RESUMO
UNLABELLED: The complement system plays an important role in tissue inflammation and damage in SLE patients. High levels of C3d were detected on the surface of erythrocytes and lymphocytes of SLE patients. The objective of this study was to assess the functional consequences of C3d fragments deposited on the surface membrane of SLE T cells. METHODS: 46 SLE patients, 43 patients with other autoimmune diseases (OAD) and 33 healthy individuals (N) were enrolled in this study. T cells were isolated from peripheral blood and flow cytometry studies were conducted to assess the levels of C3d fragments, Ca++ influx responses and cytokine production. Confocal microscopy was used to study co-localized molecules. Student's t-test was performed to determine statistical significance among study groups. RESULTS: A significant percentage of the SLE T cells were found to be positive for C3d (13.58 ± 3.92%) when compared with normal T cells (4.52 ± 2.92%) (p < 0.0000547) and T cells from patients with other autoimmune diseases (6.31 ± 4.57%) (p < 0.00513). Peak Ca++ influx responses were significantly higher in C3d- SLE T cells compared with C3d+ SLE T cells (p < 0.011). C3d+ T cells produced significantly more IL-2, IFN-gamma, IL-4 and IL-17. In contrast to the increased production of IL-2 by the C3d+ T cells, the overall SLE T cell population produced less IL-2 when compared with T cells from normal individuals or patients with other autoimmune disease. The C3d fragments were found to be localized within the lipid rafts. CONCLUSION: C3d fragments are localized in the lipid rafts of SLE T cells and contribute to abnormal T cell function by modulating Ca++ influx responses and increased cytokine production.
Assuntos
Complemento C3d/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Microdomínios da Membrana/metabolismo , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Cálcio/metabolismo , Estudos de Casos e Controles , Criança , Estudos de Coortes , Estudos Transversais , Citocinas/biossíntese , Feminino , Citometria de Fluxo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Adulto JovemRESUMO
Complement activation plays a key role in the pathogenesis of lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE). We prospectively evaluated 15 LN subjects and two control groups: 13 non-SLE renal subjects (control A) and 239 SLE subjects without LN (control B). All had C4d levels on circulating erythrocytes (E-C4d), reticulocytes (R-C4d) and platelets (P-C4d) measured by flow cytometry, while C4d deposition in renal tissue was semiquantitatively assessed in LN subjects and control A using immunoperoxidase staining. Compared with control A, LN biopsies had higher glomerular-C4d scores (p = 0.003), which were associated with more frequent granular glomerular immunofluorescence staining and electron dense deposits (p < 0.001). Compared with control A and B groups, LN subjects had higher E-C4d (p = 0.002 and p = 0.005) and R-C4d levels (p = 0.002 and p = 0.008), respectively. LN subjects were more likely to have P-C4d compared with control A (p = 0.016). In LN, only E-C4d correlated with National Institutes of Health (NIH) activity index (r = 0.55, p = 0.04). In conclusion, LN biopsies showed frequent glomerular-C4d staining associated with immune complex deposits. LN subjects had higher E-C4d and R-C4d levels compared with both control groups. E-C4d levels also correlated with NIH activity index. These findings suggest a potential role of C4d on circulating cells as a biomarker for lupus nephritis.
Assuntos
Plaquetas/patologia , Complemento C4b/imunologia , Eritrócitos/patologia , Nefrite Lúpica/sangue , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Fragmentos de Peptídeos/imunologia , Reticulócitos/patologia , Adulto , Idoso , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Lúpus Eritematoso Sistêmico/complicações , Nefrite Lúpica/etiologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Adulto JovemRESUMO
While vascular stiffness is universally studied using pulse wave velocity, this method overestimates the stiffness of small calibre blood vessels. We have developed and rigorously validated an ex vivo system for measuring stiffness of the mouse aorta. The system consists of a temperature-controlled tissue bath, a pressurization loop and a helium-neon laser micrometer. We harvested thoracic aortas from 8 (n = 56), 11 (n = 6) and 14 (n = 6) week male C57BL/6J mice, mounted them within a tissue chamber and applied an intraluminal pressure waveform while measuring mid vessel outer diameter. Vessel stiffness (E(p), mmHg) was calculated from the pressure-diameter response. Vessels were then stained for endothelial cells, smooth muscle cells, elastin fibres and collagen. The data indicate highly reproducible stiffness measurements in 8 week mice (E(p) = 602.4 +/- 160.2; p = 0.934), age-related stiffening between 11 and 14 week mice (11 week E(p) = 646.9 +/- 62.4, 14 week E(p) = 795.4 +/- 87.5, p = 0.008), and a morphologically intact vessel wall. These results represent the first ex vivo measurements of murine aortic stiffness and illustrate that our methods are feasible and reliable. Since we demonstrate that the system is sensitive to age-related stiffening and does not damage the vessel, this approach is useful for investigating the pathophysiology of vascular disease from biomechanical and histological perspectives.
