RESUMO
The electrical activity of macrophages derived from human blood monocytes was recorded in vitro with intracellular microelectrodes and was analyzed with computer-assisted data acquisition and analysis techniques. In cells impaled 6 to 8 days after the cultures were prepared, the resting potentials reached a maximum value of -72 millivolts. The cells were electrically excitable; spikes exhibited a slow upstroke, a fast downstroke, a discrete threshold, a large overshoot, and a brief undershoot. Repetitive firing was induced by a maintained depolarizing current. A positive relation was observed between transmembrane currents and resting potential. Voltage-current relations were nonrectifying for subthreshold current injections. Since these cells had not been treated with any specific activation factors, the electrical activity recorded is evidence for the presence of voltage-dependent inward and outward currents in the membranes of mature macrophages. The electrical signals generated by these cells may be useful for the assay of sensor and effector functions of macrophages, such as chemotaxis, receptor-ligand interactions, and phagocytosis.
Assuntos
Macrófagos/fisiologia , Potenciais de Ação , Diferenciação Celular , Células Cultivadas , Humanos , Monócitos/citologiaRESUMO
Almost any kind of threat to homeostasis or stress will cause plasma glucocorticoid levels to rise. The increased levels have traditionally been ascribed the physiological function of enhancing the organism's resistance to stress, a role well recognized in glucocorticoid therapy. How the known physiological and pharmacological effects of glucocorticoids might accomplish this function, however, remains a mystery. A generalization that is beginning to emerge is that many of these effects may be secondary to modulation by glucocorticoids of the actions of numerous intercellular mediators, including established hormones, prostaglandins and other arachidonic acid metabolites, certain secreted neutral proteinases, lymphokines, and a variety of bioactive peptides. These mediators participate in physiological mechanisms--endocrine, renal, immune, neural, etc.--that mount a first line of defense against such challenges to homeostasis as hemorrhage, metabolic disturbances, infection, anxiety, and others. Contrary to the traditional view that glucocorticoids enhance these defense mechanisms, however, it has become increasingly clear that glucocorticoids at moderate to high levels generally suppress them. This paradox, which first emerged when glucocorticoids were discovered to be antiinflammatory agents, remains a major obstacle to a unified picture of glucocorticoid function. We propose that stress-induced increases in glucocorticoid levels protect not against the source of stress itself but rather against the body's normal reactions to stress, preventing those reactions from overshooting and themselves threatening homeostasis. This hypothesis, the seeds of which are to be found in many discussions of particular glucocorticoid effects, immediately accounts for the paradox noted above. Furthermore, it provides glucocorticoid physiology with a unified conceptual framework that can accommodate such apparently unrelated physiological and pharmacological effects as those on carbohydrate metabolism, inflammatory processes, shock, and water balance. It also leads us to suggest that some of the enzymes rapidly induced by glucocorticoids, such as glutamine synthetase, detoxify mediators released during stress-induced activation of primary defense mechanisms. These mediators would themselves lead to tissue damage if left unchecked.
Assuntos
Glucocorticoides/fisiologia , Estresse Fisiológico/fisiopatologia , Animais , Anti-Inflamatórios , Água Corporal/metabolismo , Metabolismo dos Carboidratos , Síndrome de Adaptação Geral/fisiopatologia , Glucocorticoides/farmacologia , Hormônios/metabolismo , Humanos , Imunossupressores , Interleucina-1/fisiologia , Interleucina-2/fisiologia , Linfocinas/metabolismo , Modelos Biológicos , Choque/fisiopatologiaRESUMO
Although recent studies suggest that interferons can increase the number of IgG Fc receptor (FcR gamma) sites on mouse macrophages, direct assessment of similar effects on human mononuclear phagocytes is lacking. We therefore measured the specific binding of 125I- and fluorescein-labeled IgG1 to human monocytes and leukemic cell lines after culture in vitro with highly purified human interferons. We report that natural and recombinant human gamma-interferon causes a dramatic (nearly 10-fold) increase in the number of FcR gamma on normal human monocytes and on the human cell lines HL-60 and U-937. Alpha and beta-interferons cause a modest but significant increase in these receptors. This report demonstrates that gamma-interferon acts directly on human mononuclear phagocytes to increase FcR gamma sites, it identifies a qualitative difference in the physiologic actions of human type I and type II interferons, and it suggests that HL-60 and U-937 cells will be important models for further study of the molecular mechanisms of interferon action. The results reported here could also be the basis for a bioassay to assess the pharmacokinetics and variability of gamma-interferon action on monocytes of individual patients during treatment in vitro and in vivo.
