RESUMO
Endothelial-cell-specific molecule 1 (ESM-1) is a recently identified endothelial cell molecule. As ESM-1 mRNA is preferentially expressed in human lung and kidney tissues, and as ESM-1 mRNA expression is regulated by inflammatory cytokines, ESM-1 is thought to play a role in the vascular contribution to organ-specific inflammation. In order to define its behavior, mouse anti-ESM-1 monoclonal antibodies were developed, and three distinct epitopes were mapped, which allowed development of a specific ELISA assay, immunohistological staining and immunoblot analysis. Here, we demonstrate that ESM-1 is present in cell lysates of human endothelial cells (human umbilical vein endothelial cells) with an apparent molecular weight of 20 kD. In contrast, the secreted form of ESM-1 is shifted to an apparent molecular weight of 50 kD, indicating that the secreted form of ESM-1 is posttranslationally modified. By ELISA, we show that the secretion of ESM-1 is significantly enhanced in the presence of TNFalpha. In contrast, the spontaneous as well as TNFalpha-induced secretion of ESM-1 is strongly inhibited by IFNgamma. Moreover, ESM-1 was detected in the serum of healthy subjects at an average concentration of 1.08 ng/ml, and we demonstrated that the serum level of ESM-1 is dramatically increased in patients presenting a septic shock. Analysis of ESM-1 expression in normal human tissues by immunohistochemistry showed that ESM-1 is localized in the vascular network, but also in the bronchial and renal epithelia. Our results demonstrate that ESM-1 is mainly expressed in the vascular endothelium both in vitro and in vivo, but also by different epithelia. ESM-1 may represent a new marker of endothelial cell activation, and may have a functional role in endothelium-dependent pathological disorders.
Assuntos
Proteínas de Neoplasias , Proteínas/análise , Proteoglicanas , Adulto , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Western Blotting , Endotélio Vascular/química , Ensaio de Imunoadsorção Enzimática , Epitélio/química , Mapeamento de Epitopos , Feminino , Rejeição de Enxerto , Humanos , Técnicas de Imunoadsorção , Rim/irrigação sanguínea , Transplante de Rim , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas/imunologia , Sepse/sangue , Veias UmbilicaisRESUMO
CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the alpha-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Fragmentos de Imunoglobulinas/genética , Isoanticorpos/genética , Receptores de Complemento 3b/deficiência , Proteínas Recombinantes/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/genética , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células CHO/metabolismo , Linhagem Celular Transformada , Proteínas Inativadoras do Complemento/farmacologia , Cricetinae , Citometria de Fluxo , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Isoanticorpos/química , Isoanticorpos/metabolismo , Microscopia de Fluorescência , Receptores de Complemento 3b/antagonistas & inibidores , Receptores de Complemento 3b/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , Imunoglobulina rho(D) , SolubilidadeRESUMO
The density of CR1, the C3b/C4b receptor (CD35), on erythrocytes (E) (CR1/E) is genetically determined. However, the broad distribution of CR1/E within a given genotype suggests that other genetic elements might contribute to the regulation of CR1/E. In some pathological conditions, including systemic lupus erythematosus (SLE), AIDS and hemolytic anemia, CR1 deficiency parallels the severity of the disease. When compared to healthy individuals, an accelerated decrease in CR1/E in these patients has been demonstrated, but other mechanisms interfering with CR1 density regulation during erythropoiesis might also contribute. In exceptional circumstances, CR1/E can be dramatically decreased in healthy individuals by the effect of a regulatory gene, In(Lu), that switches off various surface molecules on E, the structure genes of which are located on four different chromosomes, suggesting a transcription regulatory role for In(Lu) gene products. The hypothesis that products of this gene could physiologically regulate the surface density of all these molecules has been tested by determining Lub density on E (Lub/E) using quantitative flow cytometry. Lub antigenic sites were then compared to CR1/E among healthy individuals of the different CR1 density phenotypes, SLE patients with and without CR1 deficiency, and an exceptional SLE patient totally lacking CR1/E and reticulocytes. No quantitative relationship was found between CR1 and Lub expression in either normal or pathological conditions. These data establish that In(Lu) products are not involved in normal or pathological CR1 density regulation.
