ARTHRITIS & RHEUMATISMVol. 58, No. 3, March 2008, pp 888–894DOI 10.1002/art.23286© 2008, American College of Rheumatology

Gene Polymorphisms in the NALP3 Inflammasome AreAssociated With Interleukin-1 Production and

Severe Inflammation

Relation to Common Inflammatory Diseases?

Deepti Verma,1 Maria Lerm,1 Robert Blomgran Julinder,1 Per Eriksson,1

Peter Soderkvist,1 and Eva Sarndahl2

Objective. NALP3, ASC, and TUCAN are compo-nents of the NALP3 inflammasome, which triggerscaspase 1–mediated interleukin-1� (IL-1�) release. Ac-tivating mutations in the gene encoding NALP3(NLRP3) have recently been linked to familial periodicfever syndromes. We undertook this study to determinewhether a patient with arthritis and antibiotic-resistantfever carried mutations in the genes encoding theNALP3 inflammasome.

Methods. Genetic analysis of NLRP3 and the geneencoding TUCAN (CARD-8) was performed on genomicDNA from the patient and from a population-basedcollection of DNA (806 subjects). For in vitro studies ofIL-1� production and caspase 1 activity, blood wasobtained from the patient at different time points afteradministration of anakinra, an IL-1 receptor antago-nist, as well as from 5 healthy age- and sex-matchedcontrol subjects.

Results. Mutation analysis of the patient’s genesencoding NALP3, ASC, and TUCAN revealed variationsin the NLRP3 (Q705K) and CARD-8 (C10X) genes. Theallele frequencies of these single-nucleotide polymor-phisms (SNPs) in the population were 6.5% and 34%,

respectively. The elevated activity of caspase 1 and thehigh levels of IL-1� measured in samples from thepatient returned to normal levels after treatment withanakinra.

Conclusion. Our results indicate that the pa-tient’s symptoms were due to elevated levels of IL-1�,since treatment with anakinra effectively abolished thesymptoms. The compound SNPs may explain the in-creased IL-1� levels and inflammatory symptoms ob-served, but further studies are needed to reveal afunctional relationship. The prevalence of the polymor-phisms (4% of the population carry both SNPs) in thegeneral population may suggest a genetic predispositionfor common inflammatory disorders.

The innate immune system has evolved as asystem for identifying and controlling microbial infec-tions. Following the discovery of the Toll-like receptorfamily, a large number of studies on innate immunereceptors have during the last decade brought newknowledge into the field of innate immunity. Mostrecently, members of the nucleotide-binding oligomer-ization domain–like receptor (NLR) family were de-scribed as intracellular sensors of microbial componentsand cell injury (1). NALP3 (also known as cryopyrin orPYPAF1) is a member of the NLR family that associateswith the adaptor proteins ASC and TUCAN (alsoknown as Cardinal) to form a multiprotein complextermed the “inflammasome” (1) (Figure 1). This inter-action results in processing of procaspase 1 to caspase 1,which processes pro–interleukin-1� (proIL-1�) andproIL-18 (1). IL-1� is a proinflammatory cytokineknown as a potent mediator of inflammation and leuko-cytosis.

Supported by grants from the Medical Research Council ofSoutheast Sweden (FORSS-6622). Dr. Lerm is recipient of an Assist-ing Professor fellowship and a project grant from the Swedish Re-search Council (529-2003-5994 and 521-2005-7046, respectively).

1Deepti Verma, MSc, Maria Lerm, PhD, Robert BlomgranJulinder, PhD, Per Eriksson, MD, PhD, Peter Soderkvist, PhD:Linkoping University, Linkoping, Sweden; 2Eva Sarndahl, PhD: Ore-bro University, Orebro, Sweden.

Address correspondence and reprint requests to Eva Sarn-dahl, PhD, Department of Biomedicine, Orebro University Hospital,SE-701 85 Orebro, Sweden. E-mail: eva.sarndahl@ikm.oru.se.

Submitted for publication June 15, 2007; accepted in revisedform November 30, 2007.

