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

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.

The promotion and inhibition of collagen-breakdown in organ cultures of pig synovium: the requirement for serum components and the involvement of cyclic adenosine 3':5'-monophosphate (cAMP).

In many pathological situations connective tissue cells acquire the ability to degrade the macromolecular components of their extracellular matrix. To study the destruction of collagen we used organ cultures of porcine synovial tissue. In the presence of 15% rabbit serum explants shrink considerably during 10-14 days, owing to early loss of interfibrillar material followed by retraction and local destruction of collagen fibres, partly by phagocytosis. These changes, and the release of latent collagenase into the medium, are largely inhibited by cortisol and partially by indomethacin. Collagen destruction can be greatly accelerated by the addition to the culture medium of one of the following: sodium fluoride, 3-isobutyl-1-methylxanthine, dibutyryl cyclic adenosine 3':5'-monophosphate or forskolin; these agents are known to affect cyclic adenosine monophosphate metabolism and our results suggest strongly that a change in the intracellular levels of cyclic adenosine monophosphate is a key-step in the process leading to the increased catabolism of collagen. With these compounds the destruction of collagen is largely extracellular; the histological changes and the increased levels of collagenase associated with the destruction can be prevented by cortisol and, except in the case of dibutyryl cyclic adenosine monophosphate, at least partially by indomethacin. Without serum only 3-isobutyl-1-methylxanthine sometimes causes drastic breakdown of collagen. This model system should be of great benefit in exploring the mechanisms involved in collagen destruction.

Ultrastructural observations on the interaction in vitro between bovine eosinophils and juvenile Fasciola hepatica.

This ultrastructural study has shown that there is a layer of dense flocculent material on the surface of juvenile Fasciola hepatica incubated in vitro with specific bovine antiserum. This material corresponds to the complexes of secreted glycocalyx and bovine antibody previously characterized by fluorescence microscopy. Bovine eosinophils attach closely to those regions of the parasite's surface that are free of flocculent precipitates. This close attachment is followed by degranulation of the eosinophils into the narrow zone between the cells and the parasite. Only in these regions is damage, in the form of vacuolation of the tegument, seen within the juvenile F. hepatica. It is concluded that the inability of bovine eosinophils to kill juvenile F. hepatica in the presence of specific antiserum results from the presence of a protective layer, consisting of antigen/antibody complexes, on the parasite's surface.

The response of human monocytes to interaction with immobilized immune complexes.

The interaction between human peripheral blood monocytes and immobilized immune complexes has been monitored by morphological and biochemical criteria using an established model system previously used with human eosinophils. The model consists of an agar layer containing immune complexes. Monocytes flatten extensively on these layers and make very close contact with the agar surface; cells incubated on control layers, without antibody, are more loosely attached. After 15 min incubation on immobilized immune complexes there is a 'pulse' of lysosomal enzyme release from the monocytes and electron micrographs indicate that extracellular secretion occurs. Simultaneously, two proteins with apparent molecular weights of 50 000 and 25 000 appear at the cell surface as a specific consequence of the interaction with immune complexes. These newly accessible proteins were detected by lactoperoxidase-catalysed iodination, and the major of the two (labelled M3) has similarities to a protein with an apparent molecular weight of 55 000 of human eosinophils (labelled protein 3), which has been shown to be closely associated with the eosonophil Fc receptor. It will be of interest to establish whether a similar association occurs in monocytes.

Mechanism of Fc-mediated interaction of eosinophils with immobilized immune complexes: I. Effects of inhibitors and activators of eosinophil function.

