Which action makes microbes more susceptible to phagocytosis

5.06.1 Introduction

Phagocytic cells of the immune system consist predominantly of macrophages and neutrophils. These cells represent the major cellular effectors of nonspecific host defense and inflammation. Through their ability to phagocytize foreign substances and release cytotoxic and proinflammatory mediators, neutrophils and macrophages protect the body from a wide array of pathogens and xenobiotics and play a central role in the host response to tissue injury. However, these phagocytic leukocytes also possess significant cytotoxic potential. Many of the mediators released by phagocytes to protect the host also have the capacity to damage normal tissue. Thus, overproduction or unregulated release of cytotoxic mediators can lead to increased or prolonged injurious tissue reactions. This chapter reviews the physiological and pathophysiological activity of phagocytic cells of the immune system. The contribution of macrophages to tissue repair is also discussed.

Introduction

Phagocytes and phagocytosis were first described by Elie Metchnikoff in the late 1800s. He identified two groups of phagocytic cells during his studies of bacterial infection and named these cells macrophages and microphages. These cells could be differentiated further by staining methods developed by Paul Ehrlich and colleagues. Metchnikoff reported that the cells he named “microphages” were the same leukocytes Ehrlich described as polynuclear cells (later “cells with polymorphous nuclei”). These microphages are now known as granulocytes or polymorphonuclear leukocytes. Metchnikoff reported the ability of phagocytes to ingest invading microbes and thereby contribute to natural or innate immunity. Our understanding of phagocyte biology, including development, function, and turnover, has increased significantly since the early work by Metchnikoff and Ehrlich. Many of the most recent discoveries, especially those related to the origins and development of phagocytes, have been made possible by advances in technology and improved methodology. Inasmuch as information and detailed knowledge related to phagocyte biology is extensive, this article is not intended to provide a comprehensive discourse on phagocytes and/or phagocyte biology. Rather, it provides an overview of selected areas of phagocyte biology that can serve as a starting point for the interested reader.

Genetic Defects That Impact Development or Function of Phagocytic Cells

Evaluation of Phagocyte Function

Phagocytes are bone marrow-derived cells of myeloid origin, including neutrophils, eosinophils, basophils, monocytes, and the mature form of the monocyte. They are pivotal cells for controlling the initial response to infection, and initiating, sustaining, or resolving inflammation, and their responses must be tightly coordinated and highly regulated to prevent infection while not damaging host tissues. Professional phagocytes, namely neutrophils, monocytes, and macrophages, are the primary cell populations that have been historically investigated by immunotoxicologists, as their mechanisms of response to infection and roles in the inflammatory process have been most closely studied in humans, and in the standard preclinical toxicology models. Phagocytosis is a process initiated by the binding of opsonized microbes or particles to opsonic receptors on the surface of the phagocyte. These include receptors for the constant regions of immunoglobulins, as well as receptors for components of the complement cascade. The engagement of opsonic receptors activates the phagocyte to rearrange cytoskeletal elements, thus altering plasma-membrane folding, allowing invagination and allowing cytoplasmic granules to fuse with the internalized target forming a phagosome. Inside the phagosome a variety of microbicidal and microbiostatic proteins are activated by changes in pH and ion concentration. Activation of NADPH oxidase generates antimicrobial concentrations of hydrogen peroxide, superoxide anion, hypochlorous acid, and other reactive species through the rapid metabolism of oxygen, the respiratory burst. A great body of literature is available concerning the processes of phagocyte transmigration, chemotaxis, phagocytosis, and oxygen-dependent and independent killing mechanisms briefly described above; the reader is referred to the following selected references on these subjects [59,60].

Immunotoxological investigation of phagocyte phenotype or function primarily involves assessment of phenotype, phagocytosis, and respiratory burst. Microscopic methods including direct staining, immunohistochemistry, and confocal microscopy are available [61]. In addition, assessment of peripheral blood phagocyte population phenotype and number, as well as spleen resident phagocyte populations, is routinely performed by flow cytometry [62]. Phenotypic assessment of phagocyte populations should be accompanied by functional assessment, as indicated by the test-article mechanism of action and the weight of evidence.

Assessment of phagocytosis can be performed by flow cytometry utilizing an array of fluorescently labeled targets including beads, bacteria, and fungi using a semiquantitative assay format [63]. A typical flow cytometric assay to measure phagocytosis would utilize whole blood, peripheral blood mononuclear cells, or isolated phagocytes incubated with fluorescently labeled targets for a period to time followed by appropriate gating by forward and side scatter or using combination gating to separate monocytes and neutrophils from lymphocytes. Typically, results would be presented as the fluorescent intensity of the internalized target normalized to unstained control and compensated with appropriate staining controls. An additional aspect of testing that provides useful information is the use of opsonized and unopsonized targets to determine the pathway by which phagocytosis is occurring. Assessment of respiratory burst activity can be assessed by multiple methods including plate-based luminescent and colorimetric methods and flow cytometry.