Assuntos
Aorta Torácica/fisiologia , Vasos Sanguíneos/fisiologia , Músculo Liso Vascular/fisiologia , Algoritmos , Animais , Aorta Torácica/anatomia & histologia , Vasos Sanguíneos/anatomia & histologia , Interpretação Estatística de Dados , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/anatomia & histologia , Reprodutibilidade dos TestesRESUMO
Studies performed during the past several decades have demonstrated a role for the complement system in both the etiology and pathogenesis of systemic lupus erythematosus (SLE). However the specifically defective molecular and cellular pathways responsible for the disease and its complications have generally not been identified. In this report, we describe two recent advances in complement pathobiology that highlight future directions for promising investigation toward enhancing our capacity to diagnose SLE, to monitor activity of the disease, and to identify molecular and cellular defects in SLE that can be targeted by therapeutic inhibitors of complement activation. In the first example, we describe recently developed assays to detect erythrocyte C4d and complement receptor 1 for diagnosis and monitoring of disease activity in SLE. In the second example, we describe a recently discovered role for complement in mediating fetal loss in antiphospholipid syndrome and discuss the potential for this observation to facilitate identification and development of complement based biomarkers to predict poor fetal outcome in pregnant patients with SLE. These two examples are meant to underscore the importance of complement in the etiology and pathogenesis of SLE and its complications, and to stress the need for further investigation focused on the link between the complement system and SLE.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Biomarcadores/sangue , Ativação do Complemento , HumanosRESUMO
Apoptosis is a genetically controlled form of cell death that occurs in many biologic processes including embryogenesis, immune cell development, and maintenance of peripheral immune tolerance. Recent studies have yielded evidence suggesting that apoptosis of parenchymal cells may play a role in providing self-antigens to initiate autoimmune reactions. Skeletal muscle cells are fully differentiated and multinucleated. Apoptosis has been described in developing myoblasts and, recently, in mature myotubes. However, the involvement of apoptosis in skeletal muscle pathologies is unclear. This article reviews the available data concerning the occurrence of skeletal muscle cell apoptosis in selected muscle diseases. It also discusses the potential role of muscle cell apoptosis in the development of autoimmune diseases such as idiopathic inflammatory myopathies.
Assuntos
Apoptose , Músculo Esquelético/citologia , Miosite/etiologia , Biópsia , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/patologia , Células Cultivadas , Humanos , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Distrofias Musculares/etiologia , Distrofias Musculares/patologia , Miosite/patologia , Miosite/fisiopatologia , Polimiosite/etiologia , Polimiosite/patologia , Linfócitos T Citotóxicos/fisiologiaRESUMO
Cell death by apoptosis is an integral part of many biologic processes, including embryonic development, T- and B-cell selection, the elimination of potentially autoreactive lymphocytes in the periphery, and maintenance of lymphocyte homeostasis through activation-induced cell death. There is also increasing evidence that apoptosis may maintain immune tolerance and that it may be the process that generates the self antigens responsible for the initial development of autoimmunity. This review discusses some of the biochemical steps involved in the apoptotic process, how potentially immunogenic self antigens are generated during apoptosis, and the mechanisms by which the products of apoptosis are cleared and processed to avoid breaking immune tolerance.
Assuntos
Apoptose/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Apoptose/fisiologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Caspases/fisiologia , Via Clássica do Complemento/imunologia , Humanos , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/fisiopatologia , Fosfotransferases/fisiologia , Transdução de Sinais , Receptor fas/fisiologiaRESUMO
Complement protein C1q is required to maintain immune tolerance. The molecular mechanism responsible for this link has not been determined. We have previously demonstrated that C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes, suggesting that it may participate in clearance of self Ags generated during programmed cell death. Here, we demonstrate that C1q also binds directly to apoptotic blebs of vascular endothelial cells and PBMC. These apoptotic cells are recognized by the globular heads of C1q, which bind specifically to the surface blebs, and deposition increases as the blebs mature on the cell surface. These observations suggest that C1q may participate in the clearance of apoptotic cells from the circulation and from the walls of the vascular lumen. The interaction of surface blebs with the globular heads of C1q suggests that surface blebs may be capable of directly activating the classical pathway of complement under certain circumstances, generating C4- and C3-derived ligands for receptors such as CR1, CR2, CR3, and CR4. Appropriate recognition of apoptotic cells by C1q and targeted clearance of the molecular contents of surface blebs to complement receptors may be critical for the maintenance of immune tolerance.