Assuntos
Imunoglobulina G/metabolismo , Interferons/farmacologia , Monócitos/imunologia , Fagócitos/imunologia , Receptores Fc/metabolismo , Linhagem Celular , Imunofluorescência , Humanos , Interferon gama/farmacologia , Leucemia/imunologia , Receptores Fc/efeitos dos fármacosRESUMO
Studies using cultured cells have shown that gamma interferon (IFN-gamma) induces the expression of Fc gamma RI (the type I Fc receptor for IgG) on human polymorphonuclear neutrophils (PMN) and greatly increases the number of these receptors on human monocytes. Administration of rIFN-gamma in vivo also causes enhanced Fc gamma RI expression on these cell populations. Because streptococcal antigens are potent inducers of IFN-gamma in vitro, we postulated that IFN-gamma would be produced endogenously in vivo in patients with streptococcal infections. Such production of IFN-gamma in vivo, even at low levels, might be expected to induce the expression of Fc gamma RI on monocytes and neutrophils. To evaluate this possibility, we used monoclonal antibody 32 (mAb 32), which is specific for Fc gamma RI, to quantitate the expression of this receptor on human peripheral blood cells. We measured the binding of mAb 32 to monocytes and PMNs isolated from healthy donors and from patients with group A beta-hemolytic streptococcal (GABHS) pharyngitis. PMNs from healthy donors (n = 12) had 700 +/- 600 (mean +/- SD) mAb 32 binding sites. Patients with pharyngitis and negative throat culture for GABHS (n = 11) had 2,100 +/- 1,600 sites on their PMNs. In contrast, the PMNs from patients with documented GABHS pharyngitis (n = 12) had 11,600 +/- 7,500 mAb 32 binding sites on their surface. There was a similar change in the expression of Fc gamma RI on monocytes, with control monocytes having a mean of 19,900 +/- 3,200 mAb 32 binding sites per cell and the GABHS-positive monocytes having 47,500 +/- 21,400 sites. The GABHS-negative throat culture group had a slightly elevated number of Fc gamma RI with a mean of 28,200 +/- 8,400 sites. 10 patients with documented urinary tract infections and three patients with uncomplicated pyelonephritis had no elevation in Fc gamma RI expression. These studies demonstrate that a localized group A streptococcal infection can cause systemic activation of the entire circulating pool of phagocytes, and suggest that a similar level of activation is uncommon in localized gram-negative infections of the urinary tract.
