15-14 After attaching to a microbe, phagocytes ingest them

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After binding, phagocytes internalize their prey and fuse them to a lysosome, forming a phagolysosome.
Once inside the phagolysosome a large collection of oxygen-dependent and -independent mechanisms are used in an attempt to kill the microbe.

Attachment of the microbe to the phagocyte results in some sort of signal (the nature of which is still not clearly understood) that triggers ingestion of the microbe. Ingestion involves encircling the target particle with phagocytic membrane so that it is eventually taken inside the cytoplasm of the phagocyte engulfed in a membrane vesicle called a phagosome. This process requires ATP and is triggered by the attachment of the target to the phagocyte's cytoplasmic membrane. Contact between a microbe and a phagocyte also changes the phagocyte's metabolism from an aerobic respiratory to anaerobic fermentative, with lactic acid being the final end product. The increase in lactic acid in the phagocyte lowers the pH of the cytoplasm, including the phagolysosome and this enhances the activity of many of the degradative enzymes present.

Phagolysosome formation

The phagosome containing the microorganism migrates into the cytoplasm and soon collides with a series of lysosomes forming a phagolysosome. When the membranes of the phagosome and lysosome meet, the contents of the lysosome explosively discharge, releasing a large number of toxic macromolecules and other compounds into the phagosome. The killing processes inside the phagolysosome are confined to the organelle of the phagolysosome, thus protecting the cytoplasm of the phagocyte from these toxic activities.

Intracellular killing and digestion

Several minutes after phagolysosome formation, the first detectable effect on the microorganism is the loss of the ability to reproduce. Inhibition of macromolecular synthesis occurs sometime later and many pathogenic and non-pathogenic bacteria are dead 10 to 30 minutes after ingestion. The mechanisms phagocytes use to carry out this killing are diverse and complex, consisting of both metabolic products and lysosomal constituents. Each type of phagocyte (neutrophils, monocytes or macrophages) has a slightly different mix of killing methods. The killing mechanisms that phagocytes use can be organized into two broad groups: oxygen-dependent and oxygen-independent mechanisms. Figure 15-13 shows the killing mechanisms of phagocytes.

Figure 15-13 Killing mechanisms of phagocytes

Killing mechanisms of phagocytes

The figure shows the many different techniques used by phagocytes to kill pathogens. Some require oxygen while others do not.

Oxygen-dependent mechanisms

As will be discussed later in section 15-18, antibodies have constant regions on them that phagocytes can bind using Fc receptors. Binding of Fc receptors on neutrophils, monocytes and macrophages (also binding of mannose receptors on macrophages) causes an increase in oxygen uptake by the phagocyte called the respiratory burst. This influx of oxygen is used in a variety of mechanisms to cause damage to microbes inside the phagolysosome, but the common theme is the creation of highly reactive small molecules that damage the biomolecules of the pathogen. Binding of these receptors activates an NADPH oxidase that reduces O₂ to O₂^- (superoxide). Superoxide can further decay to hydroxide radical (OH^.) or be converted into hydrogen peroxide (H₂O₂) by the enzyme superoxide dismutase. In neutrophils, these oxygen species can act in concert with the enzyme myeloperoxidase to form hypochlorous acid (HOCl) from H₂O₂ and chloride ion (Cl^-). HOCl then reacts with a second molecule of H₂O₂ to form singlet oxygen (¹O₂), another reactive oxygen species. Macrophages in some mammalian species catalyze the production of nitric oxide (NO) by the enzyme nitric oxide synthase. NO is toxic to bacteria and directly inhibits viral replication. It may also combine with other oxygen species to form highly reactive peroxynitrate radicals. All of these toxic oxygen species are potent oxidizers and attack many targets in the pathogen. At high enough levels, reactive oxygen species overwhelm the protective mechanisms of the microbes, leading to their death.

Oxygen-independent mechanisms

The pH of the phagolysosome can be as low as 4.0 and this alone can inhibit the growth of many types of microorganisms. This low pH also enhances the activity of lysozyme, glycosylases, phospholipases and nucleases present in the phagolysosome that degrade various parts of the microbe. A variety of extremely basic proteins present in lysosomal granules strongly inhibit bacteria, yeast and even some viruses. In fact, a few molecules of any one of these cationic proteins can damage the membranes of a bacterial cell, causing death by an unknown mechanism. The phagolysosome of neutrophils also contains lactoferrin, an extremely powerful iron-chelating agent that sequesters most of the iron present, potentially inhibiting bacterial growth.

Monocytes and macrophages also secrete a number of the following substances that may play a role in killing pathogens.

Oxygen metabolites such as O₂^-, H₂O₂, NO and OH^. are also secreted into the local environment when the cells bind an Fc receptor on an antibody or when activated by α-interferon.
Prostaglandins are vasodilators and may increase vascular permeability that accompanies infection and inflammation.
Monocytes are responsible for the synthesis and secretion of all the complement components, which then circulate in the blood and lymph as potent antibacterial factors.
Stimulation of macrophages by endotoxin causes the synthesis and secretion of interleukin-1 (IL-1). IL-1 along with other cytokines, such as IL-6, stimulates T-lymphocytes to proliferate, thus assisting in the activation of the rest of the adaptive immune system. IL-1 also serves as a chemoattractant for neutrophils and keeps them at the site of inflammation by enhancing the adhesiveness of endothelial cells for neutrophils.

Once a microbe is killed, the phagolysosome employs a large collection of digestive enzymes (e.g., lysozyme, proteases, lipases, nucleases, and glycosylases) that break bacterial macromolecules into low molecular weight components. In neutrophils, spent phagolysosomes accumulate in the cytoplasm and the phagocyte eventually lyses and dies. Dead neutrophils make up most of the material in pus.

Egestion and antigen presentation

Macrophages and monocytes live much longer than neutrophils and must dispose of their bacterial components. Once microorganisms are destroyed, the unwanted organic material is expelled from the cell in a process called egestion. Egestion is the opposite of ingestion and the molecular mechanism is basically the reverse of phagocytosis with the microbial leftovers being dumped into the blood and lymph. Some of this microbial debris is not egested, but binds to special protein complexes (called Major Histocompatibility Complex molecules) on the membranes of macrophages for presentation to the immune system, as we will discuss in the following sections.

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