A fluorescence assay demonstrating stimulation of phagocytosis by haemolymph molecules of Gallerta mellonella

An in vitro microscopic fluorescence assay determining the phagocytic activity of isolated plasmatocytes of Galleria mellonella is described. It was developed to quantify insect cellular immune reactions. The assay, a modification of a method originally established for vertebrate blood cells, is based on the quenching effect of trypan blue on fluorescein-isothiocyanate-labelled yeast cells. Only ingested yeast cells retain their fluorescence after quenching and can be easily distinguished from adhering ones. The technique is highly reproducible and easy to perform. Using this method a phagocytosis-stimulating effect caused by a haemolymph fraction > 100 kDa isolated from G. mellonella is demonstrated.     

Effect of age on phagocytosis of carbon in the rat

The half-life of intravenously injected colloidal carbon increases with age of the rat if the dose of carbon is in proportion to body weight. An equation was found for the change in half-life as a function of body weight. By the use of the equation of Gompertz we have derived a curve relating half-life of colloidal carbon to age of the rat. A large part of the variation of phagocytic activity with age can be removed by allowing for the change in relative spleen weight that occurs with age, or by the use of a constant dose of carbon instead of a dose proportionate to body weight.     

CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages

The ubiquitously expressed cell surface glycoprotein CD47 on host cells can inhibit phagocytosis of unopsonized or opsonized viable host target cells. Here we studied the role of target cell CD47 in macrophage uptake of viable or apoptotic murine thymocytes. As expected, IgG-opsonized viable CD47^−/− thymocytes were taken up more efficiently than equally opsonized Wt thymocytes. However IgG-opsonized apoptotic thymocytes from Wt and CD47^−/− mice were taken up equally. Although uptake of apoptotic thymocytes by non-activated bone marrow-derived macrophages was phosphatidylserine (PS)-independent, while uptake by non-activated resident peritoneal macrophages was PS-dependent, both macrophage populations showed a reduced uptake of non-opsonized apoptotic CD47^−/− thymocytes, as compared with the uptake of apoptotic Wt thymocytes. This difference was only seen with non-activated macrophages, and not with β-1,3-glucan-activated macrophages. CD47 promoted binding of thymocytes to macrophages, which did not require F-actin polymerization. CD47 became clustered on apoptotic thymocytes, both co-localized with or separated from, clustered PS and cholesterol-rich GM-1 domains. Thus, CD47 does not inhibit, but rather support, both PS-independent and PS-dependent uptake of apoptotic cells in the murine system. This mechanism only comes into play in non-activated macrophages.     

Cellular and extracellular changes following mast-cell secretion in avascular rat mesentery

Summary Mast cell (MC) secretion induces local cell proliferation lasting 48–72 h in fibroblasts and mesothelial cells in the almost avascular true mesentery of the rat. We studied this membranous tissue by transmission electron microscopy with regard to cellular and extracellular features occurring during the first 72 h following MC secretion.After MC secretion elicited by compound 48/80, apparently all individual tissuebound cells (i.e. fibroblasts, mesothelial cells, and macrophages) show signs of accelerated metabolic activity. In fibroblasts, conspicuous increases in the volume of Golgi apparatus and rough endoplasmic reticulum and in the amount of plasmalemmal indentations suggest an increased production and secretion of the extracellular matrix. Released MC granules lying close to projections of nearby phagocytosing cells cause areas free from electrondense material in the extracellular matrix. MC secretion therefore appears to produce a remodelling of extracellular matrix. Most of the activities initiated by MC secretion start to subside within (48-)72 h.The findings indicate a close functional relationship between the tissue MC and all its neighbouring cells and the surrounding extracellular matrix. The striking chain of events that it induces emphasizes strongly that the secreting MC plays a prominent although as yet in many respects enigmatic role in normal tissue.Key to Abbreviations CM cytoplasmic matrix - GA Golgi apparatus - MC mast cell - PL primary lysosome - RER rough endoplasmic reticulum - TEM transmission electron microscopySupported by grants from the Swedish Medical Research Council, Project 5942     

Isolation and characterization of internalized glioma cell membranes

Summary A method for isolating plasma membranes based on the ability of cultured C6-glioma cells to phagocytize inert material such as polystyrene (latex) beads is described. The beads (Ø 1.1 m) were incubated for 16 h or 5 h. After several washings and homogenization of the cells, the beads with the surrounding membranes were isolated by use of a sucrose density gradient. The membranes were analyzed morphologically and biochemically. Morphological studies by means of light- and electron microscopy confirmed the intracellular localization of the beads. Enzymatic studies revealed that the specific activity of acid phosphatase decreased with shorter incubation periods (from 268.00±38.56 U x mg protein^-1 x min^-1 after 16 h to 125.12 ± 9.10 after 5 h), whereas that of Na, K-ATPase showed the opposite trend (3.63±0.41 and 4.73±0.78 moles phosphate x mg protein^-1 x h^-1, respectively), indicating a lesser contamination with lysosomes. The main advantages of this procedure for membrane studies lie in purity and definite orientation (inside-out) of the membranes.     