888

In recent years, mutations in the gene encodingNALP3 (i.e., NLRP3 [previously known as CIAS1]) havebeen linked to familial periodic fever syndromes includ-ing Muckle-Wells syndrome (MWS)/familial cold-induced autoinflammatory syndrome (FCAS) andneonatal-onset multisystem inflammatory disease(NOMID) (2,3). Recurrent fever, joint pain, skin rashes,and systemic inflammation are common in these disor-ders. A connection between elevated levels of IL-1� andmutations in NLRP3 has been reported (3), and NLRP3-knockout mice show a clear deficiency in caspase1–mediated IL-1� activation (4). Recent studies showthat the IL-1� receptor antagonist anakinra is an effec-tive treatment for patients with mutations in NLRP3 (5).

The role of the other components of the NALP3inflammasome (i.e., ASC and TUCAN) has not beenaddressed in the context of periodic fever syndromes.ASC is an adaptor protein that regulates apoptosis andcaspase 1 activation (4). TUCAN has been identified asan antiapoptotic protein that is overexpressed in sometypes of cancer (6) and that inhibits the activation ofprocaspases (6). The connection to inflammatory disor-

ders recently became evident when genetic variants inCARD-8 (the gene encoding TUCAN) were found to beassociated with Crohn’s disease (7).

The present report describes the successful use ofanakinra, an IL-1 receptor antagonist, in a patient witharthritis and chronic febrile illness. We identified agenetic variant, previously reported not to be associatedwith disease, in the patient’s NLRP3 gene. In addition,we identified a variant in the gene encoding TUCANthat yields a premature, truncated protein. Monocytesisolated from the patient displayed increased spontane-ous caspase 1 activity and IL-1� production, indicatingthat these variants result in a constitutively activeNALP3 inflammasome. These 2 variants occur simulta-neously (compound polymorphisms) in 4% of the Swed-ish population, indicating that they play a role as sus-ceptibility factors in chronic inflammatory disorders.

PATIENTS AND METHODS

Study subjects. A patient with a long history of inflam-matory disease (for medical background, see Results) was

Figure 1. Schematic drawing of the NALP3 inflammasome. The NALP3 inflammasome is composed of NALP3,which is a member of the family of nucleotide-binding oligomerization domain–like receptors, and 2 adaptorproteins, TUCAN and ASC. When activated, the protein complex recruits and activates caspase 1, which cleavespro–interleukin-1� (proIL-1�) to active IL-1�. IL-1Ra � IL-1 receptor antagonist; TLR � Toll-like receptor.

POLYMORPHISMS IN NLRP3 AND CARD-8 GENES AND INFLAMMATION 889

evaluated at 4, 24, and 34 hours after administration ofanakinra (Kineret; Amgen, Thousand Oaks, CA), when allother immunosuppressive drugs had been withdrawn (exceptfor �10 mg/day hydrocortisone). Five healthy age- and sex-matched control subjects with normal results on routine labo-ratory test (Table 1) were selected for the in vitro studies. Thepopulation used for genetic analysis consisted of 806 individ-uals randomly selected from a population registry representingthe hospital recruitment area.

Mutation analysis. Genomic DNA was isolated fromperipheral leukocytes using the Wizard Genomic DNA purifi-cation kit (Promega, Madison, WI). The coding regions ofNLRP3, CARD-8, and the ASC gene, including intron/exonborders, were amplified by polymerase chain reaction (PCR)(primer sequences are available online at http://www.liu.se/content/1/c6/10/37/86/Supplementary_Table_Verma%20et%20al.pdf). The PCR products were sequenced usingDYEnamic ET, Dye Terminator cycle sequencing kit forMegaBACE (GE Healthcare, Piscataway, NJ). To specificallydetect the identified single-nucleotide polymorphisms (SNPs)in the population, the MegaBACE SNuPe genotyping kit (GEHealthcare) was used.

Preparation of human monocytes. Peripheral bloodwas obtained from the patient at 4, 24, and 34 hours afteranakinra administration, as well as from 5 healthy age- andsex-matched control subjects, and mononuclear cells wereisolated by density-gradient centrifugation (8). The isolatedmononuclear cells were plated for 1–2 hours at 37°C, washedto eliminate nonadherent cells (e.g., lymphocytes), and cul-tured overnight (8).

