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The process of phagocytosis by human platelets was elucidated by monitoring the uptake of small unilamellar liposomes. The results showed that liposome uptake is proportional to the number of liposomes and the incubation time. Further analysis revealed that liposomes are taken up through the open-channel system, and are localized within the acid- and esterase-containing vesicles, where they are degraded, resulting in the secretion of the aqueous components, while the lipids are retained. These results indicate that human platelets are capable of phagocytosis rather than simply sequestering particles.

Phagocytosis in the solitary ascidian Ciona intestinalis was investigated using mixed and separated populations of blood cells in vitro. Only the vacuolar and granular amoebocytes were seen to ingest bacteria, and when the serine protease inhibitors, STI, or benzamidine were added to monolayers of mixed cell types, uptake was significantly reduced. Analyses carried out with isolated cells revealed that phagocytosis was enhanced by incubation of the bacteria in blood cell lysate supernatants (CLS) that had been pre-treated with LPS. By contrast, pre-incubation of the bacteria in CLS preparations that were inhibited by benzamidine produced lower levels of phagocytosis. Treatment of the bacteria with plasma also failed to promote uptake, and there was no detectable agglutination of the bacteria by CLS. As lysate supernatants made from morula cells, but not other cell types, were effective in promoting phagocytosis, we propose that opsonins are derived from the morula cells, and that phagocytosis involves cooperation between different cell populations. Moreover, as the morula cells are the principal repositories of prophenoloxidase and an associated serine protease (factors which are activated by LPS but blocked by STI or benzamidine), prophenoloxidase or the protease may be involved in the opsonic phenomenon in a similar way to that previously reported for arthropods.

Immunocytochemical techniques reveal that phagocytic and endocytic processes in macrophages pass through the successive stages of early endosome, endosome carrier vesicle, mannose-6-phosphate (MPR) enriched endosome/prelysosomal compartment and MPR-negative lysosome. These two processes finally meet at the level of the tubulo-reticular compartment. The addition of a lectin such as wheat germ agglutinin causes gross dilatations and flattening in the Golgi cisternae. Consequently, the transport of secretory proteins is inhibited.

The binding of multivalent immunoglobulin G complexes to Fc receptors ([Fc.sub.γ.Rs]) on macrophages activates multiple immune functions. A murine macrophage cell line, but not a fibroblast cell line, that was transfected with human [Fc.sub.γ.RIIA] mediated phagocytosis and an intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) flux upon cross-linking of human [Fc.sub.γ.RIIA]. Transfected macrophages that expressed a truncated receptor lacking 17 carboxy-terminal amino acids phagocytosed small antibody complexes. However, only wild-type transfectants phagocytosed labeled erythrocytes and fluxed [[Ca.sup.2+]].sub.i]. Thus, the cytoplasmic domain of human [Fc.sub.γ.RIIA] conatins distinct functional regions., FC RECEPTORS ([Fc.sub.γ.Rs]) FOR IMMUNO-globulin G (IgG) link humoral and cell-mediated immune responses. Activation of [Fc.sub.γ.Rs] requires cross-linking of multiple receptors by immune complexes. In neutrophils and macrophages, the binding [...]

Subcellular gradients of cytosolic free [Ca.sup.2]+ concentration, [[Ca.sup.2]+].sub.i], are thought critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [[Ca.sup.2].sub.i] is the accumulation of [Ca.sup.2]+ stores at the site of [Ca.sup.2] action. The distribution of the [Ca.sup.2]+ store marke [Ca.sup.2]+-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [[Ca.sup.2]+].sub.i]. Thus, accumulation of [Ca.sup.2]+ stores in a restricted area of t contribute to the generation of localized increases in [[Ca.sup.2]+].sub.i]., The control of [[Ca.sup.2]+].sub.i] is crucial for the regulation of cellular activity. In many cellular systems, focal, rather than generalized, changes in [Ca.sup.2]+].sub.i] occur, presumably serving to localize functional responses [...]

