Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Stimulation of rat peritoneal mast cells induces phagocytosis of adriamycin by rat peritoneal macrophages

Rat and beige mouse peritoneal mast cells, induced to exocytose with the antineoplastic agent adriamycin, extrude their granule remnants in the extracellular medium. These granules are loaded with the fluorescent drug adriamycin and exhibit intense yellow-reddish fluorescent staining. Granules extruded from mast cells were ultimately phagocytosed and could be observed inside the macrophages by fluorescence microscopy. All stages of the internalization process could be followed by electron microscopy. Granules adhering to the cell surface of macrophages were first embraced by short superficial projections, then enveloped by deep surface infoldings, and finally engulfed into the macrophage cytoplasm. Phagocytosis occurred exclusively in macrophages; granules were observed also on the surface of eosinophils and lymphocytes, but never inside these cells. The concentrations of adriamycin in macrophages, measured by spectrofluorimetry, were significantly higher when these cells were incubated with adriamycin and granule remnants in comparison with adriamycin alone. Preincubation with the endocytosis inhibitor cytochalasin B significantly reduced the granule mediated adriamycin uptake. As a consequence of the phagocytosis of adriamycin loaded mast cell granules, macrophages can concentrate the antineoplastic drug. These cells act as reservoirs of adriamycin and could have an important role in both the antitumor and toxic effects of the drug.     

Effect of bicarbonate-based dialysis solutions on intracellular pH (pHi) and TNFalpha production by peritoneal macrophages

The first evidence that higher plants contain annexins was presented in 1989. Since that time, annexins have been purfied and characterized from a variety of plant sources. Analyses of the deduced proteins encoded by annexin cDNAs indicate that the majority of these plant annexins possess the characteristic four repeats of 70 to 75 amino acids and possess motifs proposed to be involved in Ca2+ binding. Like animal annexins, plant annexins bind Ca2+ and phospholipids and are abundant proteins, but there are indications that the number of distinct plant annexin genes may be considerably fewer than that found in animals. Regarding function, a number of studies show that various members of the annexin family of plants may play roles in secretion and/or fruit ripening, show interaction with the enzyme callose (1.3-beta-glucan) synthase, possess intrinsic nucleotide phosphodiesterase activity, bind to F-actin, and/or have peroxidase activity.     

Effects of phagocytosis of mineral dusts on elastase secretion by alveolar and peritoneal exudative macrophages

Peritoneal exudative and alveolar macrophages secrete a nonlysosomal neutral protease which hydrolyses particulate elastin suspended in agar. A variety of particles were administered to macrophages in culture to determine their effect on the secretion of this elastase. Among the particles were silica, two types of asbestos, and kaolinite--all minerals implicated in the production of lung diseases accompanied by reorganization of connective tissue. Peritoneal exudative macrophages increased their secretion of elastase in response to phagocytosis of all the pathogenic particles examined. The increase, however, was not as great as that observed with latex beads, the most inert particles. Although these same particle types were phagocytized by cultured alveolar macrophages, none of them augmented the elastase secretion of alveolar macrophages above the resting level, and many decreased it. The lessened stimulation of elastase secretion by peritoneal macrophages and the decrease in the resting level of elastase secretion of alveolar macrophages probably reflect the cytotoxicity of the particles.     

Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL+/+ lupus-prone mice

The nucleosome, the basic structure of chromatin and normal product of cell apoptosis, plays a pivotal role both in the induction and the pathogenesis of systemic lupus erythematosus (SLE). Nucleosomes have been found to circulate at high levels in patients with SLE and apoptosis of lymphoid cells is increased during human and murine lupus. In this study, we examined the presence of possible defects in clearance mechanisms of apoptotic cells in murine lupus, and questioned further whether nucleosomes could compromise this phagocytic process. There did not appear to be any intrinsic functional defect of macrophages from young MRL+/+ lupus-prone mice to recognize and phagocytose apoptotic thymocytes. Nucleosomes, as a mimic of increased cell apoptotsis in vivo, induced a strong, dose-dependent, inhibition of phagocytosis of apoptotic thymocytes by young, pre-autoimmune, macrophages of MRL+/+ mice, whereas macrophages of non-autoimmune C3H mice only exhibited a trend to inhibition. The nucleosome-elicited inhibitory effect persisted during the development of the autoimmune response and appeared to be specific for the molecular mechanisms involved in macrophage phagocytosis of apoptotic cells. Our data suggest that nucleosome elicited inhibition of phagocytosis of apoptotic cells by MRL+/+ macrophages before the onset of the autoimmune response contribute, in a positive loop, to sustain and/or augment the levels of circulating (and potentially immunogenic) nucleosomes in lupus.     

