Chronic infantile neurological cutaneous articular syndrome: CD10 over-expression in neutrophils is a possible key to the pathogenesis of the disease
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- Leone, V., Presani, G., Perticarari, S. et al. Eur J Pediatr (2003) 162: 669. doi:10.1007/s00431-003-1284-x
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Chronic, Infantile, Neurological, Cutaneous and Articular Syndrome (CINCA) or Neonatal/Infantile Onset Multisystem Inflammatory Disease (NOMID/IOMID) is a rare, multisystem inflammatory disease characterised by neonatal onset of urticarial symptoms, persistent rash, ocular inflammatory lesions, progressive articular and neurological involvement and associated with characteristic overgrowth of the ossification nucleus of the patella. The tissues involved are extensively infiltrated by inflammatory cells, mostly neutrophils. This paper describes the clinical features of three new cases as well as a study of activation markers in neutrophils and search for mutations of the CIAS1gene in these patients. Clinical records of three cases of CINCA are reported. For genetic analysis, exon 3 of the CIAS1gene was amplified and sequenced. Immunophenotype, oxidative burst and phagocytosis were analysed in neutrophils obtained from all the three CINCA patients as well as from eight juvenile idiopathic arthritis (JIA) patients and eight healthy controls. Functional assays in neutrophils were normal in all three patients with CINCA syndrome and did not differ from those of JIA patients and healthy controls. The surface density of CD10 was significantly higher on neutrophils from CINCA patients as compared to those of JIA and controls ( P <0.0005). In one subject a new missense mutation in the CIAS1gene was identified. Conclusion: the hyper expression of the activation antigen CD10/NEP in neutrophils from these three cases of CINCA, as compared to JIA patients and healthy controls, irrespective of the presence of mutations in CIAS1, could be a marker of the inflammatory disorder typical of some patients with CINCA syndrome.
KeywordsChronic, infantile, neurological, cutaneous and articular syndrome (CINCA)Neonatal onset multisystem inflammatory disease (NOMID)CD10/neutral endopeptidaseNeutrophil activation markers
chronic, infantile, neurological, cutaneous and articular
juvenile idiopathic arthritis
mean fluorescence intensity
Chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome, also known as infantile-onset multisystemic inflammatory disease or neonatal onset multisystemic inflammatory disease (OMIM 607115), is a rare multisystemic disease characterized by early onset in the 1st year of life, first described in 1981  and recognised as a distinct clinical entity in 1987 . Approximately 100 cases have been identified in the world [2, 5, 6, 7, 8, 9, 10, 13,19]. The main clinical features consist of persistent urticaria-like skin manifestations, recurrent articular symptoms, fever, neurological manifestations, mental impairment, ocular inflammatory lesions, skeletal deformations and persistently abnormal laboratory findings (increased acute phase reactants, hypergammaglobulinaemia, and anaemia) . Previous observations indicated that neutrophils could play a central role in the pathogenesis of this condition [7,15]; recently, mutations in the CIAS1gene, whose expression is restricted in neutrophils and chondrocytes, have been related to the disease .
The aim of the study was to evaluate the functional profile (oxidative burst and phagocytosis) and some markers of activation of neutrophils (CD10, CD11b, CD18 and CD69), in three patients with CINCA syndrome as compared to a group of patients with active juvenile idiopathic arthritis (JIA) and to healthy controls. Moreover, we searched for mutations in the CIAS1gene by analysing exon 3, where all mutations so far have been identified [1,4].
This 4-year-old girl presented a diffuse erythematous maculopapular rash immediately at birth. During her first days of life, she also developed spiking fever and papilloedema. Her mental and motor development was adequate for age. Laboratory findings constantly showed leucocytosis, increased levels of ESR and CRP, serum immunoglobulin and mild anaemia. Serum complement components were within the normal range. antinuclear antibodies (ANA) and anti-DNA antibodies were negative.
During the following months, she presented two self-limited limping episodes, involving the left hip, and arthritis of her left knee, treated with flurbiprophen with complete response. Three months later, four small, mobile, non-tender lymph nodes appeared in the cervical, inguinal regions and the right armpit. A nodal biopsy was performed; the histological findings showed reactive adenopathy with mixed hyperplasia. A recent X-ray film showed an extensive lytic lesion with sclerotic perilesional reaction in the metaphyses of the proximal extremity of the left tibia where the child presents a painful swelling. Steroids at low dosage (5 mg/day) are sufficient to control the pain.
This 4-year-old girl presented a generalised rash at birth characterised by non-pruritic, maculopapular, highly hyperemic and well-delimited lesions, a few of which had a pale central zone. Cutaneous manifestations waxed and waned in intensity and extension, with many morphological changes, but never disappeared.
