Phagocytic Digestion Research Articles

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

Digestive exophagy: Phagocyte digestion of objects too large for phagocytosis.

Mammalian phagocytes carry out several essential functions, including killing and digesting infectious organisms, clearing denatured proteins, removing dead cells and removing several types of debris from the extracellular space. Many of these functions involve phagocytosis, the engulfment of a target in a specialized endocytic process and then fusion of the new phagosome with lysosomes. Phagocytes such as macrophages can phagocytose targets that are several micrometers in diameter (eg, dead cells), but in some cases they encounter much larger objects. We have studied two such examples in some detail: large deposits of lipoproteins such as those in the wall of blood vessels associated with atherosclerosis, and dead adipocytes, which are dozens of micrometers in diameter. We describe a process, which we call digestive exophagy, in which macrophages create a tight seal in contact with the target, acidify the sealed zone and secrete lysosomal contents into the contact zone. By this process, hydrolysis by lysosomal enzymes occurs in a compartment that is outside the cell. We compare this process to the well characterized digestion of bone by osteoclasts, and we point out key similarities and differences.

Phagocytic intracellular digestion in amphioxus (Branchiostoma).

The digestive methods employed by amphioxus (Branchiostoma)—both intracellular phagocytic digestion and extracellular digestion—have been discussed since 1937. Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes. Here, in Branchiostoma belcheri, using a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy throughout all stages of its life. Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostoma, these cells accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles. We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion in Branchiostoma, explain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver.

Dual Role of Mechanisms Involved in Resistance to Predation by Protozoa and Virulence to Humans.

Most opportunistic pathogens transit in the environment between hosts and the environment plays a significant role in the evolution of protective traits. The coincidental evolution hypothesis suggests that virulence factors arose as a response to other selective pressures rather for virulence per se. This idea is strongly supported by the elucidation of bacterial-protozoal interactions. In response to protozoan predation, bacteria have evolved various defensive mechanisms which may also function as virulence factors. In this review, we summarize the dual role of factors involved in both grazing resistance and human pathogenesis, and compare the traits using model intracellular and extracellular pathogens. Intracellular pathogens rely on active invasion, blocking of the phagosome and lysosome fusion and resistance to phagocytic digestion to successfully invade host cells. In contrast, extracellular pathogens utilize toxin secretion and biofilm formation to avoid internalization by phagocytes. The complexity and diversity of bacterial virulence factors whose evolution is driven by protozoan predation, highlights the importance of protozoa in evolution of opportunistic pathogens.

Express FRET Labeling and Analysis of Phagocytic Clearance.

Lysosomal DNase II in phagocytic digestion produces DNA ends with 3'PO4/5'OH, which differ from those created in apoptotic DNA fragmentation, and can be used to label phagocytic clearance of cell death. Here, we describe the use of these specific DNA ends as selective markers of phagocytic reaction in cell suspensions. The approach does not require cell fixation. It selectively labels blunt-ended DNA breaks with terminal 5'OH. The detection is performed by ultra-fast FRET probes in a single step, closed-tube procedure. It takes 3 min and is signaled by fluorescence. The full step-by-step protocol is presented as well as instructions on analysis and representation of the results.The described DNA-end-based phagocytosis marker and the new rapid FRET assay can be useful in studies of phagocytosis, apoptosis and in immune system assessments.

Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium discoideum: social amoebae can also package bacteria.

Many bacteria can resist phagocytic digestion by various protozoa. Some of these bacteria (all human pathogens) are known to be packaged in multilamellar bodies produced in the phagocytic pathway of the protozoa and that are secreted into the extracellular milieu. Packaged bacteria are protected from harsh conditions, and the packaging process is suspected to promote bacterial persistence in the environment. To date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, have been shown to perform bacteria packaging. It is still unknown if social amoebae can do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the grazing of the social amoeba Dictyostelium discoideum and to be packaged by this amoeba was investigated in the present study. The 45 bacterial isolates displaying a resisting phenotype were tested for their capacity to be packaged. A total of seven isolates from Cupriavidus, Micrococcus, Microbacterium and Rathayibacter genera seemed to be packaged and secreted by D. discoideum based on immunofluorescence results. Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were formally packaged. These results show that social amoebae can package some bacteria from the environment revealing a new aspect of microbial ecology.

Permissiveness of freshly isolated environmental strains of amoebae for growth ofLegionella pneumophila

Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera.