Concentration-dependent effects of pentoxifylline on migration and myelin phagocytosis by macrophages

The effects of pentoxifylline (POX) on macrophage migration and myelin uptake were studied in an in vitro model of myelin phagocytosis. The POX is a phosphodiesterase inhibitor which inhibits TNF-alpha (tumor necrosis factor alpha) production and reduces ICAM-1 (intercellular adhesion molecule-1) expression by macrophages. Both of these molecules have earlier been shown to be involved in the process of myelin recognition and degradation. In the present series of experiments, cocultured peripheral nerves and macrophages were treated with different concentrations of POX. Untreated controls were massively invaded by macrophages which ingested the degenerating myelin sheaths. High concentrations of POX (100 microg ml(-1)) inhibited macrophage invasion of the nerves. Lower POX concentrations (50 microg ml(-1)), in contrast, lead to an increased myelin uptake by phagocytic cells without affecting macrophage migration. These data indicate that POX may regulate different effector functions of macrophages such as migration and myelin phagocytosis during Wallerian degeneration. This is important for inflammatory demyelinating conditions in the central or peripheral nervous system (PNS) in which macrophages are also important effector cells. Since POX is used as an immunomodulatory drug in demyelinating diseases, its effects on the described macrophage functions may be of high relevance. An increased myelin uptake during Wallerian degeneration may also support a more efficient axonal regeneration by removing axonal outgrowth inhibitors.     

Effect of aging on the tumoricidal functions of murine peritoneal macrophages

The present investigation was carried out to study the effect of aging on the activation of murine peritoneal macrophages by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) to tumoricidal state. Age-dependent alteration in macrophage-mediated tumor cell binding, cytotoxicity, cytostasis and production of the tumoricidal effector molecules oncostatin M, tumor necrosis factor and nitric oxide (NO) was studied. Macrophages (1.5 x 10(5)) obtained from mice of different age groups were separated on the basis of the reproductive status of the mice into prereproductive (young), reproductive (adult) and postreproductive (old) for studying their activation. Macrophages obtained from the old mice were found to be least responsive to the activation signal of LPS + IFN-gamma for macrophage-mediated tumoricidal activity and production of effector molecules. The macrophages of the old mice did not express inducible nitric oxide synthase, an enzyme responsible for the production of NO by activated macrophages. Macrophages of the adult and young groups showed better response; however, optimal response was observed in the macrophages of adult mice. The reasons for the observed difference in the response of macrophages are discussed.     

Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.     

Effect of carotenoids on the respiratory burst of rat peritoneal macrophages

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral-caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.     

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.     

Effect of sodium thiopentone anesthesia on the phagocytic activity of rat peritoneal macrophages

To elucidate the effect of sodium thiopentone anesthesia on the function of phagocytic cells, albino rats were anesthetized with 60 mg/kg. of sodium thiopentone. After 90 min., peritoneal macrophages were harvested and their capacity for superoxide anion generation was detected. Following anesthesia for 90 min. latex particles were injected intraperitoneally, and after additional 30 min. the macrophages were derived, embedded in agar and the number of cells engaged in phagocytosis, as well as the number of latex particles engulfed by each individual cell were counted in semi-thick sections. Macrophages of anesthetized animals showed a statistically significant decrease of both superoxide anion generation and mean number of phagocytic cells, and engulfed fewer particles than those of the controls. Similar results were obtained following incubation of the cells with sodium thiopentone in vitro. The serum corticosterone level in anesthetized rats was significantly higher than that of the control animals. The results indicate that impaired phagocytosis following anesthesia induced by sodium thiopentone, in addition to alterations of the immune system caused by surgical trauma, may be one of the reasons for increased susceptibility to infections of surgical patients during the postoperative period.     

