Surface Of Phagocytes Research Articles

Professional and non-professional phagocytes: an introduction

Phagocytosis is the uptake by the cell of relatively large particles (>-OS pm) into vacuoles, by mechanisms that are clathrin independent and usually re- quire actin polymerization’ J. It is trig- gered by the interaction of certain mol- ecules on the particle surface with re- ceptors, lectins or other molecules on the phagocyte surface. Particles may be recognized directly by these receptors, but in many instances recognition is mediated by opsonins, such as anli- bodies, which coat the particles and are themselves recognized by specific receptorG. The vesicles containing the ingested material - phagosomes- ma- ture into phagolysosomes, which are acidic and rich in hydrolytic enzymes. In the Metazoa, phagocytosis underlies the uptake and degradation of microorganisms, damaged or senescent cells, and particulates such as pollutants, by specialized phago- cytic cells. Thus, phagocytasis is cen- tral to host defence against infective agents, and to tissue remodelling and inflammation. Paradoxically, phago- cytosis is also a common mechanism by which ‘ intracellular’ microorganisms invade host cells and thus avoid direct destruction by serum antibodies and complementor by cytotoxic celW. Phagocytosis is related to pino- cytosis, the process responsible for the cellular uptake of fluid, solutes, col- loids, macromolecules, virusesorsmall particles, and for bulk internalization of the plasma membrane. In contrast to phagocytosis, pinocytosis takes place constitutively, is usually clathrin dependent and generally does not require actin polymerization. The minimum particle size necessary to trigger phagocytosis or the maximum size for pinocytosis has not been defined, but size alone is probably only one of the features that deter- mine the way a particle is taken UP’ ,~. The molecular mechanisms of receptor-mediated pinocytosis are much better known than those of phagocytosis. One reason for this is that molecular ligands are usually quite specific, whereas complex par- ticles such as bacteria are often rec- ognized by several receptors, some of them incompletely characterized. In addition, whereas a wide range of cells can be used to study pinocytosis, work on phagocytosis has been primarily performed with polymorphonuclear granulocytes (PMNs), monocytes, pri- mary rmacrophage cultures or mono- cyte-macrophage-like cell lines; all these cells are difficult to transfect, dis- play several phagocytic receptors that can act cooperatively, and respond to

Phagocytosis.

Phagocytosis is the process of recognition and engulfment of microorganisms or tissue debris that accumulate during infection, inflammation or wound repair. This ingestion, which is performed most efficiently by migrating, bone marrow-derived cells called 'professional phagocytes', is essential for successful host defense. Ingestion results when an invading microorganism is recognized by specific receptors on the phagocyte surface and requires multiple, successive interactions between the phagocyte and the target. Each of these interactions results in a signal transduction event, which is confined to the membrane and cytoskeleton around the ligated receptor and which is required for successful phagocytosis. Many molecules found at sites of inflammation or infection stimulate phagocytosis, so that efficient ingestion is confined to the site of infection or inflammation, which in turn limits the proinflammatory and tissue-destructive processes that accompany phagocytosis. This review summarizes current understanding of this critical component of host defense and of its regulation.

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): An ultrastructural study

The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: 1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. 2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlapping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. 3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. 4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. 5) Intracellular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals.

Fibronectin, hemostasis and phagocytosis

On the basis of experimental and literature data a hypothesis is proposed that plasma fibronectin serves as a mediator between hemostasis and RES. Fibronectin is thought to be an opsonic factor in the elimination of throm- bogenic particles from blood flow. As an example of thrombogenic particles soluble fibrin precursors covalently bound to plasma fibronectin are examined. Interrelated chages of plasma fibronectin level, DIC signes, and alterations of the Kupffer cells morphologic- and-functional state are in favor of the hypothesis. The idea is also proved by the presence of specific protein receptors for fibronectin on the surface of mononuclear phagocytes and neutrophils. It is suggested that plasma fibronectin and phagocytes represent a united antithrombotic mechanism which prevents excessive intravascular activation of hemostatic system in pathology.

Measurement of opsonic phagocytosis by human polymorphonuclear neutrophils

Publisher Summary This chapter discusses the measurement of opsonic phagocytosis by human polymorphonuclear neutrophil (PMN). Phagocytosis is recognized as a primary and critical cell function in host defense. In higher vertebrates, such as humans, this function is performed largely by “professional” phagocytes like the PMN. Termed opsonins (from the Greek opsonion , “a relish, seasoning, or sauce”), these humoral factors bind to the surface of foreign material and, engage receptors on the phagocytic cell surface that initiate the endocytic (phagocytic) process. Measurements of phagocytosis of microbial parasites by phagocytic cells such as PMNs have employed a wide variety of techniques that can be broadly divided into two main categories: (1) direct measures of phagocyte-target interactions and (2) indirect measures that quantitate metabolic events or biochemical markers associated with ingestion and cellular activation, such as lysosomal enzyme release, superoxide anion (O 2 -) production, and chemiluminescence. The three direct techniques for the measurement of phagocytosis are presented. The first is a novel flow cytometric assay that uses unfractionated whole blood. The second, a fluorescence microscopic method compatible with a wide variety of microorganisms, employs cytocentrifugation and cell fixation. The third is an adaptation of the oil red O method originally described by Stossel in 1973 to the user- (and technology-) friendly 96-well microtiter plate format.