Analysis of caspase 1 activity and IL-1�. Caspase 1activity was measured in monocytes using a fluorometriccaspase 1 assay (R&D Systems, Minneapolis, MN). The fluo-rescence was recorded at 535 nm after excitation at 450 nm ina Chameleon multilabel detection platform (Hidex, Turku,Finland). IL-1� concentrations were measured in plasma or inthe supernatants of stimulated monocytes using a LINCOplex

kit (Linco Research, St. Charles, MO). The samples wereexamined using a Luminex 100 system (GMI, Ramsey, MN).The data were analyzed using the software program StarSta-tion 2.0 (Applied Cytometry Systems, Sheffield, UK).

Staphylococcus aureus strain Wood 46 (American TypeCulture Collection, Rockville, MD) was grown overnight at37°C and then diluted and cultured under agitation for anadditional 3 hours at 37°C to obtain bacteria at log phase. Livebacteria were given to the monocytes (bacteria:cell ratio 20:1)for 3 hours. Where indicated, the monocytes were preincu-bated overnight (for 18 hours) with lipopolysaccharide (LPS).

NF-�B activation. For NF-�B detection, monocytesdifferentiated on glass coverslips for 9–11 days were stimulatedwith LPS or left untreated. After fixation and permeabiliza-tion, the cells were stained with an anti–NF-�B antibody(sc-8008; Santa Cruz Biotechnology, Santa Cruz, CA) and anAlexa 594–conjugated anti-mouse antibody (MolecularProbes, Eugene, OR). The samples were analyzed with a Zeissmicroscope (Zeiss, Wetzlar, Germany) equipped with a CanonG3 digital camera (Canon USA, Lake Success, NY). Thestaining intensity of the nucleus compared with that of thecytoplasm was measured in at least 50 cells using Scion Imagesoftware (Scion, Frederick, MD), and NF-�B activity wasexpressed as the ratio of nuclear staining to cytoplasmicstaining.

Ethical considerations. Genetic analysis of NLRP3,CARD-8, and the ASC gene, as well as the cell biologyanalyses, were performed after informed consent was obtainedfrom the patient and the control subjects. The study wasconducted in accordance with the ethical guidelines of theDeclaration of Helsinki.

RESULTS

Findings in the patient. The patient, a 29-year-old man, was referred to our clinic in 2002, presentingwith a long history of inflammatory disease. In 1985,when the patient was 8 years old, gonarthritis firstappeared following a Streptococcus infection. The pa-tient was positive for HLA–B27, and his father hadankylosing spondylitis (AS). Large-joint arthritis hassince recurred several times, and occasionally, musclepain has dominated. In 1993, a small amount of pleuraland pericardial fluid appeared in association with per-sistent antibiotic-resistant fever, and both symptomsimproved with high-dose cortisone. In 1996, a transientconfluent rash on the lower part of the arms wasreported in conjunction with attacks of high fever andshivering. A significantly enlarged liver permeated bycysts also characterized the clinical picture. Duringrecent years, the dominant symptoms have been arthritisin the large joints and persistent fever with a constantlyelevated C-reactive protein (CRP) level. Characteristicsigns of NOMID and MWS/FCAS, such as urticaria,bony overgrowth of joints, aseptic meningitis and othercentral nervous system manifestations, chronic papill-

Table 1. Characteristics of the patient and of the control subjects atbaseline*

WBCs,�109/liter†

CRP level,mg/liter†

Patient 8.8 � 1.3‡ 29 � 15‡Control subjects

1 9.6 �102 2.7 �103 3.9 �104 5.1 �105 5.7 �10

* Baseline values of laboratory parameters were measured at theaccredited clinical chemical laboratory at Linkoping University Hos-pital. The patient was a 29-year-old man. The 5 healthy controlsubjects were men ages 24–28 years. None of the control subjects wastaking medication.† Measured on the days of experiment.‡ Data were compiled from 3 time points (4, 24, and 34 hours afterwithdrawal of anakinra) and are the mean � SD of 5 samples (for thewhite blood cell [WBC] count) and of 4 samples (for the C-reactiveprotein [CRP] level).

890 VERMA ET AL

edema, sensorineural hearing loss, and short stature,were absent.