Assuntos
Antígenos de Diferenciação/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Faringite/metabolismo , Receptores Fc/metabolismo , Infecções Estreptocócicas/metabolismo , Antígenos de Diferenciação/imunologia , Humanos , Interferon gama/análise , Monócitos/imunologia , Faringite/imunologia , Receptores Fc/imunologia , Receptores de IgG , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes , Infecções Urinárias/imunologiaRESUMO
A major challenge for using native or modified T cell epitopes to induce or suppress immunity relates to poor localization of peptides to antigen presenting cells (APCs) in vivo. In this study, we demonstrate enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (F(c)gammaRI, CD64) on human monocytes. A Th epitope of tetanus toxoid, TT830, and the antagonistic peptide for TT830, TT833S, were genetically grafted into the constant region of the heavy chain of the humanized anti-CD64 mAb 22 and expressed as monovalent fusion proteins, Fab22-TT830 and Fab22-TT833S. These CD64-targeted peptides were up to 1,000- and 100-fold more efficient than the parent peptides for T cell stimulation and antagonism, respectively, suggesting that such fusion proteins could effectively increase the delivery of peptides to APCs in vivo. Moreover, the F(c)gammaRI-targeted antagonistic peptide inhibited proliferation of TT830-specific T cells even when APCs were first pulsed with native peptide, a situation comparable with that which would be encountered in vivo when attempting to ameliorate an autoimmune response. These data suggest that targeted presentation of antagonistic peptides could lead to promising Ag-specific therapies for T cell-mediated autoimmune diseases.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Monócitos/imunologia , Receptores de IgG/imunologia , Proteínas Recombinantes de Fusão/imunologia , Toxoide Tetânico/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Ligação Competitiva , Células Cultivadas , Antígenos HLA-D/metabolismo , Humanos , Interferon gama/biossíntese , Interleucina-4/biossíntese , Ativação Linfocitária , Dados de Sequência Molecular , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Toxoide Tetânico/antagonistas & inibidoresRESUMO
Monocytes in the circulation of normal individuals express two receptors for the constant region of immunoglobulin, Fc gamma RI and Fc gamma RII. In contrast, we have observed that AIDS monocytes express significant levels of a third Fc gamma R, Fc gamma RIII (CD16), which is normally associated with activation or maturation of the monocyte population. By dual-fluorescence analysis using a monoclonal antibody specific for Fc gamma RIII (MAb 3G8), 38.5 +/- 3.2% of the LeuM3 (CD14)-positive monocytes in AIDS patients were CD16 positive as compared to 10.4 +/- 1.0% for healthy individuals (n = 29; P less than 0.005). Furthermore, AIDS monocytes expressed Fc gamma RIII-specific mRNA which is expressed minimally or not at all in control monocytes. As a recently identified inducer of Fc gamma RIII expression on blood monocytes, transforming growth factor-beta (TGF-beta) was found to be elevated in the serum and/or plasma of AIDS patients. Moreover, incubation of normal monocytes with AIDS serum or plasma induced CD16 expression which correlated with serum TGF-beta levels (r = 0.74, P less than 0.001) and was inhibited with a neutralizing antibody to TGF-beta. Thus, the increased CD16 expression on peripheral blood monocytes in AIDS patients may be the consequence of elevated circulating levels of the polypeptide hormone TGF-beta.
Assuntos
Síndrome da Imunodeficiência Adquirida/sangue , Antígenos CD/análise , Antígenos de Diferenciação/análise , Monócitos/imunologia , Receptores Fc/análise , Fator de Crescimento Transformador beta/sangue , Síndrome da Imunodeficiência Adquirida/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação Mielomonocítica/análise , Humanos , Receptores de Lipopolissacarídeos , Masculino , RNA Mensageiro/análise , Receptores Fc/genética , Receptores de IgGRESUMO
A novel polymorphism in the extracellular domain 2 (EC2) of FcgammaRIIIA affects ligand binding by natural killer (NK) cells and monocytes from genotyped homozygous normal donors independently of receptor expression. The nonconservative T to G substitution at nucleotide 559 predicts a change of phenylalanine (F) to valine (V) at amino acid position 176. Compared with F/F homozygotes, FcgammaRIIIa expressed on NK cells and monocytes in V/V homozygotes bound more IgG1 and IgG3 despite identical levels of receptor expression. In response to a standard aggregated human IgG stimulus, FcgammaRIIIa engagement on NK cells from V/V (high-binding) homozygotes led to a larger rise in [Ca2+]i, a greater level of NK cell activation, and a more rapid induction of activation-induced cell death (by apoptosis). Investigation of an independently phenotyped normal cohort revealed that all donors with a low binding phenotype are F/F homozygotes, while all phenotypic high binding donors have at least one V allele. Initial analysis of 200 patients with SLE indicates a strong association of the low binding phenotype with disease, especially in patients with nephritis who have an underrepresentation of the homozygous high binding phenotype. Thus, the FcgammaRIIIa polymorphism at residue 176 appears to impact directly on human biology, an effect which may extend beyond autoimmune disease characterized by immune complexes to host defense mechanisms.