A protein of apparent molecular weight 55000, designated protein 3, becomes newly detectable on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes immobilized in agar layers. The effect of various agents upon this interaction has been determined by monitoring the appearance of this protein by lactoperoxidase-catalysed iodination. Other parameters that have been measured include: the attachment of eosinophils to the agar layers and their subsequent degranulation, as measured by the release of granule peroxidase, and the degree of spreading of the eosinophils, as assessed by electron microscopy. Attachment of eosinophils to antibody-coated layers is inhibited by heat-aggregated immunoglobulin G (IgG), suggesting that this attachment is mediated via eosinophil Fc receptors. In addition, agents, such as the eosinophil chemotactic factor Ala-Gly-Ser-Glu, that enhance the expression of Fc receptors also enhance the appearance of protein 3, while agents, such as hydrocortisone, that inhibit the expression of Fc receptors reduce its appearance. It is concluded that the appearance of protein 3 parallels the expression of Fc receptors. Attempts to block the Fc region of the bound antibody with staphylococcal protein A were not successful. These experiments indicated that the Fc region of bound IgG has different binding sites for protein A and for the Fc receptor. The correlation between the appearance of protein 3 and subsequent degranulation of the eosinophils was confirmed by the use of agents, such as cytochalasin D and levamisole, that enhance both the appearance of protein 3 and degranulation. Conversely, hydrocortisone reduces the appearance of protein 3 and inhibits degranulation. Protein 3 does not appear when eosinophils adhere to agar layers coated with concanavalin A instead of antibody and the eosinophils do not degranulate. Addition of the calcium ionophore A23187, while causing the release of granule peroxidase, does not elicit the appearance of protein 3. These observations provided additional evidence that the appearance of protein 3 is a specific consequence of the interaction of eosinophils with antibody-coated surfaces. The fact that protein 3 appears at the eosinophil surface as a direct consequence of the interaction with antibody suggests that this protein is closely associated with the eosinophil Fc receptor. The enhancement of the appearance of protein 3 in the presence of cytochalasin D indicates that the movement and reorientation of both this protein and the Fc receptor are constrained by association with cytoplasmic microfilaments.

Mechanism of entry and development of Trypanosoma dionisii in non-phagocytic cells.

Cultures of 'buffalo' (bison) lung (BL) cells were infected with epimastigotes or trypomastigotes of Trypanosoma (Schizotrypanum) dionisii derived from cultures in vitro, fixed after various periods of incubation at 37 degrees C and examined by light or electron microscopy. Few if any epimastigotes entered the BL cells, but many trypomastigotes did so; they adhered to the cell surface within 2 h and then appeared to sink into furrows on the cell surface until engulfed in parasitophorous vacuoles. Cytochalasin D (5-10 micrograms ml-1) completely, but reversibly, inhibited entry of trypomastigotes without affecting parasite motility. It was concluded that entry depended on the interaction of stage-specific components on the trypomastigote's surface with receptors on the BL cells, and that this interaction induced active uptake of the protozoa by a phagocytic process not involving pseudopod formation. Soon after entry of the trypomastigotes into BL cells, the membranes of the parasitophorous vacuoles disintegrated and the parasites, which were now lying free in the cytoplasm of the host cell, transformed into amastigotes (micromastigotes) during the next 24-48 h. Replication then occurred, followed by transformation, beginning after 3 days, through a transitional promastigote phase to small intracellular trypomastigotes at 7 days. The promastigotes had a characteristic curved protrusion extending from the lip of the flagellar pocket (or reservoir) into the host cell's cytoplasm. Trypomastigotes, released into the supernatant medium by rupture of the plasma membranes of the BL cells after 8 days, could re-invade other cells.

An ultrastructural study of the interaction in vitro between Trypanosoma theileri and bovine leucocytes.

Bovine neutrophils, eosinophils and macrophages obtained from the mammary gland were cytotoxic to Trypanosoma theileri epimastigotes in vitro in the presence of specific antibody. A detailed ultrastructural study revealed that all types of effector cell phagocytosed T. theileri despite the relatively large size of this parasite (10-130 microns). Phagocytosis proceeded in the classical manner by envelopment of the trypanosome by long pseudopodia. With eosinophils and macrophages structural damage of the parasite was only observed following ingestion. There was evidence of inter-granule fusion in eosinophils prior to the discharge of granule contents into the parasitophorous vacuole; following degradation of the parasite in eosinophils some phagolysosomes opened to the exterior and released what appeared to be trypanosome debris. While eosinophils required less than 30 min to initiate serious damage to the parasite, macrophages required in excess of 2 h to produce similar damage. Conversely, neutrophils caused severe damage within 5 min. An important feature of neutrophil-mediated cytotoxicity was the extensive structural damage to the parasite that was observed before closure of the phagosome. Initially this damage was observed at the end of the trypanosome that was in contact with the neutrophil. This finding suggests that neutrophil-mediated killing of T. theileri is a combination of both extracellular and intracellular mechanisms.