Increased Fc alpha R expression and IgA-mediated function on neutrophils induced by chemoattractants.

The recent reports of receptors for IgA on blood and mucosal phagocytes suggest a more active role then previously thought for IgA in host defense. Using a mAb and flow cytometry we determined the expression of the Fc alpha R on resting and chemoattractant-stimulated blood neutrophils and compared them with mucosal neutrophils. Fc alpha R expression on blood neutrophils could be rapidly up-regulated in vitro, increasing three to fourfold within minutes of exposure to the chemoattractants FMLP (optimal at 10(-8) M) and zymosan activated serum (a source of C5a). The level of Fc alpha R expression found on neutrophils obtained from bronchoalveolar lavage of cystic fibrosis patients with chronic lung infection was almost identical to that found on blood neutrophils from the same patients maximally activated in vitro. The rise in Fc alpha R expression induced by 10(-8) M FMLP was rapid, with a plateau in 15 to 20 min, and was not inhibited by 10 mg/ml of cycloheximide or puromycin, suggesting that the mechanism of up-regulation involves translocation from intracellular storage pools. Ionomycin (2 mM) plus 1.2 mM CaCl2 also increased expression of Fc alpha R, and its effects were inhibited by EDTA. Trifluoperazine, an inhibitor of calmodulin and/or protein kinase C-dependent processes, blocked the increase in Fc alpha R expression induced by FMLP, but the effects of the chemoattractant were not blocked by EDTA, suggesting that intracellular stores of calcium are important in the physiologic regulation of Fc alpha R expression. Neutrophil superoxide production could be induced by aggregated IgA, and was increased if the neutrophils were pretreated with FMLP, correlating with the increase in Fc alpha R expression. The superoxide response to a suboptimal dose of aggregated IgG was unaffected by FMLP pretreatment, and antibodies to Fc gamma R failed to block the superoxide production induced by IgA. Thus, the increase in phagocyte surface expression of Fc alpha R induced by chemoattractants in vitro and on mucosal cells in vivo, and the concomitant increase in IgA-mediated function may greatly facilitate the defense of mucosal surfaces.

Phagocyte recognition of cells undergoing apoptosis

A key feature of apoptosis is that cells undergoing this programmed form of death are recognized by phagocytes and ingested while still intact, protecting tissues from the potentially harmful consequences of exposure to the contents of the dying cells. This article reviews recent data which indicate that phagocyte recognition of apoptotic cells as ‘senescent-self’ involves at least three classes of receptors on the phagocyte surface, while apoptotic cells may display their ‘edible’ status in a number of different ways.

Soluble CD14 participates in the response of cells to lipopolysaccharide.

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis.

C1q binds to and elicits cellular responses by several cell types, including monocytes, macrophages, neutrophils, B cells, and fibroblasts. The cell-binding domain is located within the collagen-like pepsin-resistant region of the C1q molecule (C1q tails). An affinity matrix of C1q tails coupled to Sepharose was used to select C1q-binding proteins from detergent extracts of surface-iodinated human monocytes, polymorphonuclear leukocytes, and the U937 cells. The major radiolabeled polypeptide eluted specifically from the ligand affinity column had an apparent molecular mass (Mr) of 126,000. Minor iodinated components eluted from Sepharose-tails migrated with Mr of 216,000 and 55,000. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions no change in the migration of any of these polypeptide bands was detected. None of these polypeptides reacted with antibodies directed against the integrins alpha 5 beta 1 (fibronectin receptor) or alpha v beta 3 (vitronectin receptor), LFA-1, or to several other cell adhesion molecules. The Mr 126,000 band was found to contain more than one polypeptide. Lectin binding properties, susceptibility to glycosidases and proteases, and immunoreactivity with the monoclonal antibody L-10, indicated that CD43 (sialophorin/leukosialin) is a component of this band. However, further data show that a monoclonal antibody, generated by immunization with the isolated Clq-binding fractions, recognizes a cell surface sialoglycoprotein distinct from CD43 and inhibits the C1q-mediated enhancement of phagocytosis in monocytes. These latter observations provide the first definitive connection between a specific phagocytic cell surface protein and a known C1q-mediated function. While these proteins contain sialic acid, binding assays and functional assays using neuraminidase-treated cells demonstrate that the functional interaction between C1q and the cell surface is not via sialic acid. The data taken together indicate either that the functional C1q receptor on phagocytic cells is a multi-subunit complex or that multiple proteins can interact with the fragment of C1q containing the cell-binding domain, at least one of which is involved in the C1q-mediated enhancement of phagocytosis.

