RESUMO
Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha. By using a battery of anti-LT-alpha and LT-beta mAbs, it is clear that two LT surface forms exist on the surface of PMA-activated II-23 cells, a human T cell hybridoma. Assuming that these surface forms are trimers, a minor form appears early after induction having an apparent stoichiometry of LT-alpha 2/beta 1 and is recognized by one group of anti-LT-alpha mAbs and the p55-TNF receptor. The second and predominant form has an apparent LT-alpha 1/beta 2 composition and is recognized by a second group of pantrophic anti-LT-alpha mAbs and the LT-beta receptor. Neither of the heteromeric forms nor a putative LT-beta homotrimeric form were found to be secreted. The properties of surface LT on the II-23 cell system were similar to those of the surface LT forms on Chinese hamster ovary cells transfected with both LT-alpha and LT-beta genes and a number of lymphoid tumor lines. These experiments point toward the LT-alpha 1/beta 2 complex as the predominant membrane form of LT on the lymphocyte surface, and this complex is the primary ligand for the LT-beta receptor.
Assuntos
Anticorpos Monoclonais/imunologia , Linfotoxina-alfa/imunologia , Proteínas de Membrana/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Chlorocebus aethiops , Cricetinae , Imunofluorescência , Glicosilação , Humanos , Hibridomas/imunologia , Cinética , Ativação Linfocitária , Receptor beta de Linfotoxina , Linfotoxina-alfa/química , Linfotoxina-alfa/metabolismo , Linfotoxina-beta , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Solubilidade , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.
Assuntos
Moléculas de Adesão Celular/metabolismo , Receptores de Antígeno muito Tardio/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Anticorpos Monoclonais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/ultraestrutura , Linhagem Celular , Clonagem Molecular , Epitopos , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Molécula 1 de Adesão de Célula VascularRESUMO
OBJECTIVE: The authors sought to determine whether the signs and symptoms of endotoxemia were related to the endotoxin-stimulated increase in circulating phospholipase A2 (PLA2) activity. BACKGROUND: Because hypotension and pulmonary injury have been associated with elevated PLA2 activity in septic shock and PLA2 levels are reduced with the administration of glucocorticoids, the PLA2 response to endotoxin was investigated in volunteers pretreated with and without hydrocortisone. METHODS: Carefully screened human subjects were studied under four conditions: (1) saline, (2) hydrocortisone, (3) endotoxin, and (4) hydrocortisone administration before endotoxin exposure. Pulse rate, blood pressure, temperature, and symptoms of endotoxemia were serially measured. Plasma for tumor necrosis factor concentrations and PLA2 activity was obtained. RESULTS: After lipopolysaccharide, pulse rate and tumor necrosis factor concentrations rose at 1 to 2 hours; temperature increased maximally at 4 hours. PLA2 activity reached peak levels at 24 hours. With hydrocortisone pretreatment, a 50% reduction in the concentrations of tumor necrosis factor and PLA2 occurred. Significant correlations between other variables and PLA2 activity were not observed. The enzyme identified by monoclonal antibody was the secreted nonpancreatic PLA2 (SNP-PLA2). CONCLUSIONS: The results of this study suggest that elevations in circulating SNP-PLA2 activity and systemic events associated with intravenous endotoxin administration are unrelated.
Assuntos
Fosfolipases A/sangue , Toxemia/sangue , Adulto , Ensaio de Imunoadsorção Enzimática , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfolipases A2 , Toxemia/diagnóstico , Fator de Necrose Tumoral alfa/análiseRESUMO
Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.