Evasion of the oxidative microbicidal activity of human monocytes by trypomastigotes of Trypanosoma dionisii.

Trypomastigotes of Trypanosoma dionisii, a stercorarian trypanosome from bats, are effectively killed by neutrophils from human peripheral blood but are less sensitive to the cytotoxic action of human monocytes. The mechanism of killing appears to involve peroxidase and hydrogen peroxide. Trypomastigotes are as effective as epimastigotes in inducing the formation of hydrogen peroxide by effector cells. They are, however, less sensitive than epimastigotes to the cytotoxic effect of peroxidase and hydrogen peroxide. They are therefore susceptible to the high concentrations of peroxidase found in the phagosome of the neutrophil, but resist the lower levels encountered in monocytes.

The interaction of human eosinophils and neutrophils with non-phagocytosable surfaces: a model for studying cell-mediated immunity in schistosomiasis.

A model has been developed to simulate the surface of an antibody-coated schistosomulum. It consists of a layer of agar, containing antigen (tetanus toxoid) and a chemotactic factor (ECF). Some layers were coated with human anti-tetanus immunoglobulin. The mode of adherence of human eosinophils and neutrophils to these agar layers and the subsequent degranulation of the cells exactly paralleled the interaction of these cell types with antibody-coated schistosomula of Schistosoma mansoni. In particular, eosinophils made much more intimate contact than did neutrophils, and lysosomal enzymes were secreted extracellularly by direct fusion of granules with the plasma membrane of the cell. Biochemical evidence was also obtained for the secretion of enzymes during degranulation and the rate of enzyme release was found to be enhanced in the presence of specific antibody. This model, non-phagocytosable surface has the potential to provide basic information of the mode of action of effector cells in cell-mediated cytotoxic reactions against a wide range of parasites by incorporation of different factors into the agar layers.

Eosinophil interaction with antibody-coated, non-phagocytosable surfaces: changes in cell surface proteins.

Plasma membrane changes during the interaction of human eosinophils with large, antibody-coated, non-phagocytosable surfaces have been investigated in a model system. Human peripheral blood eosinophils were incubated with layers of agar into which teranus toxoid (ECF), were incorporated. Changes in organization of the eosinophil plasma membrane proteins during interaction with the agar layer were detected by lactoperoxidase-catalysed iodination with [125]iodide. A protein of apparent mol. wt 55 000 became newly accessible on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes in the agar layer. This protein appeared in the early attachnent phase of the interaction which preceded extracellular degranulation. Cytochalasin D enhanced its appearance, while Mg2+-deficiency prevented it. A second newly accessible protein of apparent mol. wt 58 000 was blocked when ECF was present and may therefore be a receptor for ECF. Other proteins of apparent mol. wt 68 000 and 46 000 newly appeared at the surface of eosinophils even after incubation in suspension, apparently as a consequence of the rapid cycling of membrane components which occurs in eosinophils.

Phagocytosis and killing of Trypanosoma dionisii by human neutrophils, eosinophils and monocytes.