Purification, biochemical composition, and biosynthesis of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes.

Mo3 is an activation Ag expressed on the surface of human mononuclear phagocytes stimulated in vitro or in vivo by various activating factors. Mo3 is obtained by immunoprecipitation with anti-Mo3 mAb from lysates of PMA-stimulated U-937 cells. The Ag is a heterogeneous glycoprotein with a molecular mass range of 42 to 66 kDa (nonreducing conditions) containing N-linked carbohydrate chains. When the cells are treated with phosphatidylinositol-specific phospholipase C, greater than 60% of total precipitable gp42-66 Ag is released in the supernatant. This phosphatidylinositol-specific phospholipase C-sensitive linkage to the plasma membrane has provided a means for the one-step purification of Mo3 by immunoaffinity chromatography. The eluted soluble Mo3 (sMo3) was greater than 90% pure as documented by the appearance of a single major protein peak on reverse phase HPLC and SDS-PAGE. The average yield was 12.1 micrograms/10(8) cells. Sufficient quantities of sMo3 have been purified to permit the determination of amino acid and carbohydrate composition. Complex N-linked carbohydrates make up nearly 50% of the glycoprotein content and contribute to its heterogeneity. An anti-Mo3 polyclonal antiserum generated from sMo3 was used to immunoprecipitate Mo3 and its precursor from biosynthetically labeled, PMA-stimulated U-937 cells or LPS-stimulated monocytes. These 35S-methionine "pulse-chase" experiments demonstrated the existence of a 40- to 42-kDa endo-beta-N-acetylglucosaminidase-sensitive precursor, which over a period of 4 to 5 h gave rise to an endo-beta-N-acetylglucosaminidase-resistant, but N-glycanase-sensitive 42- to 66-kDa mature form.

Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.

Immunological aspects of fungal pathogenesis

The incidence of infection with the pathogenic fungi continues to escalate, especially in the era of the acquired immune deficiency syndrome. To the clinician, this heterogeneous group of organisms poses both a diagnostic and a therapeutic challenge. Consequently, growing numbers of investigators are seeking to elucidate the pathogenetic mechanisms involved in disease caused by medically important fungi. In this review, many of the recent scientific advances that have been made in the immunological aspects of the pathogenesis of fungal infections are presented. The topics covered include 1) the receptors for fungi on the surface of professional phagocytes; 2) the mechanisms for killing and growth inhibition of fungi by phagocytes; 3) the means by which fungi evade host defenses; 4) the role of humoral immunity in fungal infection; 5) immunoregulation in fungal infections; and 6) the influence of cytokines on host defenses against pathogenic fungi.

Flow cytometry for the study of phagocyte functions.

Phagocytes play a key role in host defense, and cell defects are associated with increased susceptibility to infection. Flow cytometry offers rapid and reproducible measurements of single cells in suspension and, following staining with one or more fluorochromes, simultaneous examination of several cell functions. Subpopulations of cells can be identified and sorted for morphologic, biochemical, and functional examination, and specially adapted computer systems allow storage of data for subsequent detailed analysis. Several flow-cytometric assays for the study of phagocytes and their interactions with microorganisms have been developed. These assays facilitate the study of (1) phagocyte surface receptors and regulatory molecules; (2) membrane potential; (3) phagocytosis of microorganisms, including the discrimination between attachment to the phagocyte surface and actual internalization; (4) phagosomal pH; (5) degranulation and enzymatic activity; (6) intracellular calcium; (7) oxidative metabolism; (8) intracellular killing of microorganisms; (9) degradation of microorganisms; and (10) exocytosis. In addition, the influence of serum opsonins on phagocyte-microorganism interactions can be studied. Flow-cytometric techniques are applicable to both experimental and clinical work.

Immunocytochemical discovery of the 22- to 23-Kd subunit of cytochrome b558 at the surface of human peripheral phagocytes

A monoclonal antibody raised against cytochrome b558 reacted specifically with the 22- to 23-Kd protein, the small subunit of this cytochrome. Cytochemical studies showed that the epitope was located on the surfaces of human neutrophils and monocytes. The small subunit of cytochrome b558, therefore, was expressed at least in part on the outer surface of these cells.

Immunocytochemical Discovery of the 22- to 23-Kd Subunit of Cytochrome b at the Surface of Human Peripheral Phagocytes

A monoclonal antibody raised against cytochrome b558 reacted specifically with the 22- to 23-Kd protein, the small subunit of this cytochrome. Cytochemical studies showed that the epitope was located on the surfaces of human neutrophils and monocytes. The small subunit of cytochrome b558, therefore, was expressed at least in part on the outer surface of these cells.

