Human macrophage-mediated cytotoxicity of Schistosoma mansoni. Functional and structural features of the effector cells

1989 ◽  
Vol 94 (4) ◽  
pp. 733-741
Author(s):  
B.J. Cottrell ◽  
C. Pye ◽  
A.M. Glauert ◽  
A.E. Butterworth
Keyword(s):  
Schistosoma Mansoni ◽  
Plasma Membranes ◽  
Oxygen Metabolism ◽  
In Vitro Cytotoxicity ◽  
Metabolic Inhibitors ◽  
Tissue Type ◽  
Human Macrophage ◽  
Effector Cells ◽  
Ultrastructural Studies

Human monocytes were purified from peripheral blood and cultured in vitro on hydrophobic membranes. Such cells developed into mature tissue-type macrophages after approximately 1 week in culture. During this maturation period the macrophages developed a potent cytotoxic mechanism whereby they could kill the schistosomula of Schistosoma mansoni in standard in vitro cytotoxicity assays. Cytological and ultrastructural studies of the cells grown in vitro indicated that macrophages developed many of the classical histological and ultrastructural features of ‘activated’ cells with ruffled plasma membranes and significant increases in rough endoplasmic reticulum and Golgi vesicles. Effective cytotoxicity depended upon contact of the effector cells and their parasite target. Further, experiments using metabolic inhibitors indicated that cytotoxicity was dependent upon protein synthesis. Initial results point to the macrophage factor being distinct from some of the better-characterised macrophage secretory products such as tumour necrosis factor, proteases and products of oxygen metabolism.

scholarly journals Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes.

1993 ◽  
Vol 177 (6) ◽  
pp. 1821-1826 ◽  
Author(s):  
T J Schall ◽  
K Bacon ◽  
R D Camp ◽  
J W Kaspari ◽  
D V Goeddel
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cytotoxic T Cells ◽  
Human Macrophage ◽  
Immune Effector ◽  
Effector Cells ◽  
Inflammatory Protein ◽  
And Migration ◽  
Macrophage Inflammatory Protein

Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration-dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.

Anti-EpCAM XmAb antibodies with improved cytotoxicity

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12506-12506 ◽  
Author(s):  
O. Vafa ◽  
S. Kharki ◽  
J. Vielmetter ◽  
A. Chamberlain ◽  
P. Hammond ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Clinical Efficacy ◽  
Cancer Cell Lines ◽  
In Vitro Cytotoxicity ◽  
Binding Assays ◽  
Multiple Cancer ◽  
Effector Cells ◽  
Dependent Cell

12506 Background: The epithelial cell adhesion molecule (EpCAM), also known as epithelial protein 2 (EGP-2) or 17–1A antigen, is a trans-membrane protein expressed on the surfaces of most carcinomas, including those of pancreatic, colorectal, prostate, breast, kidney, lung, and ovarian origins. Moderate affinity antibodies (Abs) such as 17–1A (Kd ∼ 10−7 nM) have been safe in humans albeit with limited clinical efficacy. Attempts to improve clinical efficacy by enhancing antigen affinity (Kd ∼ 10−9 nM) have led to serious clinical toxicity, including pancreatitis. These observations raise the question of whether a moderate affinity Ab with enhanced effector function will be both safe and clinically efficacious. Methods: We applied our proprietary XmAb™ technologies to humanize the 17–1A variable domain and engineer a human IgG1 Fc domain to increase affinity for the activating receptor FcγRIIIa. Ab binding to Ep-CAM or to Fc receptors was tested with Biacore and/or AlphaScreen binding assays. In vitro cytotoxic activity against representative cancer cell lines was measured with Antibody Dependent Cell-mediated Cytotoxicity (ADCC) assays, using human PBMC as effector cells. Results: Humanized anti-EpCAM Abs have affinity for EpCAM similar to the parent 17–1A. Affinity for the activating FcγRIIIa was increased 100-fold relative to a control Ab with an IgG1 Fc domain. As expected, these Abs exhibit dramatically enhanced ADCC against multiple cancer cell lines relative to 17–1A and IgG1 control Abs. Despite their moderate affinity for EpCAM, these novel Abs have in vitro cytotoxicity comparable to the high affinity Ab ING-1. CDC activities of these Abs were similar to chimeric 17–1A. Conclusions: We have demonstrated that antibodies with moderate affinity for EpCAM and increased FcγRIIIa affinity exhibit superior cancer cell killing via an ADCC mechanism. The humanized nature and the increased cytotoxicity of anti-EpCAM XmAb™ antibodies make them promising candidates for clinical development of a novel pan-carcinoma Ab that is superior to 17–1A. [Table: see text]

scholarly journals Rosette-forming ability of thymus-derived lymphocytes in cell-mediated immunity. I. Delayed hypersensitivity and in vitro cytotoxicity.