Immune Diversity Throughout the Animal Kingdom Comparative immunology examines the diversity of immune reactions throughout the animal kingdom. This branch of immunology compares immune mechanisms not only between various groups [...]

The article discusses importance and effects of Atg7-dependent autophagy, a process of cytoplasmic degradation and recycling. The process is found to be extremely beneficial for the adults but is not necessary for metamorphosis in Drosophila tissues.

Autophagy is an intracellular degradation system of the lysosome and also plays a variety of physiological and pathophysiological roles. The process and role of autophagy is discussed in a process-based manner.

The identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b is reported. CRIg represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (Ptdlns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3 K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of Ptdlns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte. Key Words: Drosophila immunity; hematopoiesis; hemocytes; phagocytosis; encapsulation.

Weikert, Laura F., Joseph P. Lopez, Rasul Abdolrasulnia, Zissis C. Chroneos, and Virginia L. Shepherd. Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway. Am J Physiol Lung Cell Mol Physiol 279: L216-L223, 2000.--Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-[Alpha] (TNF-[Alpha]) and nitric oxide were measured at varying times. TNF-[Alpha] and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing. phagocytosis; mycobacteria; surfactant-associated protein A

To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTP[Gamma]S and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, [Alpha]-SNAP, and NSF, while DSP mainly contained tab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)--depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATP[Gamma]S inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF-depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of tab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of [Alpha]-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes. Key words: phagocytosis * reconstitution * fusion * phagosome * Rab

The SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) adapter protein is expressed in T cells and myeloid cells, whereas its homologue BLNK (B cell linker protein) is expressed in B cells. SLP-76 and BLNK link immunoreceptor tyrosine-based activation motif-containing receptors to signaling molecules that include phospholipase C-[Gamma], mitogen-activated protein kinases, and the GTPases Ras and Rho. SLP-76 plays a critical role in T cell receptor, Fc[Epsilon]RI and gpVI collagen receptor signaling, and participates in signaling via Fc[Gamma]R and killer cell inhibitory receptors. BLNK plays a critical role in B cell receptor signaling. We show that murine bone marrow-derived macrophages express both SLP-76 and BLNK. Selective ligation of Fc[Gamma]RI and Fc[Gamma]/RII/III resulted in tyrosine phosphorylation of both SLP-76 and BLNK. [SLP-76.sup.-/-] bone marrow-derived macrophages display Fc[Gamma]R-mediated tyrosine phosphorylation of Syk, phospholipase C-[Gamma]2, and extracellular signal regulated kinases 1 and 2, and normal Fc[Gamma]R-dependent phagocytosis. These data suggest that both SLP-76 and BLNK are coupled to Fc[Gamma]R signaling in murine macrophages. monocytes | phagocytosis

Mariencheck, William I., Jordan Savov, Qun Dong, Michael James Tino, and Jo Rae Wright. Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P. aeruginosa. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L777-L786, 1999.--In this study, we investigate the interaction between surfactant protein A (SP-A) and a live, mucoid strain of Pseudomonas aeruginosa and identify a mechanism of clearance of this organism by alveolar macrophages. [sup.125]I-labeled SP-A bound live, but not heat-killed, P. aeruginosa organisms in a concentration-dependent manner. Unlabeled SP-A bound live bacteria, protein isolated from whole organisms, and specific proteins of the P. aeruginosa outer membrane. The binding of SP-A to P. aeruginosa and outer membrane components was inhibited by either EDTA or mannose. Phagocytosis assays with fluorescent microscopy demonstrated that the percentage of macrophages with internalized FITC-labeled P. aeruginosa was increased 1.8-fold (19 vs. 35%) by pretreating the live bacteria with SP-A. This finding was confirmed by direct visualization of ingested bacteria by electron microscopy. Adhering macrophages to SP-A-coated surfaces attenuated the increased uptake of P. aeruginosa pretreated with SP-A, suggesting that SP-A acts as an opsonin to stimulate macrophage phagocytosis of this strain of P. aeruginosa. Pseudomonas aeruginosa; lung infection; innate immunity; cystic fibrosis

Apoptotic cells are rapidly phagocytosed by macrophages, a process that represents a critical step in tissue remodeling, immune responses, and the resolution of inflammation. In 1998, Peter Henson, Donna Bratton, [...]