Chemotactic and Phagocytic Activity of Blood Neutrophils in Allergic Asthma

Allergic asthma is a chronic inflammatory airway disease, and has been considered a T helper-2-biased response. Studies suggest that neutrophils may be associated with exacerbation and asthma severity. We sought to evaluate the chemotactic activity and phagocytic capacity by peripheral blood neutrophils from individuals with controlled and uncontrolled allergic asthma, and compare the results with non-asthmatic controls groups. Blood neutrophils were isolated from 95 patients: 24 with controlled asthma, 24 uncontrolled asthma, 24 healthy subjects and 23 patients with IgE-mediated allergies other than asthma. The neutrophil chemotaxis, stimulated with LPS, autologous serum or homologous serum, was determined using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by NBT test. The phagocytic digestion phase and chemotaxis by neutrophils from asthmatic patients was higher than in non-asthmatic controls (p < 0.05). Autologous serum-induced neutrophil chemotaxis in patients with uncontrolled asthma was greater (p < 0.05) than in other study groups. The ingestion phase of phagocytosis showed similar values in asthmatics and non-asthmatics. We conclude that the blood neutrophil from controlled and uncontrolled asthmatic patients exhibit activation markers, particularly phagocytic digestion and chemotactic activities.

WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH-null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.

Biogenesis and Proteolytic Processing of Lysosomal DNase II

Deoxyribonuclease II (DNase II) is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected – a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.

Hydrodynamic trapping of Tetrahymena thermophila for the long-term monitoring of cell behaviors

Microfluidic trapping technology has been widely applied for single-cell observation in order to reveal characteristic cell behaviors. However, this strategy has yet to be tested for monitoring highly motile cells, which are often biologically important. In this paper, we seek the conditions that enable effective and long-term trapping of a prominent model ciliate Tetrahymena thermophila within a hydrodynamic microfluidic device. Although motility and flexibility of T. thermophila make it difficult to avoid escaping from the trap, we show that tuning some key parameters in the hydrodynamic circuit was effective to achieve approximately 40 h cell retention, which is long enough to monitor cell behaviors over several generations. Here, we demonstrate the real-time observation of cell division and phagocytic digestion, revealing interesting phenomena such as a wide distribution in doubling time in a poor synthetic medium and heterogeneous time courses in digestion processes. Our results present a strategy for trapping highly motile ciliate cells in order to study the dynamic behaviors of single cells.

The influence of cell-free Lactobacillus rhamnosus GG supernatant on the phagocytic activity of macrophages

Bacteria such as Lactobacillus spp. are classed as probiotics and are already in mainstream use both in hospitals and the consumer market. They have been shown to confer a beneficial effect upon gastrointestinal health when administered in sufficient dosage; however, little is known on how this is achieved. Past studies have shown probiotics improve a number of pathological conditions by interacting with the host immune system and have demonstrated an increase in lysozyme and production of reactive oxygen species in mononuclear phagocytes. With the aim to understand further the bacteria-host interactions, this study endeavoured to test for the up-regulation of phagocytic activity. A gentamicin protection assay was utilized to quantify Escherichia coli ingested by murine macrophages (J774) at 15, 30 and 60 min periods. A multiplicity of infection of 100:1 was adopted with a bacterial concentration of 1 × 10 7 CFU ml −1 and a macrophage cell number of 1 × 10 5 ml −1 . The assay was performed in DMEM alone or DMEM supplemented with either 20 μg ml −1 lipopolysaccharide (LPS) or 10% cell-free Lactobacillus rhamnosus GG (LGG) supernatant. Viable bacterial cells recovered from lysed macrophages at 15 min were negligible for all treatments. Following 30 min incubation, bacterial recovery was observed with 6.4, 5.3 and 3.8 E. coli recovered per macrophage from the control, LPS and LGG supernatant treatments, respectively. After 60 min incubation E. coli recovery remained the same for the control group, but declined by approximately an order of magnitude to 0.6 and 0.5 E. coli per macrophage for the LPS and LGG supernatant treatments. The reduced recovery of E. coli cells from macrophages, when treated with LPS or LGG supernatants, suggests these compounds modulate macrophage activity by enhancing phagocytic digestion of bacterial cells.

Antigen processing of Vibrio salmonicida by fish ( Salmo salar L.) macrophages in vitro

Macrophages from Atlantic salmon (Salmo salar L.) were cultured in vitro and their ability to phagocytose the fish pathogen Vibrio salmonicida was studied. The intracellular degradation of the pathogen was detected by use of monoclonal antibodies against two different surface components of the bacterium, LPS and a 24 kDa molecule. The bacterium itself and the 24 kDa protein antigen were shown to be highly susceptible to phagocytic digestion compared to the more resistant LPS components of the bacterial membrane, which persisted internally for several weeks. The phagocytic and degradative processes were also shown to be inhibited by the ionophore monensin whereas NH4Cl blocked only the intracellular processing of V. salmonicida.