Assuntos
Apoptose/imunologia , Complemento C1q/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Células Cultivadas , Complemento C1q/química , Via Clássica do Complemento , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Microscopia Confocal , Microscopia de Fluorescência , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Apoptosis may have a dual role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. First, this process may be integral in the clonal deletion of self-reactive lymphocytes and maintenance of peripheral tolerance. Second, apoptosis generates altered self-antigens with the potential for breaking self-tolerance. This review will discuss these two aspects of apoptosis and autoimmunity, and explore the potential role of the classical complement pathway in this context.
Assuntos
Apoptose/imunologia , Autoimunidade/fisiologia , Via Clássica do Complemento/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Receptores de Complemento/deficiência , Feminino , Humanos , Imunidade Celular/fisiologia , Masculino , Sensibilidade e EspecificidadeRESUMO
The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.
Assuntos
Ativação do Complemento/imunologia , Receptores de Complemento 3d/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vaccinia virus/imunologia , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Células COS , Receptores de Complemento 3d/genética , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Transdução de Sinais/imunologia , Relação Estrutura-Atividade , Vaccinia virus/genéticaRESUMO
Vaccinia virus secretes a 35 kD protein, vaccinia virus complement control protein (VCP), that inhibits the classical and alternative pathways of complement at several points, indicating that it may be a viral analogue of human complement receptor type 1 (CR1; CD35). Structurally, however, CR1 is composed of 30 short consensus repeats (SCRs), whereas VCP consists entirely of four SCRs. We have begun a structure-function analysis of VCP to define the minimum number of SCRs necessary for function, the functional differences between VCP and CR1, and the potential therapeutic roles for VCP. We addressed these questions by creating and characterizing recombinant soluble and membrane-bound forms of VCP. We have determined that (1) VCP requires all four SCRs to bind C3b, (2) whereas CR1 binds C3b and iC3b, VCP binds C3b but not iC3b, and (3) although normally secreted, if expressed on the membrane of mammalian cells, VCP effectively protects the cells from complement-mediated lysis. Thus, VCP appears to be a compact and unique complement regulatory protein with the ability to inhibit both arms of the complement cascade, but lacking affinity for iC3b. By releasing rather than capturing iC3b-bearing complexes following inactivation of C3b, VCP may 'recycle' its active site locally among infected cells, and thereby enable the virus to evade more efficiently host immune and inflammatory responses. The unique function, compact structure, and capacity of VCP to protect mammalian cells from complement-mediated attack, suggests that it could be used both to better understand the structure-function relationship of complement regulatory proteins, in general, and also to rationally design and develop novel therapeutic agents.
Assuntos
Vaccinia virus/fisiologia , Proteínas Virais/fisiologia , Animais , Membrana Celular , Complemento C3b/imunologia , Proteínas Inativadoras do Complemento , Proteínas do Sistema Complemento/imunologia , Sequência Consenso , Humanos , Células L , Camundongos , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/imunologia , Solubilidade , Vaccinia virus/química , Vaccinia virus/imunologia , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
Complete deficiency of C1q, the first component of the classical pathway of complement activation, is almost invariably associated with the development of systemic lupus erythematosus. Understanding why complement deficiency results in the specific autoimmune phenotype of SLE may provide valuable clues to the role of complement in the maintenance of immune tolerance. The following review will focus on the characteristics of complement-deficient SLE and the experimental evidence in support of our hypothesis that C1q may critically influence the immune response to self-antigen contained within surface blebs generated by apoptotic cells.
Assuntos
Complemento C1q/deficiência , Via Clássica do Complemento/imunologia , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/imunologia , Animais , Humanos , Lúpus Eritematoso Sistêmico/sangueRESUMO
The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats.