The patient’s illness has been investigated repeat-edly, inter alia, by colonoscopy, leukocyte scintigraphy,radiography (small intestine), and computed tomo-graphy (CT) (thorax and abdomen), but without defini-tive diagnosis. CT of the sacroiliac joints has showngrade II bilateral sacroiliitis. Results of tests for antinu-clear antibodies, antineutrophil cytoplasmic antibodies,rheumatoid factor, and complement have been negative.In May 2005, ferritin levels were significantly elevated(1,572 �g/liter) with corresponding findings of a CRPlevel of 117 mg/liter, an erythrocyte sedimentation rate(ESR) of 80 mm/hour, and a white blood cell (WBC)count of 27 � 109/liter (the patient’s maximum value was89 � 109/liter) (Figure 2). The patient’s bone marrowprofile has been reactive. Investigations for infectionincluding serology for Echinococcus, as well as tests forsoluble tumor necrosis factor receptor and serum IgD,have yielded normal findings or negative results. Un-specified spondylarthritis was diagnosed according tothe criteria of Amor et al (9). The New York criteria forAS were not fulfilled, since the patient never had anyback pain or spinal stiffness. Still’s disease was consid-ered but was excluded by other rheumatic diseasesaccording to the criteria of Yamaguchi et al (10).

Over the years, azathioprine, sulfasalazine, meth-otrexate, cyclosporin A, colchicine, intravenous admin-istration of immunoglobulin, moderate doses of corti-sone, and nonsteroidal antiinflammatory drugs havebeen ineffective. Infliximab produced side effects, whilethe effects of adalimumab and etanercept have beeninadequate. Immediately before the start of anakinratreatment, the patient was taking methylprednisolone 12mg daily, methotrexate 12.5 mg weekly, celecoxib 400 mgdaily, and etanercept 25 mg twice weekly.

In early August 2005, treatment with anakinra(100 mg/day subcutaneously) was initiated. Within 24hours, the patient was afebrile and free of arthritis. TheCRP level dropped from 129 mg/liter to 31 mg/liter, andthe WBC count dropped from 23.3 � 109/liter to 10.9 �109/liter within 48 hours (Figure 2). These parametersbecame entirely normal within 1 week. Except for sebor-rheic dermatitis, no severe side effects have occurred.Four months after introducing anakinra, hip replace-ment was performed due to severe coxarthrosis second-ary to previous arthritis. During 2 years of followup, thepatient has felt quite well, and CRP levels and ESRshave been normal or slightly elevated (Figure 2). Today,anakinra is administered to the patient daily, and he is

taking no antiinflammatory drugs, with the exception ofhydrocortisone 10 mg daily.

Genetic analysis of the patient and of a represen-tative Swedish population. MegaBACE sequencing ofthe genes of the NALP3 inflammasome revealed thatthe patient carried a heterozygous base change,2107C�A (Q705K), in exon 3 (corresponding to theNACHT domain) of the NLRP3 gene. (The Q705Kpolymorphism has previously been reported as Q703K[11] and has then consistently been referred to asQ703K. According to the messenger RNA sequence

Figure 2. Laboratory findings in the patient. Shown are the erythro-cyte sedimentation rate (ESR; normal 1–12 mm/hour) (A), theC-reactive protein (CRP) level (normal �10 mg/liter) (B), and thewhite blood cell (WBC) count (normal 3.5–8.8 � 109/liter) (C)measured before and after the introduction of anakinra (100 mg/daysubcutaneously). The medication taken by the patient during this timeis indicated (D), as well as the time at which the patient underwent ahip replacement and the start of anakinra therapy (A–C).

POLYMORPHISMS IN NLRP3 AND CARD-8 GENES AND INFLAMMATION 891

[accession no. NM_004895], this polymorphic site will beamino acid 705 and will therefore be referred to asQ705K in this article.) A heterozygous base change wasalso detected in the CARD-8 gene at codon 10 (C10X),resulting in a premature truncation of the protein. Noneof the control subjects had a mutation in the NLRP3gene; however, 1 of the controls was heterozygous forthe C10X base change in CARD-8. The investigatedparameters (i.e., CRP level, WBC count, caspase 1activity, IL-1� production) of this individual were indis-tinguishable from those of the other control subjects,and this control subject was included in the study.