The cell-mediated resistance of human leucocytes to Trypanosoma dionisii, a bat parasite related to T. cruzi, was investigated. Human peripheral blood neutrophils and monocytes were cytotoxic to T. dionisii as assessed by electron microscopy and by induction of 99mTc release from trypanosomes pre-labelled with [99mTc] pertechnetate. The enhancement of cytotoxicity by specific antiserum varied considerably from one individual to another. Neither blood lymphocytes nor blood eosinophils induced 99mTc release from T. dionisii. The trypanosomes were readily phagocytosed by neutrophils and monocytes even in the absence of added antiserum but the rate was enchanced when antiserum was present. Eosinophils also phagocytosed T. dionisii but only in the presence of antiserum. Investigation by electron microscopy revealed that T. dionisii is rapidly destroyed in the phagocytic vacuole of enutrophils and monocytes and by eosinophils. Phagocytosis, ultrastructural damage and induction of 99mTc release occurred more rapidly in neutrophils than in monocytes.

Recent advances of high voltage electron microscopy in biology.

Recent advances in the understanding of the structure of biological material made possible by the high voltage electron microscope are reviewed by briefly summarizing some of the more important results obtained in the past few years. The examination of thick sections of selectively stained specimens has continued to be the most widely used approach and has yielded information on the three-dimensional organization of a range of organelles, including the Golgi apparatus, neurofibrils and the transverse tubular system of striated muscle. The alternative method of studying intact cells prepared by critical-point drying is becoming increasingly popular, and has already made a significant contribution in demonstrating the microtrabecular systems within the cytoplasm of cultured cells.

The mechanism of K-cell (antibody-dependent) mediated cytotoxicity. III. The ultrastructure of K cell projections and their possible role in target cell killing.

The cytotoxic interaction between lymphoid K cells from normal rat spleen and antibody-coated P815 mastocytoma cells has been studied in conditions under which the number of cytolytic events occurring at the time of observation was at a maximum. Electron micrographs of material fixed during the first 15 min after contact between the target and effector cells had been initiated by centrifugation showed that the K cells produce long projections which push deeply into the P815 cells, causing infoldings of the plasma membrane and distortion of the nucleus. The plasma membranes of the effector and target cells, and the nuclear membrane, remain intact. Subsequently the target cells undergo violent cytoplasmic blebbing (zeiosis) which is the first stage of cell lysis. The evidence for the hypothesis that projections from lymphoid K cells develop as a result of contact between receptors on the K cell surface and antibody bound to the target cell, and that the projections are involved in the cytotoxic mechanism is discussed.

The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing.

Electron micrographs of material fixed during the first 10 min of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of contact with specific antigen, and are involved in the cytotoxic mechanism is discussed.

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

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.

Demonstration by light and electron microscopy of capsules on gonococci recently grown in vivo.

A study by light microscopy, using Leishman's stain alone or Leishman's stain followed by nigrosin, showed the presence of capsules on gonococci of two strains subcultured from subcutaneous chambers in guinea pigs. With the Alcian blue method of preparation for electron microscopy, gonococci of both strains recently grown in vivo showed densely stained capsules on some cells, while others in the same preparation showed only irregular masses of dense material on their surfaces with strands connecting adjacent bacteria. Treatment with antiserum, complement and conglutinin revealed irregular masses and strands of extracellular material with fixatives that did not contain Alcian blue.

Endotoxin-induced platelet aggregation and secretion. I. Morphological changes and pharmacological effects.

Endotoxin lipopolysaccharide (LPS) from Acinetobacter 199A induced aggregation of blood platelets from immune adherence-positive species (rat, rabbit) but not from immune adherence-negative species such as pig and man. Aggregation occurred in 2 phases: the first was not accompanied by secretion of platelet constituents, was apparently a consequence of C3 activation, and was selectively inhibited by EGTA. The second phase of aggregation was associated with secretion of platelet granule contents, and with a lesser amount of cytoplasmic leakage. Secondary aggregation was abolished by the sulphydryl alkylating agent N-ethylmaleimide, and by agents which increased the level of cyclic AMP in platelets, such as prostaglandin E1 (a stimulator of adenylate cyclase) and methyl xanthines (inhibitors of phosphodiesterase). Secondary aggregation was partly inhibited by agents which block platelet prostaglandin biosynthesis (e.g. aspirin, indomethacin). Primary aggregation was unaffected by these inhibitors at concentrations which blocked secondary aggregation.