1975 ◽  
Vol 141 (3) ◽  
pp. 584-599 ◽  
Author(s):  
B E Elliott ◽  
J S Haskill ◽  
M A Axelrad
Keyword(s):  
T Cell ◽  
In Vitro Cytotoxicity ◽  
Delayed Hypersensitivity ◽  
Velocity Sedimentation ◽  
Target Cells ◽  
Cell Mediated Immunity ◽  
Small Lymphocyte ◽  
Effector Cells ◽  
Effector T Cell

Effector cells in delayed hypersensitivity and in vitro cytotoxicity were studied in lymph node cells from animals immunized with sheep erythrocytes (SRBC) in complete Freund's adjuvant. Delayed hypersensitivity response (DHR) was assayed by the increase in foot pad swelling after the intrafoot pad injection of immune cells plus antigen. Cell-mediated cytotoxicity against SRBC was assayed by a microcytotoxicity test with sheep fibroblasts as target cells. Effector cells were antigen specific, sensitive to anti-theta serum plus complement (C), and insensitive to anti-Ig serum plus C. A nonrosette-forming (non-RFC) small lymphocyte effector T cell and a rosette-forming medium lymphocyte effector T cell were isolated by velocity sedimentation. The small lymphocyte non-RFC required a longer time than the medium lymphocyte RFC effector cell to produce maximum activity. Buoyant density failed to distinguish medium lymphocyte effector cells in DHR and in vitro cytotoxicity.

scholarly journals The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937)

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 652-662 ◽  
Author(s):  
RJ Kurlander ◽  
JE Gartrell
Keyword(s):  
Cell Line ◽  
Metabolic Inhibitors ◽  
Human Macrophage ◽  
Reversible Binding ◽  
In Vitro Binding ◽  
Irreversible Reduction ◽  
Vitro Binding ◽  
Human Igg1 ◽  
The Relationship

Abstract The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I- labeled monomers, dimers, oligomers (composed of 2–4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3–7--fold, and the Ka was decreased 2–4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.

The mechansim of antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro: a study by phase-contrast and electron microscopy

1978 ◽  
Vol 34 (1) ◽  
pp. 173-192
Author(s):  
A.M. Glauert ◽  
A.E. Butterworth ◽  
R.F. Sturrock ◽  
V. Houba
Keyword(s):  
Electron Microscopy ◽  
Schistosoma Mansoni ◽  
Phase Contrast ◽  
Structural Changes ◽  
Dense Material ◽  
Muscle Fibres ◽  
Effector Cells ◽  
Sequence Of Events

A characteristic sequence of events has been identified by phase-contrast and electron microscopy during antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro. Human eosinophils initially adhere to the intact schistosomulum and then, in the presence of antibody, flatten and spread very intimately over the parasite's surface. Subsequently, dense material similar to the contents of the lysosomal granules of the eosinophils appears in the extracellular space between the eosinophil and the schistosomulum, probably following fusion of the granules with the plasma membrane of the cell. Eventually all the eosinophils adhering to the parasite are completely degranulated and large amounts of the dense material are observed on the surface of the schistosomulum. This release of granular material from the eosinophils is followed by structural changes in the schistosomulum, starting with vacuolation of the inner layer of the tegument, followed by removal of the tegument, often in the form of large sheets. Subsequently the tegument disintegrates and the fragments are phagocytosed by other eosinophils which have not degranulated. Eosinophils then attach to the exposed muscle layers of the schistosomula and participate in the further degradation of the parasites by phagocytosing fragments of muscle fibres and other cellular components. This sequence of events is compared with published observations of the damage induced by various combinations of antibody, complement and effector cells in vitro, and of cell-mediated damage to schistosomula in vivo, and it is concluded that the observations described in the present paper may reflect a process of destruction of schistosomula in the immune host.

scholarly journals Antibacterial activity of human natural killer cells.

1989 ◽  
Vol 169 (1) ◽  
pp. 99-113 ◽  
Author(s):  
P Garcia-Peñarrubia ◽  
F T Koster ◽  
R O Kelley ◽  
T D McDowell ◽  
A D Bankhurst
Keyword(s):  
Antibacterial Activity ◽  
Nk Cells ◽  
Bactericidal Activity ◽  
Nk Cell ◽  
Close Contact ◽  
Effector Cells ◽  
Ultrastructural Studies ◽  
Human Natural Killer Cells ◽  
In Vitro Effects

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

scholarly journals The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937)

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 652-662
Author(s):  
RJ Kurlander ◽  
JE Gartrell
Keyword(s):  
Cell Line ◽  
Metabolic Inhibitors ◽  
Human Macrophage ◽  
Reversible Binding ◽  
In Vitro Binding ◽  
Irreversible Reduction ◽  
Vitro Binding ◽  
Human Igg1 ◽  
The Relationship

The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I- labeled monomers, dimers, oligomers (composed of 2–4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3–7--fold, and the Ka was decreased 2–4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.

Sign in / Sign up

Export Citation Format

Share Document