Neutrophils

Neutrophilic leukocytes, particularly the polymorphonuclear (PMN) type (see Color Plate 4), provide an effective host defense against bacterial and fungal infections. The antimicrobial function of PMNs is essential in the innate immune response. Although the monocytes-macrophages and other granulocytes are also phagocytic cells, the PMN is the principal leukocyte associated with phagocytosis and a localized inflammatory response. The formation of an inflammatory exudate (pus), which develops rapidly in an inflammatory response, is composed primarily of neutrophils and monocytes.

PMNs can prolong inflammation by the release of soluble substances, such as cytokines and chemokines. The role of neutrophils in influencing the adaptive immune response is believed to include shuttling pathogens to draining lymph nodes, antigen presentation, and modulation of T helper types 1 and 2 responses. Functionality of neutrophils is no longer considered as limited as it once was because new research has discovered that PMNs have a 5.4 day lifespan.

Mature neutrophils are found in two evenly divided pools, the circulating and marginating pools. The marginating granulocytes adhere to the vascular endothelium. In the peripheral blood, these cells are only in transit to their potential sites of action in the tissues. Movement of granulocytes from the circulating pool to the peripheral tissues occurs by a process called diapedesis (movement through the vessel wall). Once in the peripheral tissues, the neutrophils are able to carry out their function of phagocytosis.

The granules of segmented neutrophils contain various antibacterial substances (Table 3-1). During the phagocytic process, the powerful antimicrobial enzymes that are released also disrupt the integrity of the cell itself. Neutrophils are also steadily lost to the respiratory, gastrointestinal (GI), and urinary systems, where they participate in generalized phagocytic activities. An alternate route for the removal of neutrophils from the circulation is phagocytosis by cells of the mononuclear phagocyte system.

Eosinophils and Basophils

Although capable of participating in phagocytosis, eosinophils and basophils possess less phagocytic activity. The ineffectiveness of these cells results from the small number of cells in the circulating blood and lack of powerful digestive enzymes. Both eosinophils and basophils, however, are functionally important in body defense.

Chemotaxis

Various phagocytic cells continually circulate throughout the blood, lymph, GI system, and respiratory tract. When trauma occurs, the neutrophils arrive at the site of injury and can be found in the initial exudate in less than 1 hour. Monocytes are slower in moving to the inflammatory site. Macrophages resident in the tissues of the body are already in place to deal with an intruding agent. Additional macrophages from the bone marrow and other tissues can be released in severe infections.

Recruitment of PMNs is an essential prerequisite in innate immune defense. Recruitment of PMNs consists of a cascade of events that allows for the capture, adhesion, and extravasation of the leukocyte. Activities such as rolling binding and diapedesis have been well characterized but receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocytes and endothelium, are poorly understood. Research has demonstrated that myeloperoxidase (MPO), a PMN-derived heme protein, facilitates PMN recruitment becaue of its positive surface charge.

Neutrophils have been shown to activate complement when stimulated by cytokines or coagulation-derived factors. Neutrophils activate the alternative complement pathway and release C5 fragments, which further amplify neutrophil proinflammatory responses. This mechanism may be relevant to complement involvement in neutrophil-mediated diseases.

Segmented neutrophils are able to gather quickly at the site of injury because they are actively motile. The marginating pool of neutrophils, adhering to the endothelial lining of nearby blood vessels, migrates through the vessel wall to the interstitial tissues. Mediators produced by microorganisms and by cells participating in the inflammatory process include interleukin-1 (IL-1), which is released by macrophages in response to infection or tissue injury. Another is histamine, released by circulating basophils, tissue mast cells, and blood platelets. Mediators cause capillary and venular dilation.

Cells are guided to the site of injury by chemoattractant substances. This event is termed chemotaxis. A chemotactic response is defined as a change in the direction of movement of a motile cell in response to a concentration gradient of a specific chemical, chemotaxin. Chemotaxins can induce a positive movement toward and a negative movement away from a chemotactic response. Antigens function as chemoattractants; when antigenic material is present in the body, phagocytes are attracted to its source by moving up its concentration gradient.

