RESUMO
BACKGROUND: Generalized pustular psoriasis is a life-threatening disease of unknown cause. It is characterized by sudden, repeated episodes of high-grade fever, generalized rash, and disseminated pustules, with hyperleukocytosis and elevated serum levels of C-reactive protein, which may be associated with plaque-type psoriasis. METHODS: We performed homozygosity mapping and direct sequencing in nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis. We assessed the effect of mutations on protein expression and conformation, stability, and function. RESULTS: We identified significant linkage to an interval of 1.2 megabases on chromosome 2q13-q14.1 and a homozygous missense mutation in IL36RN, encoding an interleukin-36-receptor antagonist (interleukin-36Ra), an antiinflammatory cytokine. This mutation predicts the substitution of a proline residue for leucine at amino acid position 27 (L27P). Homology-based structural modeling of human interleukin-36Ra suggests that the proline at position 27 affects both the stability of interleukin-36Ra and its interaction with its receptor, interleukin-1 receptor-like 2 (interleukin-1 receptor-related protein 2). Biochemical analyses showed that the L27P variant was poorly expressed and less potent than the nonvariant interleukin-36Ra in inhibiting a cytokine-induced response in an interleukin-8 reporter assay, leading to enhanced production of inflammatory cytokines (interleukin-8 in particular) by keratinocytes from the patients. CONCLUSIONS: Aberrant interleukin-36Ra structure and function lead to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis. (Funded by Agence Nationale de la Recherche and Société Française de Dermatologie.).
Assuntos
Interleucina-1/metabolismo , Psoríase/genética , Receptores de Interleucina/antagonistas & inibidores , Feminino , Genes Recessivos , Ligação Genética , Humanos , Interleucina-1/genética , Masculino , Mutação , Linhagem , Transdução de Sinais , Dermatopatias Vesiculobolhosas , TunísiaRESUMO
IL-36α, IL-36ß, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36Ra (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36Ra antagonism were unsuccessful. Here, we demonstrate that IL-36Ra antagonist activity is dependent upon removal of its N-terminal methionine. IL-36Ra starting at Val-2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36ß. Val-2 of IL-36Ra lies 9 amino acids N-terminal to an A-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36ß, and IL-36γ to this same point increased their specific activity by â¼10(3)-10(4)-fold (from EC(50) 1 µg/ml to EC(50) 1 ng/ml). Inhibition of truncated IL-36ß activity required â¼10(2)-10(3)-fold excess IL-36Ra, similar to the ratio required for IL-1Ra to inhibit IL-1ß. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36Ra bound to IL-1Rrp2, and pretreatment of IL-1Rrp2-expressing cells with IL-36Ra prevented IL-36ß-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together, these results suggest that the mechanism of IL-36Ra antagonism is analogous to that of IL-1Ra, such that IL-36Ra binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36ß, and IL-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.
Assuntos
Interleucina-1/agonistas , Interleucina-1/antagonistas & inibidores , Interleucinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucinas/química , Células Jurkat , Ligantes , Camundongos , Dados de Sequência Molecular , Receptores de Interleucina-1/metabolismo , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: Monogenic autoinflammatory diseases are disorders of Mendelian inheritance that are characterized by mutations in genes that regulate innate immunity and whose typical features are systemic inflammation without high-titer autoantibodies or antigen-specific T cells. Skin and bone inflammation in the newborn period have been described in 3 of these autoinflammatory disorders: neonatal-onset multisystem inflammatory disease, Majeed syndrome, and deficiency of interleukin-1 (IL-1) receptor antagonist (DIRA) syndrome. This study was undertaken to present the characteristics of the DIRA syndrome in 2 cases from Brazil, and describe a novel mutation in IL1RN. METHODS: Two unrelated Brazilian patients were evaluated for the clinical signs and symptoms of these 3 disorders, and peripheral blood samples were assessed for mutations in NLRP3, LPIN2, and IL1RN by DNA resequencing analysis. A mutation in IL1RN that encodes a mutant protein was identified, and the expression and function of this mutant protein were compared to those of the wild-type protein. RESULTS: Both patients presented with pustular dermatitis resembling generalized pustular psoriasis, recurrent multifocal aseptic osteomyelitis, and elevation in the levels of acute-phase reactants, all of which are features most consistent with the DIRA syndrome. Chronic lung disease was observed in 1 of the patients, and jugular venous thrombosis was observed in the other patient. Both patients showed a partial response to corticosteroid therapy, and 1 patient experienced an initial improvement of dermatitis with the use of acitretin. Both patients were homozygous for a novel 15-bp (in-frame) deletion on the IL1RN gene. The mutated protein expressed in vitro had no affinity with the IL-1 receptor, and stimulation of the patients' cells with recombinant human IL-1α or IL-1ß led to oversecretion of proinflammatory cytokines, similar to the findings obtained in previously reported patients. CONCLUSION: The presence of the same homozygous novel mutation in IL1RN in 2 unrelated Brazilian patients suggests that this genetic variant may be a founder mutation that has been introduced in the Brazilian population.