IFN-gamma plus glucocorticoids stimulate the expression of a newly identified human mononuclear phagocyte-specific antigen.

Glucocorticoid hormones, although able to exert profound immunosuppressive effects, do not suppress mononuclear phagocyte activation by IFN-gamma and may even enhance it. For example, expression and functional activity of the high affinity FcR for IgG on human mononuclear phagocytes (FcR gamma I) is increased by IFN-gamma and is maximal after co-treatment with IFN-gamma plus the glucocorticoid dexamethasone (DEX). To determine whether there are other mononuclear phagocyte surface Ag that are regulated in this manner, hybridomas were prepared using IFN-gamma-plus-DEX-treated human monocytes as immunogen. Five IgG1 mAb (Mac 2-8, 2-38, 2-48, 2-49, and 2-158) were developed that recognize a trypsin-sensitive mononuclear phagocyte-specific surface Ag of Mr 155,000. There was no detectable reactivity of these mAb to lymphocytes or granulocytes or to several cell lines, including U-937 and HL-60. The p155 Ag was detected on monocytes and increased significantly with time of culture or after treatment with DEX. Expression was maximal after co-treatment with rIFN-gamma plus DEX, but was inhibited or unaffected by treatment with IFN-gamma alone. For freshly isolated cells, expression of the p155 Ag was highest on peritoneal macrophages. Our results indicate that the p155 Ag is a newly identified Ag of the human mononuclear phagocyte lineage and may represent, in the least, a phenotypic marker of monocyte differentiation or maturation.

Resistance of spores of Aspergillus fumigatus to ingestion by phagocytic cells.

Phagocytic cells are believed to have an important role in the eradication of fungal spores from the lung. The ability of human and mouse cells to phagocytose the opportunistic fungus Aspergillus fumigatus has been examined, spores of the non-pathogenic fungus Penicillium ochrochloron being used for comparison. Most spores became associated with cells. Those of A fumigatus appeared to remain bound to the surface of the phagocyte rather than being ingested; in contrast, P ochrochloron spores appeared to be phagocytosed more readily, although they also were seen, in small numbers, o n the cell surface. In view of the subjective nature of these observations, the effects of spore diffusates on phagocytosis were examined. Diffusates from spores of A fumigatus were shown to inhibit phagocytosis of antibody coated radiolabelled sheep red blood cells by primed mouse phagocytic cells. Diffusates of spores of P ochrochloron had no such effect. These results suggest that when spores of A fumigatus become bound to the surface of phagocytes they are able to release a substance that inhibits their ingestion while having little or no effect on surface binding.

Soluble immune complex triggering of a respiratory burst in macrophages: the role of complex aggregation at the phagocyte surface.

The capacity of small, soluble immune complexes (SIC) to rearrange into larger aggregates, through additional antibody-antigen bridge formation, on a receptor-bearing cell surface has previously been shown to play an important role in enhancing the uptake and ingestion of SIC by macrophages and has been implicated in the efficient clearance of SIC in vivo. In this report, we extend the study to investigate the role of SIC aggregation in triggering a respiratory burst in macrophages. SIC were found to be more efficient trigger signals than nonaggregating, antigen-free IgG oligomers of similar average size in that they induced a more rapid respiratory burst response that was between 2- and 6-fold greater in magnitude. The implications of these findings are discussed.

T cell and mononuclear phagocyte populations of the human small and large intestine.

Considering the diverse roles subsumed by the intestinal mucosal immune system, namely defense against potential luminal pathogens and maintenance of immune tolerance to dietary antigens, our knowledge of the local adaptations of immunocyte subpopulations remains sparse. In the past few years, the range of monoclonal antibodies to lymphocyte and mononuclear phagocyte (Mph) surface differentiation antigens has greatly increased. Such reagents, often shown capable of distinguishing between functional cell subpopulations, should therefore provide potent tools for the in situ analysis of the gut mucosal immune system.

Effect of emetine and chloroquine on phagocytic processes of rat macrophages

Emetine and chloroquine caused a dose-dependent and time-dependent inhibition of bacterial phagocytosis in rat peritoneal macrophages. The effect of emetine was found to be stronger: 50% inhibition was achieved at 5 × 10 −6 M concentration. The inhibition of phagocytosis was irreversible. Neither the binding of bacteria to the surface of phagocytes nor membrane marker 5' nucleotidase was touched by this treatment. The adherence of macrophages to plastic dishes was also impaired by the drugs but only in a smaller extent. Both emetine and chloroquine blocked the amino acid incorporation into macrophage proteins. The effect of emetine was more marked; 10 −6 M decreased the incorporation to 25% of control, while 10 −4 M chloroquine produced a similar inhibition. The block of protein synthesis was also irreversible.