Effects of fluoxetine on the activity of phagocytosis in stressed mice

We studied the effects of 1, 2, 5, 10 and 20 mg/kg of fluoxetine on the activity of phagocytosis in mice subjected to a chronic auditory stressor. Both the in vitro and in vivo activity of phagocytosis, measured using the zymosan-particle uptake method and the carbon clearance test, respectively, were reduced after 2, 4, 8 and 16 days of stress exposure. A partial recovery on the in vivo activity of phagocytosis was found on day 16th. Daily treatment with fluoxetine partially reversed the adverse effects of stress in a dose-dependent manner on both parameters but did not significantly affect the activity of phagocytosis in unstressed mice. Significant differences appeared when fluoxetine was administered at 2 mg/kg. Maximum effect was reached at 5 mg/kg.     

Functional impairment of rat Kupffer cells induced by aflatoxin B1 and its metabolites

Abstract Contamination of food with mycotoxins is a major health problem. Impairment of several immune functions has been repeatedly reported in animals fed with contaminated fodder. Since the liver is a major target of toxicity by aflatoxins, the effects of aflatoxins B1, and its hepatic metabolites Q₁ and M₁ on Kupffer cell function was investigated in vitro. Aflatoxin B₁ induced significant ( P < 0.05) inhibition of phagocytosis, intracellular killing of Candida albicans , and intrinsic anti-Herpes virus activity at concentrations as low as 0.01 pg ml⁻¹. Aflatoxin Q₁ and M₁ had similar effects on phagocytosis and microbicidal activity, but were two- to ten-fold less potent than aflatoxin B₁.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, alphaSMA, PDGFR-beta). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ(+) PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.     

Phagocytic inability of Kurloff cells in the lung and spleen of the guinea pig

Summary Adult female guinea pigs received subcutaneous implants of diethylstilbestrol-cholestrol pellets which produced splenomegaly and increased numbers of splenic Kurloff cells. Latex spheres subsequently injected intravenously were not phagocytized by Kurloff cells within the lungs and spleen as examined with the electron microscope. This is considered as evidence that Kurloff cells are probably not phagocytic. The origin of these cells is discussed.     

Plasmodium berghei: Phagocytic activity in two strains of rats

The Wistar and Sprague-Dawley strains of rats differ in their ability to maintain phagocytic hyperactivity toward colloidal carbon during infection with Plasmodium berghei. Both strains were highly stimulated by the 4th day, but on the 7th and 8th days of infection one-third of the Sprague-Dawley rats were not hyperactive while all the Wistar rats remained hyperactive. The course of infection subsequent to the carbon test was similar in hyperactive and in nonhyperactive rats.     

Ultrastructural study of the follicle cells in the freshwater gastropod Viviparus viviparus L.

Summary The morphology and the role of the follicle cells of Viviparus viviparus were examined by means of light and electron microscopy. The follicle cells appear to contain glycogen and fat, and often lysosomes or heterogeneous inclusions. Therefore, they seem to be active in phagocytosis and storage. They are probably involved in the nutrition of the oocyte. Their role in the formation of a selectively permeable barrier is discussed.The authors thank Drs. H.H. Boer, M. de Jong-Brink and J. Wijdenes of the Free University of Amsterdam for their assistance in the translation of this paper     

Degeneration of the tail notochord of Rana temporaria at metamorphic climax

Summary An electron microscopical study has been made of the notochordal cells of the tail of the larval Rana temporaria, before and after they commence to degenerate at metamorphic climax.Degeneration of the tail begins at the tip; the necrotic area proceeds to extend proximally and becomes more widespread. Simultaneously during climax the tail shortens and finally disappears as the animal acquires the froglet form.Notochordal cells autolyse independently and at random, when there is disruption and disorganisation of the surrounding notochordal sheath. The release of lysosomal enzymes intracellularly elicits organelle necrosis.Evidence is provided to support the view that necrotic notochordal tissue is phagocytosed by invading mesenchymal macrophages.     

Lysozyme is stored in the granules of certain haemocyte types in Locusta

Lysozyme has been isolated from the serum of normal adult Locusta migratoria and antibodies were raised in rabbits against this molecule. Immunocytological techniques indicate that lysozyme is synthesized and stored in the two granular hemocyte types of this insect species: the typical granulocyte and the coagulocyte. The release of lysozyme appears to be associated with the process of coagulation.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.