RESUMO
Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of C1r has been observed to occur concomitantly with deficiency in C1s and 9 out of 15 reported cases presented systemic lupus erythematosus (SLE). Here, we describe a family in which all four children are deficient in C1s but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children's sera. C1s was undetectable, while in the parents' sera it was lower than in the normal controls. The levels of C1r observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of C1s synthesis was observed in the patients' fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the C1s cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of C1s mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3' splice site within intron 1 which increases the size of exon 2 by 87 nucleotides.
Assuntos
Processamento Alternativo , Complemento C1s/deficiência , Lúpus Eritematoso Sistêmico/genética , Adulto , Sequência de Bases , Células Cultivadas , Criança , Complemento C1s/genética , Complemento C1s/imunologia , Éxons , Feminino , Fibroblastos/imunologia , Humanos , Íntrons , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Dados de Sequência Molecular , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.
Assuntos
Diferenciação Celular , Complemento C3/deficiência , Células Dendríticas/citologia , Antígenos CD/imunologia , Antígenos CD1/imunologia , Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Antígeno CD11c/imunologia , Moléculas de Adesão Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Complemento C3/imunologia , Citocinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Antígenos HLA-DR/imunologia , Humanos , Imunoglobulinas/imunologia , Lectinas Tipo C/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Fenótipo , Receptores de Superfície Celular/imunologia , Soro , Antígeno CD83RESUMO
Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected.
Assuntos
Proteínas Inativadoras do Complemento/biossíntese , Proteínas do Sistema Complemento/biossíntese , Células Dendríticas/metabolismo , Monócitos/citologia , Receptores de Complemento/biossíntese , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Imunidade Inata , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismoRESUMO
C3 occupies a central position in the complement pathway, mediating such diverse functions as convertase activity, opsonization and anaphylotoxin production. The deficiency of this protein is a rare autosomal recessive inherited disease, characterized by severe recurrent infections and immune complex disorders. We looked for molecular alterations that could explain the C3 deficiency present in a Brazilian boy of consanguineous parents who suffered from recurrent bacterial infections. Using reverse-transcriptase polymerase chain reaction to amplify C3 mRNA from LPS-stimulated fibroblasts from the patient, we demonstrated that his C3 gene has no large structural aberrations. However, after sequencing the amplified and cloned products we found: (1). a L314P amino acid substitution; (2). silent mutations at codons P577, S798 and A1437; and finally, (3). an R848STer substitution that results in the production of a truncated protein. Densitometry studies revealed a lower C3 mRNA concentration in the patient's fibroblasts, suggesting an inherent instability of his C3 mRNA. Our results indicate the presence of a premature termination codon in the C3 gene that results in a lack of the protein in patient's serum, which correlates with the acceleration of C3 mRNA decay in the patient's fibroblasts. This mRNA instability is consistent with a nonsense-codon-mediated decay process that ensures the elimination of possible deleterious truncated proteins, which, in the case of constitutively expressed abundant proteins such as C3, may otherwise accumulate to significant levels, leading to toxicity.