On her first admission to hospital, when she was 1 year old, laboratory data showed leucocytosis, mild anaemia, increased inflammatory indices and immunoglobulin levels. ANA, ANCA, rheumatoid factor and circulating immunocomplexes were negative. A skin biopsy was performed showing histological findings consistent with an urticaria-like vasculitis involving the small and medium vessels of the dermis. Direct immunofluorescence showed some IgM deposits in the dermis-epidermis junction.
Clinical examination revealed a moderate patellar hypertrophy. An X-ray film of the knee confirmed the presence of a bilateral overgrowth of the ossification nucleus of the patella. During the last year the patient has developed recurrent arthralgia in the lower limbs, with no signs of arthritis. This symptom responded to non-steroidal anti-inflammatory drug therapy (ibuprofen 30 mg/kg per day).
This presently 13-year-old girl developed an urticaria-like cutaneous rash on the 9th day of life which rapidly spread over the whole body.
At 10 months of age, daily spiking fever developed. One year later, a skin biopsy was performed, the findings of which were consistent with leucocytoclastic vasculitis, with deposits of IgM and complement (C3) in the vessel walls.
During follow-up, laboratory findings remained constantly altered; in particular, haemoglobin levels were always below the normal range, whereas ESR, CRP, platelet count, complement components (C3, C4), serum immunoglobulins and circulating immunocomplexes (C1q based ELISA) were constantly high.
At 2.5 years of age, the patient's growth rate slowed down slightly and she began to develop chronic arthritis of the left knee which recovered at the age of 5 years following local steroid treatment. The fever completely resolved with low doses of oral steroids (0.2 mg/kg every other day). All attempts to cease steroids met with recurrent episodes of spiking fever until 2 years ago when the steroids could be stopped.
At 6 years of age, the patient developed a severe headache. Fundoscopy revealed a bilateral papilloedema. In the same period a saddle-back nose, frontal bossing and patellar overgrowth appeared. At the age of 8 years, perceptive deafness also occurred.
Clinical symptoms in the three patients affected by CINCA
Rash at birth
Subjects and methods
Subjects and controls
The study of neutrophil function was performed in the three CINCA patients described above and compared to eight JIA patients and eight healthy controls. The JIA patients (six females and two males; mean age 9.3 years) included two systemic, three oligoarticular and three polyarticular forms.
At the time of the study, the three patients with the oligoarticular form were given indomethacin, flurbiprofen, celecoxib respectively; one patient with the polyarticular form was given only methotrexate at a low dosage and the other patient methotrexate at a low dosage plus indomethacin; one patient with the systemic form was given methotrexate plus chetoprofen, the other steroids at a low dosage plus naproxene; however, in spite of treatment, all patients had active disease.
The therapy in CINCA cases at the time of the investigation was as follows:- case 1 prednisone (5 mg/day); case 2 no therapy; case 3 ibuprofen (30 mg/kg per day).
Investigations on neutrophils
Oxidative burst, phagocytosis and activation markers of neutrophils were investigated. A rapid and simple one-step procedure by means of two-colour flow cytometry, recently described , was used to measure simultaneously the production of reactive oxygen intermediates, referred to as oxidative burst, and phagocytosis of FITC-labelled Staphylococcus aureus. Expression of surface molecules associated with neutrophil activation (CD10, CD69, CD11b, CD18) on the cell surface of polymorphonuclear cells (PMNC) was investigated with specific monoclonal antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA). Surface density was expressed as mean fluorescence intensity (MFI) which is an arbitrary unit currently used in flow cytometry to denote how much fluorescence a given cell population carries, expressed and compared in linear numbers.
Statistical analysis of the results was performed with the Turkey-Kramer test.
DNA was extracted from peripheral blood using the GenomicPrep kit (Pharmacia-Amersham, Piscataway, NJ), following the manufacturer's instructions. Exon 3 of the CIAS1gene was amplified using the following primers: forward 5'-GACAGGCCCCAGCTGAGAG-3', reverse 5'-CATGATCAGGTCCCCAGG-3, forward 5'-GAGCATTCTGAGCCTGTGCA-3', reverse 5'-AATCAGACTGAAGGCTGCCCT-3', forward 5'-GAGACCTGTGGCCCTGGAG-3', reverse 5'-AGCAGGTAGTACATGGCGGC-3', forward 5'-TGAGGAGTCCGACCTCAGGA-3', reverse 5'-CCCAGGGACAGTGACTCCAC-3', forward 5'-GAGGAGGACTTCGTGCAAAGG-3', reverse 5'-AGGCAAGCGCCAAGAAGAA-3'. Thirty-five cycles of PCR were performed using the GeneAmp PCR System 9700 (Applied Biosystems Foster City, CA) with 1 U of Taq Gold (Applied Biosystems), 2 mM MgCl2 and an annealing temperature of 53°C.