The prevalence rates of the mutations of NLRP3

and CARD-8 were assessed in a population of 806randomly selected individuals. These variants werefound to be common polymorphisms, present at allelefrequencies of 6.5% for Q705K in NLRP3 and 34% forC10X in the CARD-8 gene. Approximately 4% of thepopulation was found to be compound heterozygotes(i.e., carriers of both single SNPs), and these SNPs werefound to be in Hardy-Weinberg equilibrium in thepopulation.

Inflammatory activity of leukocytes. Accordingto the manufacturer, the half-life of anakinra is 4–6hours. The baseline caspase 1 activity of monocytes wastherefore investigated 4, 24, and 34 hours after admin-

Figure 3. Caspase 1 activity and interleukin-1� (IL-1�) level in the patient (4, 24, and 34 hours after administration of anakinra) and in healthycontrol subjects. A, Relative mean baseline caspase 1 activity in monocytes from the patient (n � 2), expressed as a percentage of that in monocytesfrom the controls (n � 5). Dotted lines indicate the SD of the mean value in monocytes from the controls. B, Mean baseline levels of IL-1� in plasmafrom the patient (n � 2) and from controls (n � 5). Dotted line indicates maximum normal IL-1� value. Error bar indicates the SD. C and D, MeanIL-1� levels in response to live Staphylococcus aureus (C) or mean IL-1� levels in response to lipopolysaccharide (LPS) and live S aureus (D)measured in the supernatant of monocytes from the patient (n � 2) or from controls (n � 5). Error bars indicate the SD.

892 VERMA ET AL

istration of the drug. Four hours after administration ofanakinra, the patient demonstrated spontaneous caspase1 activity and IL-1� plasma levels similar to those in thecontrol subjects (Figures 3A and B). Twenty-four hoursafter administration of anakinra, the patient’s IL-1�level was elevated (Figure 3B), and 34 hours afteradministration of anakinra, high spontaneous caspase 1activity in the patient’s monocytes (Figure 3A) and ele-vated plasma levels of IL-1� (Figure 3B) were evident.

Next, we measured the basal and LPS-stimulatedNF-�B activity of macrophages. Following stimulationwith LPS (for 0 minutes, 6 hours, or 18 hours), the cellswere fixed and stained with an antibody directed towardthe p65 subunit of NF-�B. LPS induced a transientincrease of NF-�B activity, which was 1.4-fold that in theunstimulated cells at 30 minutes and which decreased tobasal levels at 6 hours (data not shown). However, nodifference between the control subjects and the patientwas observed.

IL-1� production in the presence of microbes.The release of IL-1� upon challenge with microbes wasmeasured in the supernatants of monocytes (Figures 3Cand D). S aureus alone increased by 3-fold the amount ofIL-1� in the supernatant of monocytes from controlsubjects, but this effect was not seen in the supernatantof monocytes from the patient 4 hours after anakinratreatment. However, at 24 hours or 34 hours, high levelsof IL-1� (64 pg/ml and 140 pg/ml, respectively) weredetected in the supernatant of monocytes from thepatient (Figure 3C), and LPS pretreatment of mono-cytes from control subjects or from the patient 34 hoursafter administration of anakinra strongly enhancedIL-1� secretion upon S aureus challenge (to 6,400 pg/mland 5,100 pg/ml, respectively) (Figure 3D). The enhanc-ing effect of LPS was less obvious 4 hours and 24 hoursafter anakinra administration (Figure 3D).

DISCUSSION

We report the case of a patient with a longhistory of inflammatory disease resulting from excessiveIL-1� production. The patient was found to be a het-erozygous carrier of 2 common polymorphisms in theNLRP3 and CARD-8 genes, both of which are compo-nents of the NALP3 inflammasome. Administration ofanakinra proved effective in treating this patient.

The Q705K polymorphism in NLRP3 found inthe patient has previously been described as a low-penetrance mutation found at an allele frequency of 3%in a Caucasian population (11). However, in a randomlyselected population of 806 individuals from the south-

eastern region of Sweden, the Q705K polymorphism wasfound to be more prevalent (allele frequency 6.5%).Sequencing of other components of the patient’s NALP3inflammasome revealed that ASC was wild type, while acodon change from cysteine to a premature stop codonwas found in the CARD-8 gene.

