The role of plasma membrane receptors and the kinetics of macrophage activation by lymphokines encapsulated in liposomes.

The kinetics of activation of tumoricidal functions in mouse macrophages incubated with macrophage-activating factors (MAF) released by mitogen-stimulated lymphocytes (free MAF) and MAF encapsulated with liposomes (liposome-MAF) have been compared. Development of tumoricidal activity requires incubation of macrophages with free or liposome-encapsulated MAF for a minimum of 4 hr. Macrophages incubated with MAF for 4 hr were not cytotoxic when tumor target cells were added immediately after removal of MAF, but they were highly cytotoxic when allowed to complete a "lag" phase before being exposed to tumor cells. The duration of the lag phase varied with different activation protocols. The levels of cytotoxic activity induced by liposome-encapsulated MAF was consistently higher than that obtained with free MAF. Studies using inhibitors of endocytosis demonstrated that internalization of the liposome carrier is required for activation by liposome-MAF and that activation does not result from MAF leaking from liposomes and binding to MAF receptors on either the plasma membrane or the membrane of endocytic vesicles. Comparison of the efficiency of macrophage activation by MAF encapsulated in liposomes of differing internal volume revealed that large multilamellar and large unioligolamellar liposomes were more efficient in activating peritoneal exudate macrophages than were small unilamellar liposomes. Measurement of the volume of liposome contents internalized by macrophages from these three types of liposomes revealed that maximum cytotoxicity required internalization of a given volume of MAF-containing lymphocyte supernatants, after which no further increase in cytotoxicity occurred.

Morphological evidence for the translocation of lysosomal organelles from cytotoxic macrophages into the cytoplasm of tumor target cells.

In this study we have recorded in detail the morphological sequence of interactions between activated macrophages and tumor target cells in vitro. The study is unique because it involves the combination of several microscopic techniques that are used sequentially to study a single cell-to-cell interaction. Many such cellular interactions were examined first by time lapse cinematography; then the effector cell was identified by specific immunofluorescence, and the areas of interaction were processed for scanning and then transmission electron microscopy. The tumor target cells were guinea pig line 10 hepatocarcinoma cells. The effectors were peritoneal exudate cells (PEC) harvested from syngeneic strain 2 guinea pigs that had been cured of a line 10 tumor by intratumoral injection of Bacillus Calmette-Guérin. Host-target cell interactions between (a) line 10 cells and PEC from Bacillus Calmette Guérin-tumor-cured animals, (b) line 10 cells and PEC from normal animals and (c) syngeneic guinea pig embryo cells and PEC from Bacillus Calmette-Guérin-tumor-cured animals were studied. These comparisons demonstrate that the mechanism of tumor cell killing by activated macrophages is a nonphagocytic process. Our results suggested that the macrophage-tumor cell interaction is initiated by a recognition phase that results in extracellular release of lysosomes through macrophage exocytosis and clasmatosis. Neoplastic target cell susceptibility may be the result of an active or passive uptake of lysosomes and consequently cytolysis.

Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

Viral diseases of fish: first report of carp pox in golden ide (Leuciscus idus) in North America.

Carp pox, a putative viral disease exotic to North America, occurred in golden ide 1 yr after the fish were imported into the United States from the Federal Republic of Germany. The raised, white, plaque-like lesions, which occurred on about 5% of the fish, healed spontaneously and caused no mortality. Electron micrographs showed herpesvirus-like particles associated with lesion specimens; however, no infectious viruses were detected in tests with seven warmwater fish cell lines.

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

Movement of cell surface immunoglobulin (Ig) in guinea pig B cells and lymphocytic leukemia cells as observed by light microscopy and scanning and transmission electron microscopy.

The distribution and regeneration of immunoglobulin (Ig) of guinea pig leukemia cells were investigated through the use of ferritin labeling and scanning electron microscopy. Throughout this work, correlative light microscopy using fluorescein label and transmission electron microscopy using ferritin label were used. The cells used in this study were lymphocytic leukemia cells, an acute (L2C) and a chronic (KSL) form, and normal B cells obtained from Sewall Wright strain 2 guinea pigs. Distinct differences in the movement and regeneration of cell surface Ig were observed when these cells were compared. Both L2C and KSL cells were slower to cap than normal B cells which formed a well-organized single patch. The cells endocytosed the label rapidly and either processed or shed the label within 24 hours except for the L2C cells which had retained internalized label after 24 hours. Regeneration of surface Ig was clearly demonstrated in all three cell types. The apparent similarities between these two lymphocytic guinea pig leukemias and similar reports of human leukemias strongly suggest that the L2C and KSL cells could provide excellent models for future studies of acute and chronic forms of this disease.

Preservation of multilamellar lipid vesicles (liposomes) for ultrastructural studies.