Phagocytes detect antigens using various cell surface receptors. The speed of phagocytosis can be greatly increased by recruiting the following two attachment devices present on the surface of phagocytic cells:

This coating of the organisms by molecules that speed up phagocytosis is termed opsonization; the Fc portions of antibody and C3 are called opsonins. The steps in opsonization are as follows:

Necrotic cells release an independent chemoattractant of necrotaxis signal, which directs PMN migration beyond the intravascular chemokine gradient. This intravascular danger sensing and recruitment mechanisms have evolved to limit the collateral damage during a response to sterile injury. In this process, PMNs are allowed to migrate intravascularly as they navigate through healthy tissue to sites of injury. Necrotaxis signals promote localization of neutrophils directly into existing areas of injury to focus the innate immune response on damaged areas and away from healthy tissue, which provides an additional safeguard against collateral damage during sterile inflammatory responses. The innate immune system can clean up the dead by killing the living.

Adherence

The leukocyte adhesion cascade is a sequence of adhesion and activation events that ends with the cell exerting its effects on the inflamed site (see later, “Acute Inflammation”). At least five steps appear to be necessary for effective leukocyte recruitment to the site of injury—capture, rolling, slow rolling, firm adhesion, and transmigration.

The process known as capture (tethering) represents the first contact of a leukocyte with the activated endothelium. Capture occurs after margination, which allows phagocytes to move in a position close to the endothelium. P-selectin on endothelial cells is the primary adhesion molecule for capture and the initiation of rolling. Functional E-selectin ligands include CD44.

In addition, many studies have suggested that L-selectin also has an important role in capture. Other cell adhesion molecules (CAMs) have been implicated in capture (e.g., PECAM-1, ICAM-1, VE-cadherin, LFA-1 [CD11a/CD18], IAP [CD47], VLA-4 [4β₁–integrin]), although their level of actual involvement varies.

The inflammatory response begins with a release of inflammatory chemicals into the extracellular fluid. Sources of these inflammatory mediators, the most important of which are histamine, prostaglandins, and cytokines, are injured tissue cells, lymphocytes, mast cells, and blood proteins. The presence of these chemicals promotes the reactions to inflammation (redness, heat, swelling, pain).

The transit time through the microcirculation and, more specifically, the contact time during which the leukocyte is close to the endothelium, appears to be a key parameter in determining the success of the recruitment process, as reflected in firm adhesion.

Engulfment

On reaching the site of infection, phagocytes engulf and destroy the foreign matter (Fig. 3-3). Eosinophils can also undergo this process, except that they kill parasites. After the phagocytic cells have arrived at the site of injury, the bacteria can be engulfed through active membrane invagination. Pseudopodia are extended around the pathogen, pulled by interactions between the Fc receptors and Fc antibody portions on the opsonized bacterium. Pseudopodia meet and fuse, thereby internalizing the bacterium and enclosing it in a phagocytic vacuole, or phagosome.

The principal factor in determining whether phagocytosis can occur is the physical nature of the surface of the bacteria and phagocytic cell. The bacteria must be more hydrophobic than the phagocyte. Some bacteria, such as Diplococcus pneumoniae, possess a hydrophilic capsule and are not normally phagocytized. Most nonpathogenic bacteria are easily phagocytized because they are very hydrophobic. The presence of certain soluble factors such as complement, a plasma protein, coupled with antibodies and chemicals such as acetylcholine enhance the phagocytic process. Enhancement of phagocytosis through opsonization can speed up the ingestion of particles. If the surface tensions are conducive to engulfment, the phagocytic cell membrane invaginates. This invagination leads to the formation of an isolated vacuole (phagosome) within the cell.