Assuntos
Eritrócitos/metabolismo , Sistema do Grupo Sanguíneo Lutheran/genética , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3b/genética , Anticorpos Monoclonais , Tipagem e Reações Cruzadas Sanguíneas , Eritrócitos/química , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Sistema do Grupo Sanguíneo Lutheran/imunologia , Receptores de Complemento 3b/sangue , Coloração e RotulagemRESUMO
BACKGROUND: The alveolar macrophage (AM) expresses the low affinity IgE receptor and has the ability to produce not only several proinflammatory cytokines (TNF-alpha, IL-1, IL-6) but also antiinflammatory cytokines (IL-1 receptor antagonist [IL-lra], IL-10), chemokines (IL-8, monocyte chemotactic protein-1 [MCP-1]), and macrophage inflammatory protein-1alpha (MIP-1alpha). OBJECTIVE: The aim of this study was to evaluate the capacity of the AM from patients with allergic asthma and control subjects to produce chemokines and antiinflammatory versus proinflammatory cytokines after activation by IgE receptors and to define the role of CD23 in this activation. METHODS: AMs were collected by bronchoalveolar lavage from 13 patients with allergic asthma and 14 healthy subjects. Adherent AMs were activated either by the successive addition of IgE and anti-IgE or by monoclonal mouse IgG anti-CD23 or by a control monoclonal mouse antibody. TNF, IL-1beta, IL-1ra, IL-10, IL-8, MCP-1, and MIP-1alpha levels were evaluated in supernatants of AMs incubated for 18 hours and in some cases after 4 hours of incubation. RESULTS: Activation by IgE and anti-IgE antibodies significantly increased the production of TNF, IL-1beta, IL-8, MCP-1, MIP-lalpha, and IL-10 in both control subjects and patients with asthma, whereas the increase for IL-1ra was only significant for the control subjects. Whereas F(ab) fragments of anti-CD23 antibodies inhibited IgE plus anti-IgE-induced cytokine production, activation by monoclonal IgG anti-CD23 antibodies reproduced the effect of IgE immune complexes. At 4 hours, the secretion of proinflammatory cytokines was increased by activation by IgE receptors, in contrast to antiinflammatory cytokines. In addition, analysis of the balance between proinflammatory and antiinflammatory cytokines showed that IgE-dependent activation largely favored the proinflammatory cytokines, particularly in patients with asthma. CONCLUSION: IgE-dependent activation by the FcepsilonRII receptor upregulates the synthesis of both chemokines and antiinflammatory cytokines in addition to proinflammatory cytokines. However, AMs from patients with allergic asthma may promote airway inflammation after activation by IgE receptors through its preferential effect on proinflammatory cytokines.
Assuntos
Quimiocinas/biossíntese , Citocinas/biossíntese , Ativação de Macrófagos/fisiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/ultraestrutura , Receptores de IgE/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Quimiocina CCL2/biossíntese , Quimiocina CCL3 , Quimiocina CCL4 , Humanos , Imunoglobulina E/fisiologia , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/biossíntese , Interleucina-1/metabolismo , Interleucinas/biossíntese , Proteínas Inflamatórias de Macrófagos/biossíntese , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Receptores de IgE/imunologia , Sialoglicoproteínas/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.
Assuntos
Eritrócitos/imunologia , Técnicas Genéticas , Técnicas Imunológicas , Polimorfismo Genético , Receptores de Complemento 3b/genética , Animais , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/imunologia , Southern Blotting , Mapeamento Cromossômico , Ativação do Complemento/genética , DNA/genética , Humanos , Processamento de Imagem Assistida por Computador , Família Multigênica , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Receptores de Complemento 3b/análise , Receptores de Complemento 3b/metabolismo , SolubilidadeRESUMO
Monomeric recombinant molecules prove generally unsatisfactory for in vivo use. Most biological systems are indeed multivalent either structurally, associating different chains, or functionally, when cross-linked by their ligands. Mimicking natural molecules for immune intervention implies the need for multimerizing systems to create multivalent molecules capable of interfering with physiological processing. A multivalent anti-Rh(D) recombinant protein has been designed by reconstructing the antibody binding site of a human monoclonal anti-Rh(D) antibody as a single chain Fv mini antibody, then multimerizing it by inserting at its C-terminal end the C-terminal part of the C4 binding protein (C4bp) alpha chain, which is responsible for the octamer multimerization of that molecule. This soluble multivalent recombinant molecule was functional, bound red blood cells (RBCs), agglutinated them, and did not activate complement. This demonstration model opens the way for future in vivo use of multivalent molecules associating antibody valences and other functional molecules for cell targeting, imaging, or removal of cells such as Rh(D)-positive RBCs for preventing Rh alloimmunization.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Transporte/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Sequência de Aminoácidos , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/genética , Sequência de Bases , Linhagem Celular , Complemento C4/imunologia , Humanos , Integrina alfaXbeta2 , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Human erythrocytes (E) react by exocytosis of membrane vesicles to various stresses including the fixation of the membrane attack complex of Complement. E from normal individuals loose a notable proportion of their initial number of surface CR1 molecules during the ageing process. An acquired decrease of CR1 on E also occurs in pathological conditions such as Systemic Lupus Erythematosus or AIDS. The present study investigated whether calcium ionophore A23187 (Ca-ion) induced vesicle formation of human E in vitro is responsible for a preferential loss of CR1 as well as whether CR1 molecules at the surface of Ca-ion treated E or vesicles are: (i) functional, (ii) native or protease degraded, or (iii) more clustered than CR1 on native E. A study of E from 137 normal individuals showed that a one-hour Ca-ion induced vesicle formation preferentially removed one third of E surface CR1. Kinetic experiments suggested that all surface CR1 could be removed from E upon longer incubation times. CR1 molecules on vesicles were still able to inhibit Complement activation, and were found in larger clusters than on native E. These data suggest that a significant part of surface CR1 molecules may be removed from E by vesicle formation during the life of E in normal individuals. This phenomenon could be exacerbated in pathological conditions.