Resistance of Neisseria gonorrhoeae to ingestion and digestion by phagocytes of human buffy coat.

In tests in vitro with the phagocytes of human buffy coat, a recent isolate of Neisseria gonorrhoeae, which was pilated, formed small colonies and resembled the virulent Kellogg type 2 (strain BS), resisted ingestion more than did another isolate (strain AL), which was non-pilated, formed large colonies and resembled the avirulent Kellogg type 4. Some members of both strains survived for significant periods within the phagocytes in test conditions that tended to minimise rather than maximise such survival; and strain BS had a greater capacity for intracellular survival than strain AL, with some of its members surviving for at least 8 h. Resistance to phagocytic ingestion is one important invasive mechanism of gonococci, and resistance to phagocytic digestion may also play a role in pathogenicity.

In-vivo and in-vitro studies of the effect of specific antitoxin on the phagocytosis of cells of Clostridium botulinum type A.

SUMMARY The conversion of heat-resistant spores of Clostridium botulinum type A into germinated heat-sensitive cells in the peritoneal cavity of mice has been observed. This in-vivo germination of spores was depressed in mice passively immunised with type-A botulinal antitoxin. Studies, in which the release of free 45Ca in the urine of mice challenged with labelled Cl. botulinum spores provided an indicator of spore germination, showed that spore germination occurred normally at 8 hr in vivo. However, the rate of germination as determined from 45Ca excretion was substantially inhibited when antibody-treated spores were used as the challenge. In in-vitro studies with guinea-pig leucocytes, spore germination was similarly assayed by monitoring the release of 45Ca from labelled spores. Spore germination in these leucocyte systems was also suppressed when the spores were first treated with Cl. botulinum type-A antitoxic serum, although phagocytic indices for antibody-treated spores and vegetative cells were increased. Release of type-A botulinal toxin was either completely prevented or substantially suppressed when Cl. botulinum spores and vegetative cells were treated with specific botulinal antibody and incubated with leucocytes. These results demonstrate that combination of spores with type-specific antibody prompted phagocytic engulfment of antibody-coated spores, but strongly inhibited subsequent phagocytic digestion of engulfed spores.

Effect of Pyrogen on Phagocytic Digestion and Survival of X-Irradiated Mice

Piromen in doses of 1 mu g per injection administered twice daily for 3, 5, or 7 days significantly increased intracellular digestion of chicken erythrocytes by mouse phagocytes. Similar treatment with 0.1 mu g of Piromen for 5 to 7 days also increased intracellular digestion. However, Piromen in doses of 0.1, 1, or 2 mu g per injection administered twice daily for various intervals prior to and after x irradiation failed to increase survival of x- irradiated mice. (auth)

The Influence of Immunization and Total Body X-Irradiation on Intracellular Digestion by Peritoneal Phagocytes

Summary Total body x-irradiation suppresses intracellular digestion of chicken erythrocytes by the peritoneal phagocytes of mice and rabbits. Such digestive function was normal on the second and eighteenth postirradiation days but was depressed on the sixth through the fourteenth day following 350-r total body irradiation of mice. When the irradiation dose was increased to 450 r, digestion of chicken erythrocytes by mouse phagocytes did not return to normal until the twenty-sixth day. The inhibition of phagocyte digestion of Candida guilliermondi followed the same pattern as observed with chicken red blood cells. Immunization with chicken erythrocytes caused an increase in the digestion of these cells by the phagocytes from mice and rabbits. The immunization-induced increase in digestive capacity was reversed by irradiation procedures that did not appreciably alter serum antibody titers. In vitro tests carried out in the presence of serum from immunized and immunized-irradiated rabbits revealed that the beneficial effect of immunization and the detrimental effect of irradiation on phagocyte digestion were due to alterations in the phagocytic cell and not due to the humoral changes that follow irradiation or immunization.

Source and Fate of the Zooxanthellæ of the Visceral Mass of Tridacna elongata

ZOOXANTHELLÆ have been recorded from the visceral mass of Tridacna by Yonge1, who assigned to them together with those of the mantle edge a very significant role in nutrition. According to this author the zooxanthellæ are transported, by means of the blood-cells via the blood stream, from the mantle edge where they are ‘farmed’ to the visceral mass where they undergo phagocytic digestion.