DNA sequencing of the amplicons was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 2.0 (Applied Biosystems). DNA sequences were detected and analysed on an automated ABI Prism 3100 Genetic Analyser (Applied Biosystems). DNA sequences were processed using the SeqScape 1.0 Software (Applied Biosystems).
Oxidative burst and phagocytosis were normal in all three patients with CINCA syndrome and did not differ from those of JIA patients and healthy controls (data not shown).
Surface density of CD69, CD11 and CD18, expressed as MFI values, on PMN of patients with CINCA, JIA and healthy controls
No CINCA associated mutation described in literature was found in our three patients, but we identified a new allelic variant associated to CINCA phenotype in case 3, who was heterozygous for a C-to-A transition at nucleotide 2859 in exon 3 of the CIAS1gene, resulting in a glutamine-to-lysine substitution at codon 703 (Q439K). The other two cases showed no variation of their CIAS1exon 3 nucleotide sequence.
Urticaria-like or maculopapular rash at birth or within the first days of life in all three patient is the most particular manifestation of CINCA syndrome. In the skin biopsy performed in one of our cases, complement and immunoglobulin deposits were detected by indirect immunofluorescence; this finding has not been described in previous reports  but in our opinion it might constitute a variant of this syndrome which is still not completely defined in all its aspects.
The aetiopathogenesis of this syndrome is still not completely understood. The occurrence of familial cases, although much rarer than sporadic cases, had suggested a genetic background. Recently, Feldmann et al.  described seven patients with CINCA syndrome with a distinct missense mutation in CIAS1, a gene encoding cryopyrin, known to cause Muckle-Wells syndrome and cold urticaria, whose expression was found to be restricted to PMNC and chondrocytes. Peripheral neutrophilia and a non-specific inflammatory reaction with PMN infiltrates of multiple organs had been observed, suggesting a possible defect in neutrophil function or migration, but previous reports had shown normal neutrophil function . Our results confirm that the oxidative burst following PMA stimulation and the phagocytosis of FITC-labelled Staphylococcus aureus, did not differ in patients with CINCA syndrome from JIA patients and healthy controls. On the contrary, the surface density of CD10 (also called common acute lymphoblastic leukaemia antigen or neutral endopeptidase) expressed as MFI was significantly higher in CINCA patients ( P <0.0005). CD10 is an activation marker of neutrophils  and its over-expression in leukocytes from subjects with CINCA may be associated with a specific inflammatory phenotype of the disease, as it has not been found in JIA and controls. Functionally, CD10 is one of the best-known peptidases; it is a member of a family of cell-surface zinc-metallopeptidases involved in vivo in the degradation of several bioactive peptides controlling important physiological processes.
How mutations in CIAS1 result in the CINCA inflammatory phenotype and in CD10 over-expression is not clear. CIAS1 is an adapter protein with a pyrin domain, which exerts its action through the protein ASC, a second adapter which has the dual capacity of activating NF-kB leading to inflammation  through the production of pro-inflammatory cytokines such as IL1 and Il-1beta, IL6 and TNFbeta1 , or of activating the caspase cascade resulting in apoptosis [16,18].
Over-expression of CD10 may represent a specific disorder in the balance between the processes of inflammation and apoptosis. We point out that this overexpression was observed in case 3 with a proven mutation in CIAS1, and in the other two cases as well, but without any detectable mutation. Although we cannot exclude that there may be mutations in other regions of the gene, this is not likely as all the mutations described so far in the literature have been located in exon 3 of CIAS1. Moreover, in a reported series , 7/13 patients showed no mutation in CIAS1. We suggest that over-expression of CD10 could be related not simply to the defect in CIAS1 but also to defects in other proteins influencing the same pathway of neutrophil activation.
Furthermore, because osteogenic growth peptide, PTH-related peptide and α-calcitonin gene-related peptide are all peptides known to promote differentiation and proliferation of osteoblasts and are also CD10 substrates activated by this enzyme, CD10 is thought to act as a modulator of proliferation and differentiation of cells of osteoblastic lineage . Finally, CD10 may also modulate osteoclast-mediated bone resorption by cleaving osteostatin and calcitonin, two peptides with inhibitory effects on the activity of osteoclasts. Further studies are needed to link CD10 over-expression with the recently described genetic defect.