Assuntos
Moléculas de Adesão Celular/genética , Adesão Celular/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/farmacologia , Linhagem Celular , Membrana Celular/fisiologia , Códon , Citoplasma/fisiologia , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/fisiologia , Transfecção , Molécula 1 de Adesão de Célula VascularRESUMO
Endothelial leukocyte adhesion molecule 1 (ELAM1) is a leukocyte adhesion molecule induced on human venular endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for ELAM1 has been constructed, stably expressed in Chinese hamster ovary cells, and the secreted recombinant soluble form of ELAM1 (rsELAM1) purified to homogeneity by immunoaffinity chromatography. rsELAM1, when immobilized on plastic, is fully functional as an adhesion protein, and selectively binds only cells known to bind cell-surface ELAM1 expressed on human endothelial cells, including the myelomonocytic cell line HL60 and the colon carcinoma cell line HT29. Immobilized rsELAM1 also binds human PMN, monocytes, NK cells, and T cells. T cell subset analyses indicate preferential binding of CD4+ T memory cells. However, rsELAM1 is only a weak inhibitor of ELAM1-mediated adhesion. rsELAM1 should prove valuable for the further study of the role of ELAM1 expressed on the vascular wall during the inflammatory response.
Assuntos
Moléculas de Adesão Celular/genética , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Adesão Celular , Moléculas de Adesão Celular/química , Linhagem Celular , Clonagem Molecular , Selectina E , Humanos , Leucócitos/citologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Recombinantes , SolubilidadeRESUMO
ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.
Assuntos
Anticorpos Monoclonais/imunologia , Moléculas de Adesão Celular/imunologia , Endotélio Vascular/imunologia , Adesão Celular , Selectina E , Endotélio Vascular/citologia , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/imunologia , Neutrófilos/citologia , Proteínas Recombinantes/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
We have developed two monoclonal antibodies to human lipocortin-1 (103 and 105) as reagents for quantitating the protein in biological systems and neutralizing its activity. Lipo 105 is a high affinity antibody that is functional in ELISA and Western blot formats. The antibody recognizes a site between amino acids 30 and 55 in the lipocortin-1 sequence and can be used on native or denatured protein. Lipo 103 is an antibody that neutralizes the phospholipase A2 inhibitory activity of lipocortin-1 by blocking binding of the protein to phospholipid surfaces. The antibody is specific for native human lipocortin-1. Lipo 103 was recently shown to block lipocortin-1-dependent differentiation of a squamous carcinoma cell line, demonstrating its usefulness as a probe for function.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Animais , Anexinas , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Western Blotting , Proteínas de Ligação ao Cálcio/farmacologia , Brometo de Cianogênio , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Proteínas RecombinantesRESUMO
An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.
Assuntos
Glicoproteínas , Inibidores do Crescimento/sangue , Hormônios Testiculares/sangue , Adolescente , Adulto , Fatores Etários , Hormônio Antimülleriano , Anticorpos Monoclonais , Formação de Anticorpos , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Inibidores do Crescimento/imunologia , Inibidores do Crescimento/uso terapêutico , Humanos , Lactente , Recém-Nascido , Masculino , Valores de Referência , Hormônios Testiculares/imunologia , Hormônios Testiculares/uso terapêuticoRESUMO
Tumor necrosis factor is a monokine, which causes cytolysis of many transformed cells. In this study we have found that in addition to cytotoxicity recombinant Escherichia coli-derived human tumor necrosis factor, like cachectin, inhibited the lipoprotein lipase of 3T3-L1 preadipocytes. Both effects were inhibited by monoclonal anti-tumor necrosis factor antibodies. Monoclonal antibodies against recombinant human tumor necrosis factor were produced by fusing splenocytes of immune mice with P3X63Ag8 653 myeloma cells. The monoclonal antibodies, namely BG 2-4, were of IgG2a, IgG, and IgG2a subclasses. These monoclonal antibodies neutralized the cytotoxicity of natural and recombinant human tumor necrosis factor but not that of rabbit or mouse tumor necrosis factor. They also neutralized the cachectin activity of human tumor necrosis factor in the 3T3-L1 embryonic cell assay. These results indicate that the functional structure(s) of human tumor necrosis factor responsible for the cytotoxicity and cachectin activities are likely to be closely related.