Assuntos
Doenças Hereditárias Autoinflamatórias/diagnóstico , Doenças Hereditárias Autoinflamatórias/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Mutação/genética , Brasil , Proteínas de Transporte/genética , Pré-Escolar , Feminino , Doenças Hereditárias Autoinflamatórias/patologia , Homozigoto , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/genética , Osteomielite/diagnóstico , Osteomielite/genética , Osteomielite/patologia , Psoríase/diagnóstico , Psoríase/genética , Psoríase/patologiaRESUMO
The recently discovered IL-1F9 (IL-1H1) is a putative member of the interleukin (IL)-1 family of cytokines that has been shown to activate nuclear factor-kappa B (NFkappaB) in Jurkat cells transfected with the orphan receptor IL-1 receptor-related protein (IL-1Rrp)2. The aim of the present study was therefore to investigate expression of IL-1Rrp2 and to determine if IL-1F9 induces known IL-1 signaling pathways in the different cell types of the mouse brain in culture. Messenger RNA for IL-1Rrp2 was not detected in primary neurones by RT-PCR, but significant constitutive expression was found in mixed glial cells, particularly in astrocytes and microglia, which was strongly decreased by exposure to bacterial lipopolysaccharide (LPS). LPS induced the release of IL-6, and activated NFkappaB and the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated protein kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) in microglial cultures. IL-1beta induced release of IL-6 and activated NFkappaB, p38, JNK and ERK1/2 in mixed glial cultures, which was completely abolished in the presence of IL-1 receptor antagonist (IL-1ra). When injected intracerebroventrically in the rat, IL-1beta increased core body temperature, and reduced body weight and food intake. In contrast, IL-1F9 failed to induce any of these responses either in vivo or in vitro. These results demonstrate that glial cells may be a target for the new ligand IL-1F9, since high expression of IL-1Rrp2 mRNA was detected in these cells. However, IL-1F9 failed to induce any of the classical IL-1beta responses, suggesting that it may trigger alternative pathway(s).
Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Interleucina-1/metabolismo , Neuroglia/metabolismo , Proteínas/genética , Receptores de Interleucina/genética , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Astrócitos/metabolismo , Encéfalo/imunologia , Encefalopatias/imunologia , Encefalopatias/fisiopatologia , Células Cultivadas , Técnicas de Cocultura , Feto , Regulação da Expressão Gênica/imunologia , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/imunologia , Interleucina-1/farmacologia , Subunidade alfa de Receptor de Interleucina-18 , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/metabolismo , NF-kappa B/metabolismo , Neuroglia/imunologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-18 , Sialoglicoproteínas/farmacologiaRESUMO
The putative new interleukin (IL)-1 family member IL-1F8 (IL-1eta, IL-1H2) has been shown recently to activate mitogen activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK1/2) and c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NFkappa B) via a mechanism that requires IL-1Rrp2 expression in cell lines. The aim of this study was to test the hypothesis that IL-1F8 contributes to brain inflammation and injury, by studying its expression and actions in the different cell types of the mouse brain in culture. Messenger RNA for IL-1F8 was detected in neurons and glia (microglial cells, oligodendrocytes progenitor cells and to a lesser extent astrocytes) by RT-PCR. Bacterial lipopolysaccharide (LPS) had no effect on IL-1F8 mRNA levels in mixed glial cultures. Recombinant mouse IL-1beta induced strong activation of ERK1/2, p38, JNK and NFkappa B, and significant release of IL-6 and PGE2, which was blocked by IL-1ra. In contrast, recombinant mouse IL-1F8 did not influence any of these parameters. These results demonstrate that CNS cells may be a source of IL-1F8, but the failure of LPS to modulate IL-1F8 mRNA expression, and of recombinant IL-1F8 to induce any of the classical IL-1 responses, suggest that this cytokine has restricted activities in the brain, or that it may act via alternative pathway(s).