The engulfment and elimination of microorganisms by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, involves extensive remodeling of the actin cytoskeleton as well as membrane fusion and fission events. These responses are rapid, transient, and highly localized, which complicates their analysis by conventional biochemical means. We therefore studied the signals involved in phagosome generation by digital imaging of live cells. In particular, we analyzed the role of phosphoinositides using chimeric constructs of PH, PX, FYVE, of C1 domains fused to fluorescent proteins. [PIP.sub.2] was found to undergo a biphasic change at the phagosomal cup: an early accumulation that was rapidly superseded by complete disappearance of the phosphoinositide. The former phase was associated with the accumulation of actin at the cup. Elimination of [PIP.sub.2] was accompanied by generation of [PIP.sub.3] and also by the appearance of diacylglycerol in the sealed phagosome. Of note, disappearance of [PIP.sub.2] coincided with the dissociation of actin from the nascent phagosome. Conditions that precluded the hydrolysis of [PIP.sub.2] resulted in thickening of the submembranous F-actin layer, prevented the dissociation of actin from the cup and arrested particle internalization by impairing scission of the phagosomal vacuole. Some bacteria, including Salmonella, gain entry to nonphagocytic cells by an invasion process akin to phagocytosis. Invading Salmonella elicit massive ruffling of the membrane of the host cell, followed by pinching off of small vacuoles at the base of the ruffles. We found that while [PIP.sub.2] is abundant at the tip of the ruffles, the phosphoinositide is absent from their base, where scission of bacteria-containing vacuoles is occurring. The focal depletion of [PIP.sub.2] from the base of the ruffles is induced by a bacterial protein, SigD/SopB, which is injected into the host cell cytosol by the microorganism. When transfected into mammalian cells, SigD/SopB suffices to deplete cellular [PIP.sub.2] and induces the spontaneous formation of blebs and of large intracellular vacuoles. Transfection of SigD/SopB also alters the cellular F-actin skeleton. Jointly, these experiments suggest that uptake of bacteria or large particles by animal cells requires two phases of actin remodeling: an early stage of actin recruitment and remodeling at the tip of pseudopodia or ruffles, and a secondary phase of actin dissociation which is necessary for membrane fission and vacuole sealing. The early phase requires [PIP.sub.2] and is associated with a local increase in the concentration of the inositide. The second phase is accompanied by, and likely requires removal of [PIP.sub.2]. Thus, localized phosphoinositide metabolism appears to play a critical role in both phagocytosis and bacterial invasion.

Certain species of Chlorella have exploited an intracellular habitat and occur naturally as cytobionts in Hydra viridissima. The algae evoke phagocytosis by Hydra digestive cells and are sequestered in individual phagosomes that migrate to the base of the host cells and resist fusion with lysosomes. The ability to resist digestion is closely correlated with release of extracellular carbohydrate (maltose) by the algae. The established population of algae grows at an average rate equal to or greater than that of the host and a constant population density is maintained. The host regulates algal population density by expelling or digesting excess algae, or by controlling algal cell division. The control mechanism is unknown but can be breached by addition of inorganic ions to the Hydra culture medicum with the result that the algae overgrow the Hydra. The Hydra-Chlorella symbiosis is probably mutually beneficial, but conditions such as prolonged darkness (with or without feeling) can reduce the competitive fitness of the host since this condition results in heterotrophy by the algae at the expense of selected host substrates. The evolution of selective permeability to organic substrates is a major feature of the successful colonization of the intracellular habitat by symbiotic Chlorella.

Determination of Junction Avidity of Cytolytic T Cell and Target Cell THE MOLECULAR MECHANISM OF cell-mediated cytolysis has been the subject of several investigations (1-5). Long-term allospecific human cytolytic T [...]