Phagocytes and Their Receptors

Phagocytes include neutrophils, macrophages, and dendritic cells (DCs), which have the capacity to engulf and digest relatively large particles on the order of 1–10 µm and even larger. In adults, these cells are generated from hematopoietic stem cells in the bone marrow. Phagocytosis can be activated by receptors that share structural features with T and B cell antigen receptors, in which case the process takes on characteristics of cell–cell interactions with specialized junctions referred to as phagocytic synapses. Phagocytes express many type of ‘scavenger’ receptors that allow them to participate in clearance of aged host proteins and cells or microbe-derived materials. Pathogens may also exploit phagocytes as a port of entry to colonize the host. One example is the gram positive bacterium Listeria monocytogenes, which generates a number of interesting virulence factors. One is call listeriolysin O, which allows the baceterium to lyse the phagocytic vacuole and directly enter the phagocyte’s cytoplasm. A second virulance factor, called Act A, then induces dynamic actin polymerization by activating the Actin-related protein 2/3 complex to generate ‘actin rockets’ that allow Listeria to move from cell to cell without passing through the extracellular space, thus avoiding extracellular immune effectors. Due to their ability to take up extracellular particles, phagocytes such as macrophages and DCs are excellent antigen presenting cells (APCs) that activate lymphocytes. B cells are also potent APCs because they can internalize antigens bound to the B cell receptor, which is essentially a plasma membrane-bound antibody. Therefore, although phagocytosis can lead to destruction of internalized microbes, the process also functions as a bridge between innate and adaptive immunity.

4.2 Encapsulation

Circulating phagocytes usually encapsulate any foreign material too large to be ingested by a single cell. The formation of capsule around parasitic crustaceans has been reported in Ascidiella aspersa,69 Microcosmus savignyi,70 and Styela gibbsii.71 In Molgula manhattensis, both phagocytes and MCs are recruited in the infectious area during capsule formation.72 In C. intestinalis, the injection of mammalian erythrocytes, BSA, or LPS in the tunic leads to capsule formation following the massive recruitment of hemocytes to the inoculum site.73–75

Botryllus scalaris is the only botryllid species reported so far in which capsule formation by circulating phagocytes is involved in allorecognition between contacting, incompatible colonies. In this case, after the fusion of the ampullae and the initial blood-exchange between contacting colonies, circulating phagocytes crowd inside the fused vessels and stimulate the aggregation of hemocytes into large clusters, which are encapsulated by other phagocytes. In this way, the hemocyte-mass plugs the lumen of the fused ampullae and the blood flow is interrupted within a few minutes.76

Abstract

Phagocytes such as neutrophils, monocytes, and macrophages are the major cells of innate immunity. They produce high amounts of reactive oxygen species (ROS) to kill phagocytized pathogens. The enzyme responsible for ROS production is called the phagocyte nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) NOX2. NOX2 is a multicomponent enzyme system built by the assembly of cytosolic proteins (p47phox, p67phox, p40phox, and rac1/2) and transmembrane proteins (p22phox and gp91phox which form cytochrome b558). NOX2-derived ROS are essential for innate immunity, however, excessive ROS production induces tissue injury. Thus NADPH oxidase activation must be tightly regulated. This article describes the regulation of this enzyme complex.

1.14.3.5 Phagocytes

Phagocytic cells (e.g., neutrophils and monocytes) are well-established sources of free radicals. When activated by appropriate stimuli, these cells exhibit marked increases in oxygen consumption that are due to the rapid reduction of oxygen to superoxide by a plasma membrane-bound NADPH oxidase. The net result is the release of large amounts of ROS extracellularly (Figure 5). This process is critical for the normal functioning of the immune system. Phagocytes also generate nitric oxide (•NO), a free radical species (Moncada and Higgs 1993). The contributions of this radical in signaling, microbicidal activities, and as a cause of tissue injury are ongoing areas of research (Halliwell 2006).

Concluding Remarks

Phagocytes exert their important immunological functions in the absence of acquired immunity through their ability to sense, through PRRs, and through changes in the physiological status of tissues induced by infectious agents and/or cellular stress. PRR engagement induces an inflammatory response that will endeavor to clear the initial insult and repair tissue. This effective, although unspecific, response is greatly boosted by the tools offered by the acquired immunity that include the presence of antigen-specific antibodies and activated T cells. Activating FcRs expressed by phagocytes promotes antigen uptake and cellular activation, leading to increased phagocytosis, cellular recruitment, and T-cell activation. This positive feedback circle of activation is kept under control by inhibitory FcγRs, which function as buffers of the immune system and negatively regulate cellular activation in response to PRRs and activating FcRs. In this manner, inhibitory FcγRs minimize the potential for pathological immune responses that could compromise organ function. Further understanding of the interplay between phagocyte receptors will enable the design of novel therapeutic agents aimed at blunting immune complex-induced pathologies and vaccination strategies that could exploit activating FcγRs for the elicitation of robust humoral and T cell-mediated immune responses.

Abstract

Most immunodeficiencies affect phagocytes as well as lymphocytes, reflecting their shared hematopoietic origin. However, several are regarded as primarily phagocytic, largely reflecting not only their major manifestations and infections but also the state of knowledge when they were identified and named. Phagocyte immunodeficiencies are those that affect the number or function of phagocytes, reflected primarily in their associated infections. Infections most typical of phagocyte defects are caused by bacteria and fungi, which are typically invasive and severe. One of the hallmarks of phagocyte immune deficiencies is that they sum the effects of both infection susceptibility and immune dysregulation, reflecting the dual nature of phagocytic cells in both controlling local invaders and in regulating and “mopping up” the havoc that those invaders cause. This complex mixture of too little infection prevention and too little inflammation regulation makes the phagocyte immunodeficiencies complex to manage clinically.