Assuntos
Interleucina-1/biossíntese , Interleucina-1/farmacologia , Neuroglia/imunologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Dinoprostona/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Interleucina-1/genética , Interleucina-6/biossíntese , Camundongos , NF-kappa B/metabolismo , Neuroglia/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
Recent additions have expanded the interleukin (IL)-1 gene family to 10 members. We have determined the order, orientation, and intergenic distance of the nine IL-1 family genes that lie on human chromosome 2. We report cDNA sequences for the mouse orthologs of three of these genes. The order and orientation of the mouse genes have been mapped, and the mouse locus compared with the human locus. There is a break in the mouse locus of > 100 kb, compared with the human locus, located between Il1b and the most centromere-proximal of the novel mouse genes. The mouse seems to be missing an ortholog of human IL1F7.
Assuntos
Interleucina-1/genética , Família Multigênica/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Ordem dos Genes , Genes/genética , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SinteniaRESUMO
Interleukin 1 (IL-1) plays a prominent role in immune and inflammatory reactions. Our understanding of the IL-1 family has recently expanded to include six novel members named IL-1F5 to IL-1F10. Recently, it was reported that IL-1F9 activated NF-kappaB through the orphan receptor IL-1 receptor (IL-1R)-related protein 2 (IL-1Rrp2) in Jurkat cells (Debets, R., Timans, J. C., Homey, B., Zurawski, S., Sana, T. R., Lo, S., Wagner, J., Edwards, G., Clifford, T., Menon, S., Bazan, J. F., and Kastelein, R. A. (2001) J. Immunol. 167, 1440-1446). In this study, we demonstrate that IL-1F6 and IL-1F8, in addition to IL-1F9, activate the pathway leading to NF-kappaB in an IL-1Rrp2-dependent manner in Jurkat cells as well as in multiple other human and mouse cell lines. Activation of the pathway leading to NF-kappaB by IL-1F6 and IL-1F8 follows a similar time course to activation by IL-1beta, suggesting that signaling by the novel family members occurs through a direct mechanism. In a mammary epithelial cell line, NCI/ADR-RES, which naturally expresses IL-1Rrp2, all three cytokines signal without further receptor transfection. IL-1Rrp2 antibodies block activation of the pathway leading to NF-kappaB by IL-1F6, IL-1F8, and IL-1F9 in both Jurkat and NCI/ADR-RES cells. In NCI/ADR-RES cells, the three IL-1 homologs activated the MAPKs, JNK and ERK1/2, and activated downstream targets as well, including an IL-8 promoter reporter and the secretion of IL-6. We also provide evidence that IL-1RAcP, in addition to IL-1Rrp2, is required for signaling by all three cytokines. Antibodies directed against IL-1RAcP and transfection of cytoplasmically deleted IL-1RAcP both blocked activation of the pathway leading to NF-kappaB by the three cytokines. We conclude that IL-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP.
Assuntos
Interleucina-1/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Família Multigênica , NF-kappa B/metabolismo , Proteínas/metabolismo , Animais , Humanos , Interleucina-1/genética , Interleucina-1/farmacologia , Proteína Acessória do Receptor de Interleucina-1 , Subunidade alfa de Receptor de Interleucina-18 , Células Jurkat , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas , Proteínas/genética , Receptores de Interleucina-18 , TransfecçãoRESUMO
Soluble type II interleukin (IL)-1 receptor (sIL1R-II) binds human IL-1beta with high affinity and neutralizes its activity. Recombinant sIL1R-II is considered a potentially useful anti-IL-1 therapeutic, and preclinical studies have been undertaken with this molecule in primates. To better understand the cytokine-receptor interactions occurring in this nonhuman context, monkey IL-1 and IL1R-II were cloned, and their binding abilities were examined in vitro. IL-1beta from cynomolgus monkey was capable of binding and activating the human type I IL-1 receptor. However, unlike human IL-1beta, it was unable to effectively bind and become neutralized by sIL1R-II. Human and cynomolgus IL-1beta proteins are 96% identical, differing by only six amino acids. Structural and mutational analysis revealed that the unique sIL1R-II binding ability of human IL-1beta is due to a single amino acid difference compared with monkey IL-1beta.