Assuntos
Doenças Autoimunes/etiologia , Receptores de IgG/fisiologia , Citotoxicidade Celular Dependente de Anticorpos , Epitopos , Humanos , Imunoglobulina G/metabolismo , Lúpus Eritematoso Sistêmico/etiologia , Polimorfismo Genético , Receptores de IgG/químicaRESUMO
We investigated the ability of purified, recombinant DNA-derived interferons (IFN) to induce phenotypic changes in cells of the HL-60 promyelocytic leukemia cell line. Changes in cell surface markers detected by monoclonal antibodies as well as morphologic, histochemical, and functional changes were monitored. We found that gamma-IFN, but not alpha- or beta-IFN, induced the expression of antigens characteristic of monocytes and granulocytes (AML-2-23, 63D3, and 61D3), as well as changes in morphology consistent with monocytoid differentiation. These included induction of alpha-naphthyl acetate esterase, increased cell size, and a decrease in azurophilic granules. The gamma-IFN dose dependency and time course of the effect on antigen expression suggest that de novo protein synthesis was induced by gamma-IFN. The activity of gamma-IFN and of mixed-lymphocyte culture supernatant was blocked by a monoclonal antibody to gamma-IFN. Significant augmentation in the ability of the HL-60 cells to mediate antibody-dependent cellular cytotoxicity was induced by gamma-IFN. These findings suggest that gamma-IFN plays a role in the regulation of hematopoiesis.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Interferon gama/farmacologia , Leucemia Mieloide Aguda/patologia , Monócitos/patologia , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Sítios de Ligação de Anticorpos , Linhagem Celular , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Hematopoese/efeitos dos fármacos , Humanos , Interferon gama/imunologia , Leucemia Mieloide Aguda/imunologia , Teste de Cultura Mista de LinfócitosRESUMO
Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.
Assuntos
Formação de Anticorpos , Células Apresentadoras de Antígenos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores de IgG/metabolismo , Adjuvantes Imunológicos , Animais , Células Cultivadas , Endocitose , Humanos , Camundongos , Camundongos Transgênicos , Fagocitose , Receptores de IgG/genéticaRESUMO
Glucocorticoids are immunosuppressive and antiinflammatory agents that act through multiple mechanisms. This review highlights recent evidence that lipocortin-1, a member of the annexin family of calcium-binding proteins, can be induced by glucocorticoids, and may mediate some of the effects of glucocorticoids through putative lipocortin-1 receptors, which have been found on the surface of phagocytic cells. Recent advances in annexin biology have been drawn together to formulate a novel hypothesis for the regulation of inflammation.
Assuntos
Anexina A1/fisiologia , Glucocorticoides/fisiologia , Imunidade Celular/fisiologia , Inflamação/fisiopatologia , Animais , Anexina A1/biossíntese , Regulação da Expressão Gênica , Humanos , Sistema Hipotálamo-Hipofisário/fisiopatologia , Modelos Biológicos , Fagócitos/fisiologia , Sistema Hipófise-Suprarrenal/fisiopatologia , Receptores Fc/antagonistas & inibidores , Proteínas Recombinantes/farmacologiaRESUMO
We investigated the influence of glucocorticoids on two effects of interferons (IFNs) which are thought to relate to their antitumor actions: cytotoxic activity and induction of HLA antigen expression. We treated human myeloid cell lines (U-937, HL-60, THP-1, K-562, and KG-1a), and T-(MOLT-4) and B- (Daudi) lymphoblastic cell lines with concentrations of IFN-alpha, IFN-gamma, and dexamethasone (Dex) which are commonly achieved in the circulation following therapeutic administration. The results show that for every cell line except Daudi, the greatest inhibition of cell growth occurred when IFN-gamma and Dex treatments were combined. The advantage of combined IFN-gamma and Dex treatment over treatment with either agent alone was most dramatic for the three cell lines (U-937, HL-60, and THP-1) which have monocytoid characteristics. There was also more growth inhibition by the combination of IFN-alpha and Dex than by either agent alone for all seven cell lines tested. The induction of HLA antigen expression by IFN-alpha and IFN-gamma, an effect which could increase recognition of the tumor cells by the immune system, was as great or greater in the presence of Dex as in its absence. These results demonstrate that glucocorticoids do not inhibit, and in some cases enhance, two effects of IFNs that appear to be related to their antitumor actions: inhibition of tumor cell proliferation and enhancement of HLA antigen expression.