Assuntos
Ativação do Complemento/imunologia , Proteínas Inativadoras do Complemento/imunologia , Proteínas Virais/imunologia , Animais , HumanosRESUMO
Complete deficiency of C1q is almost invariably associated with the development of systemic lupus erythematosus. It has been suggested that this association may result from a generalized failure to clear Ag-Ab complexes. However, it has not been demonstrated how such a broad impairment results in this specific and consistent autoimmune phenotype, in which photosensitive skin disease is the most prominent manifestation. We believe there is another role for the classical pathway in maintaining immune tolerance. Surface blebs of apoptotic keratinocytes are concentrated sources of autoantigens, and these packages may define a novel immune context and challenge self-tolerance if not properly cleared and processed. We demonstrate here that when human keratinocytes are rendered apoptotic, they also develop the capacity to specifically and directly bind to C1q in the absence of Ab. C1q may mediate Ab-independent clearance of apoptotic keratinocytes, and prevent immunization with autoantigens of cutaneous origin.
Assuntos
Apoptose , Complemento C1q/metabolismo , Queratinócitos/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Células Cultivadas , Complemento C1q/deficiência , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Raios UltravioletaRESUMO
The mechanisms underlying the profound suppression of cell-mediated immunity (CMI) accompanying measles are unclear. Interleukin-12 (IL-12), derived principally from monocytes and macrophages, is critical for the generation of CMI. Measles virus (MV) infection of primary human monocytes specifically down-regulated IL-12 production. Cross-linking of CD46, a complement regulatory protein that is the cellular receptor for MV, with antibody or with the complement activation product C3b similarly inhibited monocyte IL-12 production, providing a plausible mechanism for MV-induced immunosuppression. CD46 provides a regulatory link between the complement system and cellular immune responses.
Assuntos
Antígenos CD/fisiologia , Tolerância Imunológica , Interleucina-12/biossíntese , Vírus do Sarampo/imunologia , Glicoproteínas de Membrana/fisiologia , Monócitos/imunologia , Monócitos/virologia , Receptores Virais/fisiologia , Anticorpos Monoclonais , Antígenos CD/imunologia , Sítios de Ligação , Células Cultivadas , Quimiocinas/biossíntese , Complemento C3b/imunologia , Complemento C3b/fisiologia , Citocinas/biossíntese , Regulação para Baixo , Humanos , Interleucina-10/fisiologia , Vírus do Sarampo/metabolismo , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/imunologia , Receptores Virais/imunologiaRESUMO
Several lines of evidence indicate that antibody responses to T-dependent antigens require complement receptors expressed on either B lymphocytes or follicular dendritic cells. We have used RAG-2 deficient blastocyst complementation to create mice specifically lacking B cell complement receptors. Despite normal expression of complement receptor 1 (CR1[CD35]) and CR2 (CD21) on follicular dendritic cells, these mice have a profound defect in their capacity to mount a T-dependent antibody response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This suggests that CD21 and/or CD35 on B lymphocytes may be required for cellular activation, adsorptive endocytosis of antigen, recruitment to germinal centers, and/or protection from apoptosis during the humoral response to T-dependent antigens.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Proteínas de Ligação a DNA , Células Dendríticas/imunologia , Hemocianinas/imunologia , Receptores de Complemento/imunologia , Linfócitos T/imunologia , Animais , Haptenos , Isotipos de Imunoglobulinas/sangue , Camundongos , Camundongos Mutantes , Mutagênese , Proteínas/genética , Proteínas/metabolismo , Receptores de Complemento/genética , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Baço/citologia , Baço/imunologiaRESUMO
Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens.
Assuntos
Subpopulações de Linfócitos B/imunologia , Imunoglobulina G/biossíntese , Receptores de Complemento 3d/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Antígenos Virais/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Bacteriófago phi X 174/imunologia , Transplante de Medula Óssea , Antígenos CD40/imunologia , Antígenos CD5 , Imunoglobulina G/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Síndromes de Imunodeficiência/terapia , Contagem de Linfócitos , Camundongos , Camundongos Mutantes , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Complemento 3d/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologiaRESUMO
Immune complexes formed in vitro by incubating cell-free human immunodeficiency virus type 1 (HIV-1) with sera from infected or gp160-vaccinated persons, together with normal human serum as a source of complement, readily bound to K562 cells expressing recombinant human complement receptor type 1 (CR1). However, antibodies from seronegative persons had little or no effect. This effect was absent in the presence of heat-inactivated or C3-depleted normal human sera or when wild type K562 cells were used, confirming a requirement for complement and CR1. In additional experiments, complement alone targeted HIV-1 to CR1 on red blood cells, and envelope-specific antibodies increased this effect. These results demonstrate that envelope-specific antibodies promote HIV-1 immune complex formation with complement and that these complexes readily bind CR1 on cell surfaces.