Effect of structural analogs of platelet activating factor on the intracellular signal transduction in murine peritoneal neutrophils and macrophages of P388D1 line

Direct and modulate effects of platelet activating factor (PAF), its structural analogues and ATP on primary and second processes at peritoneal neutrophils and P388D1 cells activation has been studied. The effect of compounds was evaluated on changes in Ca2+ transport and generation of reactive oxygen species. It was shown, that the synthetic analogues of MS series interact with PAF receptor, mobilize Ca2+ from thapsigargin-dependent intracellular stores and inhibit Ca2+ response on PAF in both types of cells. Unlike PAF the analogues do not induce the formations of reactive oxygen species in neutrophils and inhibit the PMA-induced respiratory burst. The activation of pyrinoreceptor of P388D1 cells by exogenous ATP does not inhibit PAF induced Ca2+ rise in cytoplasm, though partly releases Ca2+ from the same store.     

Extracellular and bacterial factors influencing staphylococcal phagocytosis and killing by human polymorphonuclear leukocytes

Extracellular and bacterial factors that influence the phagocytosis and killing of staphylococci by human polymorphonuclear leukocytes have been studied. Staphylococcus epidermidis strains were, in general, more rapidly phagocytized than were S. aureus strains. However, two strains of S. epidermidis had a very slow rate of ingestion. Although the rate of phagocytosis of S. aureus Wood 46 was greater than that of S. aureus 502A, the Wood 46 strain was more difficult to kill. Serum was essential for phagocytosis of both S. aureus and S. epidermidis. The opsonic titer of pooled serum was similar for S. aureus and S. epidermidis. In normal pooled serum, heat-labile factors were more important for effective phagocytosis than they were in immune serum. Although a saturation point for ingestion was reached, the percentage of ingested bacteria that remained alive within the leukocyte remained relatively fixed. Heat-killed and live staphylococci were igested in a similar fashion. The rate of phagocytosis was greatly reduced at 41 degrees C.     

Destruction of allogeneic tumour cells by peritoneal macrophages

The population of peritoneal macrophages from mice immunised with allogeneic tumor cells contains two types of cytotoxic cell. One lyses the specific target cell, the other initiates activation of macrophages and hence leads to inhibition of growth of the specific target cell. The yield of lytic effector macrophages was found to depend on the route and the nature of the cell used for immunisation and the condition of the mice. The yield correlated with the yield of complement-dependent cytotoxic antibodies. In contrast, production of specific "activator" macrophages did not depend critically on these factors. The results underline the difference between the two types of cell and suggest that they are produced independently of one another.     

Phagocytosis and killing of suspended and adhered bacteria by peritoneal cells after dialysis

OBJECTIVE: To determine the effect of dialysis fluid containing various glucose concentrations on the phagocytosis and killing of Staphylococcus aureus by rat peritoneal cells under conditions mimicking the in vivo situation. DESIGN: Phagocytosis and killing were evaluated by quantitation of the killing capacity of macrophages after in vivo phagocytosis of the bacteria as well as by an in vitro flow cytometric assay of the phagocytosis and killing of adhered bacteria by peritoneal cells. ANIMALS: Male Wistar rats. MAIN OUTCOME MEASURE: It was expected that the intraperitoneal administration of dialysis fluid would impair the capacity of peritoneal cells to eliminate bacteria. RESULTS: The first test revealed no effects of glucose concentration or dwell time on the killing of phagocytosed bacteria by macrophages, median percentages ranging between 29% and 64%. In the second series of experiments no effect of glucose concentration on the phagocytosis and killing of adhered bacteria was found either; however, longer dwell times significantly enhanced both the phagocytosis (at a dwell time of 1 hour, under 20%; at dwell times of 4 or 18 hours, above 20%, p < 0.02) and the killing (at a dwell time of 1 hour, under 53%; at dwell times of 4 and 18 hours, above 70%, p < 0.01). CONCLUSIONS: Glucose concentration has no effect on the phagocytosis and killing of Staphylococcus aureus, whereas the dwell time significantly enhances both of these functional capacities of peritoneal cells if the bacteria are adhered to surfaces.     