The purpose of these studies was to determine the optimal conditions for the preparation of multilamellar phospholipid vesicles (liposomes) for microscopic studies. Multilamellar vesicles (MLV) were prepared from two naturally occurring phospholipids: phosphatidylcholine and phosphatidylserine admixed at a 7:3 mol ratio. Several techniques including light microscopy, negative staining, thin sectioning, transmission and scanning electron microscopy, and freeze fracture replication were used to study the morphology of the liposomes. Rapid fixing in 1% osmium tetroxide in cacodylate buffer was most suitable for preparation of the MLV for examination and photography by light microscopy. Optimal preservation of MLV for both scanning and transmission electron microscopy was achieved by fixing with glutaraldehyde followed by osmium-thiocarbohydrazide-osmium and uranyl acetate or by glutaraldehyde-tannic acid-osmium and uranyl acetate. These techniques appear to protect the structure of the liposomes from damage by organic solvents which are often used in the preparation of samples for electron microscopy.

Scanning immunoelectron microscopy for the identification and mapping of two or more antigens on cell surfaces.

This study demonstrates the feasibility of using scanning electron microscopy for the identification and mapping of two or more antigens on cell surfaces. Three types of cells: human type B red cells, tumor cells and their associated virus, and guinea pig hepatocarcinoma cells were immunolabeled with ferritin or gold conjugated to IgG fractions of antigen-specific antisera. Gold particles, 300-500 A in diameter, were conjugated to an IgG fraction either directly or indirectly using Staphylococcal protein A. Gold particles of this size can be distinguished from ferritin label using a high resolution scanning microscope. Correlative light, transmission electron microscopy, and scanning electron microscopy studies, as well as x-ray diffraction analysis and the study of stereo micrographs, were performed to visualize the simultaneous immunolabeling by ferritin and gold of two antigens on the guinea pig hepatocarcinoma cell.

Kinetics and ultrastructural studies of the induction of rat alveolar macrophage fusion by mediators released from mitogen-stimulated lymphocytes.

Treatment of F344 rat alveolar macrophages (AMs) in vitro with cell-free supernatant fluids obtained from concanavalin-A (Con A)-stimulated syngeneic lymphocytes induced extensive fusion. The lymphokine responsible for the fusion of AMs (but not other cells) is here referred to as AM fusion factor (Con-A-MFF). Fusion is dependent on the dose of Con-A-MFF and the population density of AM cultures and occurred 10 hours after Con-A-MFF was added to cultures of normal AMs. Con-A-MFF must interact with AMs for more than 8 hours before full expression of fusion is reached at 24 hours. Using a technique allowing for sequential scanning to transmission electron microscopy analysis of cells, the authors determined the relationship of the morphologic characteristics of the surface and the internal structure of cells fusing to form multinucleate giant cells (MGCs). The process of AM fusion begins with the aggregation of AMs, followed by interdigitation of cell processes. Serial sections of MGCs showed lysosomes associated with remnants of plasma membrane in the cytoplasm. The MGCs contained numerous organelles associated with increased secretory activity of cells.

Ultrastructural studies of the interaction between liposome-activated human blood monocytes and allogeneic tumor cells in vitro.

Human blood monocytes were activated to become tumoricidal by incubation with liposomes containing muramyl tripeptide-phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide. The interaction of both tumoricidal and control monocytes with target melanoma cells was analyzed by means of light microscopy and scanning and transmission electron microscopy. The authors found increased clustering around the melanoma cells by tumoricidal monocytes as compared with the control monocytes. The initial clustering of the tumoricidal monocytes around the tumor cells was followed by the establishment of numerous focal points of contact (binding), some of which actually exhibited areas of discontinuous membrane, a finding confirmed by stereophotography. By 24-48 hours of cocultivation, many of the target cells exhibited zones of vacuolation in the immediate vicinity of the tumoricidal monocytes, suggesting target cell damage. (This finding was confirmed by time-course cytotoxicity assays.) The authors conclude that tumor cell lysis mediated by activated human blood monocytes occurs as the final step in a process that includes the establishment of a direct cell-cell contact, damage to the target cell membrane, and the development of areas of vacuolation in the target cells.

The immunoglobulin-like T-cell receptor. I. In situ demonstration of immunoglobulin Fab-region determinants of rodent T- and B-lymphocytes using chicken antibodies.

Antibodies were raised in chickens to the (Fab')2 fragment of normal murine IgG and to the k-myeloma protein MOPC 41. Following appropriate absorptions or purification by immune affinity chromatography the chicken antibodies bound specifically to both T- and B-cells of mice, rats and guinea-pigs as detected by quantitative cytofluorescence, radioactive binding assays and transmission and scanning immunoelectronmicroscopy. These antibodies are directed against polypeptide determinants of the Fab fragment, block antigen binding by purified idiotype-bearing murine antibodies, and provide useful probes for visualization and analysis of immunoglobulin-like surface receptors of rodent B- and T-cells.