Assuntos
Dexametasona/farmacologia , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Antígenos de Superfície/análise , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Antígenos HLA/análise , HumanosRESUMO
In vitro, monocyte-derived macrophages (MDM) are capable of efficient antibody-mediated phagocytosis of human nucleated tumor cells. These MDM express on their cell surface all three classes of Fc receptors for IgG (Fc gamma R). Fc gamma R specificity for murine antibody isotype allowed us to examine the phagocytic role of Fc gamma RII on control and gamma-interferon (IFN-gamma)-primed MDM. Monoclonal antibody 520C9 (IgG1) mediates phagocytosis through Fc gamma RII. This monoclonal antibody is directed against the HER-2/neu protooncogene product overexpressed on a variety of adenocarcinomas including the breast carcinoma cell line SK-BR-3. Our results showed that IFN-gamma treatment of differentiated MDM (days 8-12 in culture) inhibited Fc gamma RII-mediated phagocytosis in a dose-dependent manner with negative effects noted at doses as low as 0.1 units/ml. The percentage reduction in antibody-mediated phagocytosis observed following IFN-gamma priming (40 units/ml for 18 h) ranged from 23-89% of control. The inhibitory effect was evident when exposure to IFN-gamma was transient. Fc gamma RII expression was not altered by IFN-gamma treatment. In our model, IFN-gamma did not up-regulate or down-regulate HER-2/neu protein expression on our targets or affect the level of CD14 antigen expression on our MDM. Although IFN-gamma is a potent activator of monocytes/macrophages and can enhance certain tumoricidal mechanisms, our data show that antibody-dependent phagocytosis through the type II Fc receptor is inhibited by IFN-gamma priming. Nonspecific phagocytosis was not affected.
Assuntos
Interferon gama/farmacologia , Macrófagos/fisiologia , Fagocitose/efeitos dos fármacos , Receptores Fc/efeitos dos fármacos , Adolescente , Neoplasias da Mama , Relação Dose-Resposta Imunológica , Receptores ErbB/metabolismo , Citometria de Fluxo , Humanos , Macrófagos/efeitos dos fármacos , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2 , Receptores Fc/fisiologia , Células Tumorais CultivadasRESUMO
A bispecific antibody, MDX-H210, was developed to target cytotoxic effector cells expressing Fc gamma receptor type I (Fc gammaRI, CD64) to HER2/neu-overexpressing tumor cells. HER2/neu is an appropriate target for immunotherapy due to the high level of expression of this proto-oncogene in a variety of malignancies. The expression of Fc gammaRI is limited primarily to cytotoxic immune cells, including monocytes, macrophages, and cytokine-activated polymorphonuclear (PMN) cells. Therefore, tumor cells bound with MDX-H210 can be selectively recognized by effector cells with cytotoxic potential. MDX-H210 was prepared by chemical conjugation of Fab' fragments derived from the HER2/neu-specific monoclonal antibody, 520C9, and the Fc gammaRI-specific monoclonal antibody, H22. This bispecific molecule demonstrated specific, dose-dependent, and saturable binding to both HER2/neu- and Fc gammaRI-expressing cells. A solid-phase immunoassay that demonstrated simultaneous and specific binding to both antigens was used to confirm the bispecific nature of MDX-H210. Monocytes and PMN cells mediated MDX-H210-dependent lysis of HER2/neu-overexpressing cell lines derived from breast, ovarian, and lung carcinomas. IFN-gamma treatment of monocytes enhanced antibody-dependent cellular cytotoxicity, whereas IFN-gamma and granulocyte colony-stimulating factor were required for PMN cell-mediated tumor cell lysis. In addition, MDX-H210 elicited tumor necrosis factor-alpha secretion from monocytes when cultured in the presence of HER2/neu-positive target cells. These in vitro data suggest that targeting tumor cells to Fc gammaRI with MDX-H210 may be an effective treatment for malignancies that overexpress HER2/neu. The in vivo cytotoxic potential of MDX-H210 may be enhanced by combination therapy with the cytokines granulocyte colony-stimulating factor and IFN-gamma, which up-regulate Fc gammaRI expression on cytotoxic effector cells.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Receptor ErbB-2/imunologia , Receptores de IgG/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Citotoxicidade Imunológica , Humanos , Imunoterapia/métodos , Proto-Oncogene Mas , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.