Assuntos
Proteínas do Sistema Complemento/imunologia , Eritrócitos/metabolismo , Anticorpos Anti-HIV/imunologia , HIV-1/metabolismo , Receptores de Complemento 3b/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Ativação do Complemento , Produtos do Gene env/imunologia , Proteína gp160 do Envelope de HIV , HIV-1/imunologia , Humanos , Soros Imunes/imunologia , Precursores de Proteínas/imunologiaRESUMO
Epstein-Barr virus (EBV), a herpesvirus with oncogenic potential, is camouflaged with glycoprotein 350/220, which mimics the human ligand C3dg and thereby binds to and exploits complement receptor type 2 (CR2; CD21), the EBV receptor. It has not been possible to determine the role of CR2 during postbinding events of viral infection because all B lymphocytes express endogenous CR2, precluding an informative study of receptor mutants. We have overcome this obstacle through creation of a novel experimental system based on molecular dissection of the ligand-binding domains of human CR2 and murine CR2. Our results demonstrate first, that two discontinuous amino acid substitutions within the ligand-binding domain of murine CR2 render it capable of mediating EBV infection of human B-lymphoblastoid cells, and second, that the specific role of CR2 during EBV infection is to capture virions at the cell surface, after which cofactors not associated with CR2 mediate postbinding events. These are the first studies to be described in which a cell that is normally susceptible to viral infection can be manipulated so as to direct entry of virions via recombinant or endogenous receptors.
Assuntos
Linfócitos B/microbiologia , Herpesvirus Humano 4/fisiologia , Receptores de Complemento 3d/fisiologia , Receptores Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Humanos , Molécula 1 de Adesão Intercelular , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Receptores de Complemento/metabolismo , Replicação ViralRESUMO
The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Linfócitos B/metabolismo , Proteínas de Membrana/fisiologia , Receptores de Complemento 3d/fisiologia , Antígenos CD/química , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/química , Sequência de Bases , Linhagem Celular , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Receptores de Complemento 3b/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetraspanina 28 , Células Tumorais CultivadasRESUMO
Particulate glycoproteins lacking sialic acid, such as desialylated enveloped viruses, readily activate complement through the alternative pathway. Human immunodeficiency virus type 1 (HIV-1) contains two heavily glycosylated and partially sialylated envelope glycoproteins: a surface gp120 and a transmembrane gp41. The abilities of naturally glycosylated HIV-1 and glycosylation-modified HIV-1 to interact with the complement system were examined with a biological assay which measured the binding of whole virus particles to cells expressing CR2 (CD21), the complement receptor found naturally in abundance on follicular dendritic cells and immature B cells. HIV-1 IIIB was synthesized in the presence or absence of the mannosidase II inhibitor, swainsonine, to give rise to high-mannose-type, nonsialylated, nonfucosylated carbohydrate moieties. The virus also was treated with neuraminidase or endo-beta-galactosidase to remove terminal sialic acids. An enzyme immunoassay specific for HIV-1 p24 core protein was used to quantitate the amount of virus bound to cell surfaces. Virus particles incubated with 1:3-diluted, fresh HIV-1-negative human serum as a source of complement readily bound to MT-2 (CD4+ CR2+) and Raji-3 (CD4- CR2+) cells but not to CEM (CD4+ CR2-) cells, suggesting that the virus bound to CR2 independently of CD4. Compared with heat-inactivated or C3-deficient sera, fresh complement increased binding by as much as 62 times for naturally glycosylated virus, and 5 times more than this for glycosylation-modified virus. Similar observations were made with freshly isolated, non-mitogen-stimulated peripheral blood mononuclear cells. Additional evidence that HIV-1 bound to CR2 independently of CD4 was provided by the fact that binding was blocked by monoclonal antibody OKB7 (anti-CR2) but not by OKT4a (anti-CD4). Also, the virus bound to transfected K562 cells (CD4-) which expressed recombinant human CR2 but did not bind to untransfected K562 cells. Results obtained with complement component-deficient sera indicated that binding required the alternative complement pathway. Raji-3 and transfected K562 cells could not be infected with HIV-1 in the presence of complement, suggesting that utilization of CR2 as a receptor in the absence of CD4 does not allow virus entry. The demonstration of CR2 as a receptor for HIV-1 in the presence of complement, together with the ability to enhance binding by desialylation, provides new insights into mechanisms of HIV-1-induced immunity and immunopathogenesis.