls. Taken together, these data indicate that an overall enhanced LPS response did not occur in the patient.

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Figure 2. Synthesis of TNFα, IL-6, and IL-10. Samples of whole blood obtained from the patient (P), her mother (M), and healthy controls (C) were stimulated with increasing amounts of LPS. Eighteen hours later, TNFα (A), IL-6 (B), and IL-10 (C) were measured in the supernatant. The mean ± SD production of TNFα, IL-6, and IL-10 in healthy controls is shown. See Figure 1 for other definitions.

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To ascertain whether the enhanced IL-1β production was indeed attributable to enhanced caspase 1 activity, whole blood was stimulated with LPS in the presence of 5 μM Z-YVAD-FMK (BioVision, Mountain View, CA), a well-characterized specific caspase 1 inhibitor. After 2 and 4 hours, supernatants were collected, and IL-1β was measured. The caspase 1 inhibitor markedly blocked secretion of IL-1β, confirming that the enhanced secretion in the patient's blood cells was caspase 1 dependent (Figure 3). In the whole blood of a healthy control, the level of IL-1β secretion was much lower than that in the patient, and the secretion was also caspase 1 dependent.

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Figure 3. Production of IL-1β in the presence or absence of a caspase 1 inhibitor. Samples of whole blood obtained from the patient and a healthy control were stimulated with increasing amounts of LPS in the presence or absence of the caspase 1 inhibitor Z-YVAD-FMK. Two hours (A) and 4 hours (B) later, IL-1β was measured in the supernatant. In the absence of LPS, the levels of IL-1β were below the level of detection. See Figure 1 for definitions.

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  2. Abstract

This study is the first to show that caspase 1 activity is enhanced in a patient with the CINCA syndrome, as evidenced by enhanced IL-1β and IL-18 release by unstimulated peripheral blood cells and, more strikingly, after stimulation of these cells with LPS. Because the ELISAs used in our experiments detect only the secreted forms of both cytokines, we conclude that caspase 1 activity is markedly enhanced in this patient. In addition, we show that IL-1β secretion could be blocked by the addition of the specific caspase 1 inhibitor Z-YVAD-FMK. In the latter experiments, IL-1β release was measured 2 and 4 hours after stimulation with LPS; therefore, it is likely that new synthesis of IL-1β does not account for the enhanced IL-1β release observed in this patient. These observations were made despite the fact that the patient was being treated with prednisone. Prednisone treatment did reduce the synthesis of TNFα and IL-6, indicating that the level of caspase hyperactivation we measured is probably even an underestimate of the true effect. A similar observation was recently made in a patient with pyogenic arthritis, pyoderma gangrenosum, and acne syndrome, another inflammatory disorder, suggesting that enhanced IL-1β secretion could be a more general feature of the inflammatory syndromes (10).

Recently, Aksentijevich et al showed that proIL-1β synthesis was enhanced in a patient with the CINCA syndrome (11). Those investigators hypothesized that this was attributable to increased caspase 1 activity. However, their data (i.e., higher levels of proIL-1β in monocyte lysates) do not support their hypothesis. Higher levels of proIL-1β are a reflection of enhanced synthesis of this cytokine and not of enhanced caspase 1 activity. The higher levels of proIL-1β observed by Aksentijevich et al can be explained by the fact that secreted IL-1β will induce NF-κB activation and in that way enhance proIL-1β synthesis. In their experiments, LPS stimulation did not further increase IL-1β levels inside monocytes, presumably due to enhanced release of cleaved IL-1β. Our findings that TNFα also enhances IL-1β secretion may be mediated by a similar mechanism. Alternatively, TNFα may directly activate caspase 1 in a manner similar to that of LPS. However, more research is necessary to further investigate this observation.

Because CIAS1 is expressed in polymorphonuclear cells, and these cells are thought to mediate many of the clinical symptoms of CINCA patients, this enhanced proinflammatory response accompanied by inhibited apoptosis would provide an attractive pathophysiologic mechanism explaining the febrile episodes and dysmorphic features observed in patients with the CINCA syndrome. It would be interesting to investigate whether increased IL-1β production also leads to decreased apoptosis in our patient, although we must keep in mind that the antiapoptotic effects of IL-1β are dependent on NF-κB activation, which, in this patient, is hampered by prednisone.

Clearly, the CINCA syndrome, MWS, and FCAS are multifactorial diseases in which one gene defect may contribute to development of 3 clinically distinct syndromes. It is intriguing to speculate that various mutations in CIAS1 result in different levels of caspase 1 activation and thereby lead to a clinical spectrum ranging from the CINCA syndrome to the much less severe MWS and FCAS. Other factors are involved in caspase 1 activation as well; for instance, polymorphisms in the ASC gene, which is necessary for cryopyrin-dependent caspase 1 activation (4), may contribute to “hyperactivation” of caspase 1 and the severity of disease.

Pyrin, the familial Mediterranean fever (FMF) gene product and founder protein of the pyrin family, was shown to compete with caspase 1 for binding to the ASC gene product, and mice transgenic for the FMF form of pyrin display enhanced caspase 1 activation (12), indicating that the physiologic role of pyrin is inhibition of caspase 1. This finding fits well with the fact that FMF is a recessive disorder. In addition, pyrin was reported to modulate caspase 8 activation (13). For cryopyrin, the story may be different. Patients with the CINCA syndrome, MWS, or FCAS are heterozygous for mutations in CIAS1, suggesting that the mutated protein exerts a dominant effect. Although cryopyrin alone inhibits caspase 1 and NF-κB activation, when it is expressed together with ASC, it is a potent activator of caspase 1 and NF-κB (14–16). Recently, it was proposed that the relative ratio between ASC and cryopyrin is crucial in determining the level of caspase 1 activation (15). Because the expression levels of ASC and cryopyrin are regulated by cytokines, and because cryopyrin expression is restricted to certain cell types (1, 15), the complex interplay between these proteins and other caspase 1–activating factors may determine the outcome of the response in various cell types. In our patient with the CINCA syndrome, the outcome apparently is hyperactivation of caspase 1.

Our data and those discussed above also are consistent with the recently proposed hypothesis that inflammatory responses are controlled by “inflammasome”-like complexes (17), and our findings of increased activity of such a complex in the CINCA syndrome provide the first link between these inflammasome-like complexes and a clinical syndrome. In addition, our findings indicate that clinical studies should focus on a role for IL-1β inhibitors in the treatment of these disorders. The beneficial effects of IL-1 receptor antagonist treatment were recently demonstrated in patients with MWS (18, 19). Extending such treatment to patients with the CINCA syndrome could now be considered.


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  2. Abstract
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