Essential role of phosphatidylserine externalization in apoptosing cell phagocytosis by macrophages

In many apoptotic cells, phosphatidylserine (PS), that is normally restricted to the inner membrane layer, is externalized and subsequently recognized by phagocytes. However, it has been unclear whether PS externalization is sufficient for phagocytosis induction. In a cultured cell line undergoing Fas-mediated apoptosis, PS externalization preceded other apoptotic events. When transbilayer movement of membrane phospholipids was analyzed, a decrease of the uptake of PS and phosphatidylethanolamine and an increase of phosphatidylcholine incorporation were observed upon apoptosis induction. Apoptotic cultured cells were phagocytosed by macrophages in a manner dependent on externalized PS before plasma membrane permeability increased. Moreover, a N-ethylmaleimide treatment caused PS externalization independent of apoptosis, and such cells underwent PS-mediated phagocytosis. These results suggested that PS is externalized as a result of membrane phospholipid redistribution and externalized PS by itself induces apoptosing cell phagocytosis.     

Nitric oxide-independent killing of Francisella tularensis by IFN-gamma-stimulated murine alveolar macrophages

Alveolar macrophages (AMs) were analyzed for ability to support replication of the intracellular bacterium Francisella tularensis live vaccine strain (LVS). AM supported in vitro growth (2 to 3 logs over 5 days) of LVS with a doubling time of 8 +/- 0.8 h. AMs were analyzed for responsiveness to rIFN-gamma for destruction of this lung pathogen. AM treated with 50 U/ml rIFN-gamma allowed early growth of bacteria (six doublings over 48 h) but between 48 and 96 h rIFN-gamma-treated AM eliminated 1.5 logs of LVS. AMs were sensitive to effects of rIFN-gamma; as little as 5 U/ml rIFN-gamma stimulated AM antimicrobial activity, with half-maximal activity 0.3 U/ml. rIFN-gamma-induced antimicrobial effects in AM correlated with amount of nitrites produced, but nitric oxide played only a minimal role in antibacterial effects induced in AM, because NG-MMLA (specific inhibitor of L-arginine-dependent nitric oxide production) failed to block antimicrobial activity of IFN-gamma-stimulated AM. IL-10, TGF-beta 1, and IFN-alpha (cytokines known to regulate effector functions of activated macrophages) also did not block anti-F. tularensis activity of IFN-gamma-stimulated AM. Reactive oxygen metabolites, depletion of tryptophan, and sequestration of iron did not contribute to anti-F. tularensis activity because addition of superoxide dismutase or catalase, excess iron, or tryptophan to IFN-gamma-treated AM did not reverse the anti-F. tularensis activity observed in these cells. Regulation of AM effector activity differed from that of other macrophage populations, in that while rIFN-gamma-stimulated AM produced TNF-alpha (100 U/ml at 72 h), TNF-alpha was not required as a costimulator for induction of antimicrobial activities by rIFN-gamma because anti-TNF-alpha treatment of rIFN-gamma-stimulated AM blocked TNF-alpha but had no effect on either production of nitrites or anti-F. tularensis activity.     

Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.     

Induction of antigen presentation by macrophages after phagocytosis of tumour apoptotic cells

Due to their resistance to classical chemotherapies, most human colorectal cancers have a high incidence and a poor prognosis. Immunotherapy using interleukin 2 (IL2) has provided disappointing results in the treatment of these cancers. Recently, however, we have demonstrated that a treatment combining a cell-differentiating agent, sodium butyrate (NaBut) with IL2 resulted in a remission of established peritoneal colorectal carcinomatosis in rats. Separately, neither NaBut nor IL2 treatment cured these tumour-bearing rats. NaBut is known to induce cell differentiation and subsequent apoptosis in epithelial cells, while IL2 stimulates the immune cells capable of participating in tumour rejection. We postulated that the significant therapeutic effect of NaBut/IL2 treatment could be attributed to a NaBut-induced increase in the immunogenicity of the cancer cells. We report here that NaBut induced an apoptotic process in rat colon tumour cells in vivo and in vitro. We observed, in an efficient cure, colocalization of apoptotic bodies and monocytes/macrophages at the periphery of the tumour. We propose that these apoptotic bodies are phagocytosed in vivo by the macrophages. We also showed in vitro that a subpopulation of macrophages involved in the phagocytic clearance of apoptotic cells expresses cell surface molecules associated with antigen presentation and stimulates the proliferation of naive splenocytes. Our data suggest that therapies that recruit massive induction of the apoptotic process in tumour cells could favour tumour antigen presentation via their specific phagocytosis by antigen-presenting cells (APCs). We propose that the development of specific therapies that stimulate both tumour cell apoptosis and the immune system could offer new opportunities in anti-cancer treatments of poorly immunogenic cancer cells.