In a recent study, this CARD-8 gene polymor-phism has been shown to be present in 40% of aCaucasian population, and its association with Crohn’sdisease has been a matter of debate (7,12). Since theprotein TUCAN normally binds to procaspase 1 andrepresses activation of caspase 1 and subsequent IL-1�formation (13), the observed heterozygous inactivationof CARD-8/TUCAN may, at least in part, result in thelack of caspase 1 repression. Therefore, the increasedspontaneous caspase 1 activity and the elevated IL-1�levels observed in the patient after withdrawal of anak-inra are likely to be the consequence of either theNLRP3 gene polymorphism or the CARD-8 gene poly-morphism; alternatively, they may result from a com-bined, synergistic effect of both polymorphisms. Sincethe predicted frequency of individuals carrying thiscompound polymorphism is �4%, it may represent asignificant susceptibility factor for several chronic in-flammatory disorders. This is partly supported by ourstudy (14), in which these 2 SNPs are shown to representa genetic susceptibility factor for rheumatoid arthritiswith a more severe disease course.

Other reported patients with NLRP3 mutationshave higher baseline levels of secreted IL-1� (15) thanthose observed in our patient, but in those studies IL-1�was either measured before starting anakinra treatmentor during a disease flare when the drug was withheld.Our patient had been receiving anakinra treatment for�1 year, and for ethical reasons, this treatment wasnever withheld for more than 34 hours. The patient’selevated spontaneous caspase 1 activity and IL-1� re-lease, as well as in response to microbe challenge,returned to normal levels upon anakinra treatment.Anakinra has been shown to affect IL-1� production bydecreasing the expression of proIL-1� and its targetgenes (15), whereas withdrawal of IL-1 receptor antag-onists in patients with NOMID increases the expressionof these genes (15). The levels of NF-�B activity inresting and stimulated cells from the patient were similarto the levels found in cells from control subjects, sug-gesting that this proinflammatory signaling pathway isnot important for his disease.

NALP3 and related proteins have been suggestedto be sensors of intracellularly encountered microbialmotifs and “danger signals” (1). Polymorphisms in these

POLYMORPHISMS IN NLRP3 AND CARD-8 GENES AND INFLAMMATION 893

genes in combination with an exogenous exposure (e.g.,infection) may elicit an inflammatory response thatdevelops into a chronic condition. Prior to the outbreakof the disease, the patient had a streptococcal infectionthat might have triggered his disease. However, inaddition to having the polymorphisms of NLRP3 andCARD-8, the patient was also HLA–B27 positive. HLA–B27 is commonly found in patients with reactive arthritisand other subgroups of spondylarthropathy in whichbouts of arthritis are often preceded by infections. Theinterrelationships among HLA–B27, the gene polymor-phisms of the inflammasome, and exogenous agents maybe of importance in the pathogenesis of rheumaticdiseases or other chronic inflammatory conditions.

In addition to the familial disorders NOMID andMWS/FCAS, Still’s disease is also characterized byelevated levels of IL-1�, and treatment with anakinrahas also been efficacious in these patients (16). Still’sdisease was also considered in the present case, but ifdiagnostic criteria are strictly applied, other rheumaticdiseases exclude a diagnosis of Still’s disease (10). Webelieve that the combination of clinical, genetic, andexperimental data should be emphasized, and that thesecombined data may improve the accuracy of diseasedefinitions in the future.

In conclusion, the discovery that a significantfraction of the population carries the genetic variants ofNLRP3 and CARD-8 genes may be important for com-mon chronic inflammatory diseases. Further studiesusing constructs of the genetic variants of the compo-nents of the inflammasome are required to dissect thefunctional consequences and to evaluate their potentialcausative roles in inflammation.

ACKNOWLEDGMENTS

We thank all participants in this study. Marie Ruber,Annette Molbaek, and Åsa Schippert are acknowledged forexcellent technical assistance. Drs. Olle Stendahl and ChristinaEkerfelt are gratefully acknowledged for valuable discussions.