Assuntos
Antígenos de Diferenciação/biossíntese , LDL-Colesterol/fisiologia , Receptores Fc/biossíntese , Anticorpos Monoclonais , Antígenos de Diferenciação/metabolismo , LDL-Colesterol/sangue , Citometria de Fluxo , Humanos , Imunoglobulina G/metabolismo , Técnicas In Vitro , Interferon gama/farmacologia , Masculino , Receptores Fc/metabolismo , Receptores de IgGRESUMO
The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue.
Assuntos
Tecido Linfoide/metabolismo , Linfotoxina-alfa/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Linhagem Celular , Células Dendríticas/metabolismo , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Receptor beta de Linfotoxina , Linfotoxina-beta , Monócitos/metabolismo , Ligação Proteica , Baço/metabolismo , Timo/metabolismoRESUMO
PURPOSE: MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS: Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS: Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION: MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias da Mama/terapia , Expressão Gênica , Genes erbB-2 , Neoplasias Ovarianas/terapia , Receptor ErbB-2/imunologia , Receptores de IgG/imunologia , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Biopterinas/análogos & derivados , Biopterinas/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Estudos de Coortes , Feminino , Febre/etiologia , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Hipotensão/etiologia , Infusões Intravenosas , Interleucina-6/sangue , Pessoa de Meia-Idade , Neopterina , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Proto-Oncogene Mas , Receptor ErbB-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bispecific antibodies--molecules combining two different antigenic specificities--are currently being developed as new agents for immunotherapy and for basic studies in cell biology. Bispecific antibodies (BsAb) are prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Both of these approaches present challenges with respect to yield and purity that should eventually be solved through newer molecular genetic approaches. BsAb have been used to demonstrate that specific surface molecules can trigger leukocytes to either phagocytose or kill tumor cells, viruses, parasites, and infected cells. Such trigger molecules include CD3 on T lymphocytes and Fc receptors for IgG on monocytes, macrophages, and natural killer cells. BsAb have also been used experimentally to localize toxins to tumor sites and fibrinolytic agents to areas of thrombosis, to study the molecular specificity of particular receptors, and as adjuvants in in vitro models of vaccines for infectious disease. The limited clinical trials that have occurred to date, primarily for therapy of tumors, suggest that BsAb may offer considerable promise for therapeutic applications, including cancer, heart disease, infectious disease, allergy, and autoimmunity.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ensaios Clínicos como Assunto , Citotoxicidade Imunológica/imunologia , Fibrinolíticos/imunologia , Humanos , Hibridomas/imunologia , Imunotoxinas/imunologia , Infecções/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores Fc/imunologia , Linfócitos T/imunologia , Vacinas/imunologiaRESUMO
