RESUMO
Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains.
Assuntos
Camelídeos Americanos/imunologia , Ativação do Complemento/imunologia , Hemaglutinação/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Camelídeos Americanos/genética , Clonagem Molecular , Ativação do Complemento/genética , Sequência Conservada/genética , Hemaglutinação/genética , Testes de Hemaglutinação/veterinária , Cadeias Pesadas de Imunoglobulinas/imunologia , Alinhamento de SequênciaRESUMO
Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.
Assuntos
Artrite Infecciosa/imunologia , Brucella abortus/imunologia , Brucelose/imunologia , Articulações/microbiologia , Articulações/patologia , Metaloproteinases da Matriz/biossíntese , Membrana Sinovial/imunologia , Animais , Antígenos de Bactérias/imunologia , Artrite Infecciosa/enzimologia , Artrite Infecciosa/microbiologia , Artrite Infecciosa/patologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucella abortus/crescimento & desenvolvimento , Brucella abortus/patogenicidade , Brucelose/enzimologia , Brucelose/microbiologia , Brucelose/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Meios de Cultivo Condicionados , Citocinas/biossíntese , Citocinas/metabolismo , Indução Enzimática , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Articulação do Joelho/microbiologia , Lipoproteínas/imunologia , Ativação Linfocitária , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/fisiologia , Neutrófilos/fisiologia , Membrana Sinovial/citologia , Membrana Sinovial/microbiologia , Receptor 2 Toll-Like/metabolismoRESUMO
Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.