AUTHOR CONTRIBUTIONS

Dr. Sarndahl had full access to all of the data in the study andtakes responsibility for the integrity of the data and the accuracy of thedata analysis.Study design. Verma, Lerm, Eriksson, Soderkvist, Sarndahl.Acquisition of data. Verma, Lerm, Blomgran, Julinder, Eriksson,Soderkvist, Sarndahl.Analysis and interpretation of data. Verma, Lerm, Eriksson,Soderkvist, Sarndahl.

Manuscript preparation. Verma, Lerm, Eriksson, Soderkvist, Sarn-dahl.Statistical analysis. Verma, Soderkvist, Sarndahl.

REFERENCES1. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular

platform triggering activation of inflammatory caspases and pro-cessing of proIL-�. Mol Cell 2002;10:417–26.

2. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, CortisE, et al. Chronic infantile neurological cutaneous and articularsyndrome is caused by mutations in CIAS1, a gene highly ex-pressed in polymorphonuclear cells and chondrocytes. Am J HumGenet 2002;71:198–203.

3. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, KolodnerRD. Mutation of a new gene encoding a putative pyrin-like proteincauses familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat Genet 2001;29:301–5.

4. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichten-berger GS, Grant EP, et al. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulationof caspase-1. Immunity 2006;24:317–27.

5. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome.Nature 2006;440:237–41.

6. Pathan N, Marusawa H, Krajewska M, Matsuzawa S, Kim H,Okada K, et al. TUCAN, an antiapoptotic caspase-associatedrecruitment domain family protein overexpressed in cancer. J BiolChem 2001;276:32220–9.

7. McGovern DP, Butler H, Ahmad T, Paolucci M, van Heel DA,Negoro K, et al. TUCAN (CARD8) genetic variants and inflam-matory bowel disease. Gastroenterology 2006;131:1190–6.

8. Zheng L, He M, Long M, Blomgran R, Stendahl O. Pathogen-induced apoptotic neutrophils express heat shock proteins andelicit activation of human macrophages. J Immunol 2004;173:6319–26.

9. Amor B, Dougados M, Mijiyawa M. Criteria of the classification ofspondylarthropathies. Rev Rhum Mal Osteoartic 1990;57:85–9. InFrench.

10. Yamaguchi M, Ohta A, Tsunematsu T, Kasukawa R, MizushimaY, Kashiwagi H, et al. Preliminary criteria for classification ofadult Still’s disease. J Rheumatol 1992;19:424–30.

11. Hoffman HM, Gregory SG, Mueller JL, Tresierras M, Broide DT,Wanderer AA, et al. Fine structure mapping of CIAS1: identifi-cation of an ancestral haplotype and a common FCAS mutation,L353P. Hum Genet 2003;112:209–16.

12. Franke A, Rosenstiel P, Balschun T, Von Kampen O, Schreiber S,Sina C, et al. No association between the TUCAN (CARD8)Cys10Stop mutation and inflammatory bowel disease in a largeretrospective German and a clinically well-characterized Norwe-gian sample. Gastroenterology 2007;132:2080–1.

13. Razmara M, Srinivasula SM, Wang L, Poyet JL, Geddes BJ,DiStefano PS, et al. CARD-8 protein, a new CARD familymember that regulates caspase-1 activation and apoptosis. J BiolChem 2002;277:13952–8.

14. Kastbom A, Verma D, Eriksson P, Skogh T, Wingren G,Soderkvist P. Genetic variation in proteins of the cryopyrininflammasome influences susceptibility and severity of RA (TheSwedish TIRA project). Rheumatology. In press.

15. Goldbach-Mansky R, Dailey NJ, Canna SW, Gelabert A, Jones J,Rubin BI, et al. Neonatal-onset multisystem inflammatory diseaseresponsive to interleukin-1� inhibition. N Engl J Med 2006;355:581–92.

16. Fitzgerald AA, Leclercq SA, Yan A, Homik JE, Dinarello CA.Rapid responses to anakinra in patients with refractory adult-onset Still’s disease. Arthritis Rheum 2005;52:1794–803.

894 VERMA ET AL