Digestion

Digestion follows the ingestion of particles, with the required energy primarily provided by anaerobic glycolysis. Granules in the phagocyte cytosol then migrate to and fuse with the phagosome to form the phagolysosome. These granules contain degradatory enzymes of the following three types:

Degranulation of the neutrophil releases antibacterial substances (e.g., lactoferrin, lysozyme, defensin) from the granules; released enzymes promote bactericidal activity by increasing membrane permeability. Elastase, one of several substances that can damage host tissues, is also released. The myeloperoxidase granules are responsible for the action of the oxygen-dependent, myeloperoxidase-mediated system. Hydrogen peroxide (H₂O₂) and an oxidizable cofactor serve as major factors in the actual killing of bacteria within the vacuole. Other oxygen-independent systems, such as alterations in pH, lysozymes, lactoferrin, and the granular cationic proteins, also participate in the bactericidal process. Monocytes are particularly effective as phagocytic cells because of the large amounts of lipase in their cytoplasm. Lipase is able to attack bacteria with a lipid capsule, such as Mycobacterium tuberculosis. Monocytes are further able to bind and destroy cells coated with complement-fixing antibodies because of the presence of membrane receptors for specific components or types of immunoglobulin.

Release of lytic enzymes results in the destruction of neutrophils and their subsequent phagocytosis by macrophages. Macrophage digestion proceeds without risk to the cell unless the ingested material is toxic. If the ingested material damages the lysosomal membrane, however, the macrophage will also be destroyed because of the release of lysosomal enzymes.

During phagocytosis, cells demonstrate increased metabolic activity, referred to as a respiratory burst. This results in the production by the phagocyte of large quantities of reactive oxygen species (ROS), which are released into the phagocytic vesicle. This phenomenon is achieved by the activity of the enzyme known as reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. Together, the granule-mediated and NADPH oxidase–mediated effects elicit microbicidal results. NADPH oxidase forms the centerpiece of the phagocyte-killing mechanism and is activated in about 2 seconds. The NADPH oxidase generates ROS by generating the superoxide radical (O₂⁻); the associated cyanide-insensitive increase in oxygen consumption is the respiratory burst.

The importance of the oxygen-dependent microbicidal mechanism is dramatically illustrated by patients with chronic granulomatous disease (CGD), a severe congenital deficit in bacterial killing that results from the inability to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs). The production of residual ROIs is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected. CGD results from defects in the genes encoding individual components of the enzyme system responsible for oxidant production. Acquisition of oxidase activity occurs in the course of myeloid cell maturation, and the genes for several of its components have been identified. This system also lends itself to analysis of the transcriptional and translational events that occur during cellular differentiation and under the influence of specific cytokines.

Rather than being discarded by exocytosis, some peptides undergo an important separate process at this stage. Instead of being eliminated, they attach to a host molecule called major histocompatibility complex (MHC) class II and are expressed on the surface of the cell within a groove on the MHC molecule (antigen presentation).

Subsequent Phagocytic Activity

If invading bacteria are not phagocytized at entry into the body, they may establish themselves in secondary sites such as the lymph nodes or various body organs. These undigested bacteria produce a secondary inflammation, where neutrophils and macrophages again congregate. If bacteria escape from secondary tissue sites, a bacteremia will develop. In patients who are unresponsive to antibiotic intervention, this situation can prove fatal.

Neutrophil Extracellular Traps

In addition to phagocytosis, including the release of antimicrobial molecules at the site of infection, an additional defense mechanism has been discovered. This mechanism is the formation of neutrophil extracellular traps (NETs), which are produced following the release of the nuclear contents of the neutrophil into the extracellular space. NETs function in innate immunity. They are composed of chromatin components, including histones, and neutrophil antimicrobial proteins. Microbes are trapped in NETs, where they encounter high concentrations of antimicrobial proteins.

Mononuclear Phagocyte System

The macrophage (Fig. 3-4