Intracellular free calcium (Ca.sup.2+i) is thought to be an important secondary messenger for many phagocyte functions including chemotaxis (1), phagocytosis (2), and degranulation (3). Surface stimuli trigger transient changes in [...]

New studies of nematode worms and human cells investigates apoptosis, in which scavenger cells ingest intact dying cells to keep organisms healthy. The study examines the mechanisms of apoptosis and how apoptotic cells reach their destination within phagocytes. It is proposed that genes ced-5 and cd14 are implicated in the process, which involves the tethering of apoptotic cells before the phagocytic cells closes around them.

When the body is short on nutrients, the hydrolysis of triglycerides into fatty acids is upregulated--and so is autophagy, which provides a source of energy from the breakdown of cellular [...]

A giant virus isolated from amoebas infects macrophages by phagocytosis--an entry route typically taken by bacteria (PLoS Pathog. 4, e1000087). Acanthamoeba polyphaga mimivirus is not a typical virus: the double-stranded [...]

According to life history theory tropical birds, with relatively high adult survival and small dutch sizes in comparison with temperate zone birds, should favor their own self-maintenance and survival over that of their offspring when confronted with extra energy demands during reproduction. This trade-off has been subject of an experiment in which we handicapped adults of the tropical house wren (Troglodytes aedon) during the nestling phase by trimming their flight feathers to increase the costs of raising nestlings. To quantify parental effort we determined parental field metabolic rate, nestling feeding frequency and nestling growth. To determine investments in self-maintenance processes we measured parental basal metabolic rate, body mass, and several immune function parameters (bactericidal ability of blood, phagocytosis, and leukocyte counts). Despite similar levels of field metabolic rate, handicapped wrens fed their offspring less frequently resulting in poorer nestling condition when compared with control birds. Parental basal metabolic rate, body mass, and immune parameters did not differ between treatments. These results indicate that when confronted with extra energy demands for work during the nestling phase, tropical house wrens compromise the outcome of the current reproductive event in order to maintain their own body condition and thereby presumably chances for future reproduction.

Many an immunology undergraduate's headache can be traced to memorizing the intricacies of the complement system. Three activation pathways lead to the generation of the C3 converting enzymes, which are [...]

In fighting invading pathogens, the body's defenses include the engulfment of incoming pathogens by macrophages in the blood. This process is known as phagocytosis and involves the recruitment of intracellular [...]

In tissues and organs, epithelial cells form tight barriers that demarcate extracellular compartments. On occasion, one of these cells may undergo apoptosis (programmed cell death). Clearance of apoptotic cells by [...]

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InIB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InIB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, [LIMK1.SUP.-], or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InIB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytqsis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

Phosphoinositide 3 kinases (PI3Ks) * are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5][P.sub.3]) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes.

Phagocytosis of Candida albicans Enhances Malignant Behavior of Murine Tumor Cells PHAGOCYTOSIS OF PARTICLES THE size of red blood cells, mammalian cell nuclei, and microorganisms can be experimentally induced in [...]

Research was conducted to examine the possible role of myristoylated, alanine-rich, C kinase substrate (MARCKS) in macrophage function. Fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. Phagocytosis mediated by different receptors and fluid phase endocytosis in macrophages and mouse embryo fibroblasts were also investigated. Results indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

A study was performed that exposed tilapia, Oreochromis niloticus, to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through intraperitoneal injection for five successive days. The phagocytic cell activities in the fishes were observed since it was believed that impaired activities of phagocytic cells indicated environmental chemical exposure. Results revealed that hypocellularity of immune organs was a more accurate indicator of immunosuppressive chemical exposure in fish than phagocytic activity.

The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis. Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42 and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the different biological consequences that are associated with phagocytosis., Phagocytosis is the process by which cells recognize and engulf large particles ([is greater than] 0.5 µm) and is important to host defense mechanisms as well as to tissue repair [...]