In addition to CD4, the primary receptor to which the human immunodeficiency virus type 1 (HIV-1) binds, mononuclear phagocytes (monocytes) express three classes of Fc receptors for immunoglobulin G (Fc gamma R). We have previously shown that infection of monocytes by HIV-1 is inhibited when bispecific antibodies (BsAbs) are used to target the virus to either the type I, type II, or type III Fc gamma R on these cells. Infection of monocytes was not inhibited when HIV-1 was targeted to either human leukocyte antigen class I or CD33. We have extended these studies to examine the ability of BsAbs plus polymorphonuclear leukocytes (neutrophils, PMNs) and monocytes to reduce infectivity of HIV-1 to cells from the human T cell lymphoma line, H9. The production of HIV-1 following interaction of virus with BsAb and phagocytes was determined in an indicator cell assay by mixing BsAb, HIV-1, and phagocytes with uninfected H9 cells. Productive infection of H9 cells was quantitated on subsequent days by measuring p24 gag antigen levels in supernatants by enzyme-linked immunosorbent assay. Our findings show that the addition of interferon-gamma-activated PMNs or monocytes to cultures of HIV-1 plus H9 cells in the absence of BsAb results in a marked reduction in p24 levels equivalent to 85 to 90% of control levels. With the combination of BsAb (anti-Fc gamma RI x anti-gp120) plus IFN-gamma-activated phagocytes, levels of p24 in H9 cultures were below those at culture initiation. These findings demonstrate that IFN-gamma-activated phagocytes can affect the natural course of HIV-1 infection of T cells, a finding of potential clinical importance.
Assuntos
Especificidade de Anticorpos , Anticorpos Anti-HIV/imunologia , HIV-1/metabolismo , HIV-1/fisiologia , Fagócitos/química , Fagócitos/citologia , Receptores de IgG/análise , Linfócitos T/citologia , Linfócitos T/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Anticorpos Anti-HIV/farmacologia , Proteína do Núcleo p24 do HIV/análise , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/isolamento & purificação , Humanos , Imunidade Inata , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Interferon gama/farmacologia , Monócitos/química , Monócitos/citologia , Monócitos/ultraestrutura , Neutrófilos/química , Neutrófilos/citologia , Neutrófilos/ultraestrutura , Fagócitos/ultraestrutura , Receptores de IgG/metabolismoRESUMO
The high-affinity receptor for the constant region of immunoglobulin G IgG (Fc gamma RI; CD64) is virtually undetectable on mature polymorphonuclear neutrophils (PMNs) in healthy individuals but is expressed on PMNs in patients with certain infections and in patients treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). The induction of Fc gamma RI by rhG-CSF has previously been reported to result from effects on immature granulocyte progenitors. To evaluate the G-CSF effect on mature PMNs, we studied the correlation between G-CSF plasma concentration and expression of Fc gamma RI on PMNs in vivo as well as the effect of G-CSF on Fc gamma RI expression on mature PMNs in vitro. Fc gamma RI expression on PMNs correlated (R = 0.79; p < .001) with plasma concentrations of endogenous or recombinant G-CSF in healthy volunteers and in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation. PMNs exhibited a unimodal distribution for elevated Fc gamma RI expression, suggesting that G-CSF induced increased expression of Fc gamma RI on mature as well as on immature PMNs. In vitro, incubation of mature PMNs with G-CSF induced mRNA for Fc gamma RI. Significant Fc gamma RI surface expression was induced in a time- and dose-dependent manner. Thus, G-CSF can act on mature PMNs to increase Fc gamma RI expression and may be useful for stimulating antibody mediated immune functions of PMNs in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Transplante de Medula Óssea , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Interferon gama/farmacologia , RNA Mensageiro/genética , Transplante AutólogoRESUMO
About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.