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.

Table 3-1

Function and Types of Granules in Neutrophils

Function	Azurophilic (Primary) Granules	Specific (Secondary) Granules
Microbicidal	Myeloperoxidase	Cytochrome b558 and other respiratory burst components
	Lysozyme	Lysozyme
	Elastase	Lactoferrin
	Defensins
	Cathepsin G
	Proteinase-3
	Bacterial permeability-increasing protein (BPI)
Cell migration		Collagenase
		CD11b–CD18 (CR-3)
		N-formulated peptides (e.g., N-formyl-methionyl-leucylphenylalanine receptor [FMLP-R])

Adapted from Peakman M, Vergani D: Basic and clinical immunology, ed 2, Edinburgh, 2009, Churchill Livingstone, p 24.

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.

Eosinophils

The eosinophil (see Color Plate 5) is considered to be a homeostatic regulator of inflammation. Functionally, this means that the eosinophil attempts to suppress an inflammatory reaction to prevent the excessive spread of the inflammation. The eosinophil may also play a role in the host defense mechanism because of its ability to kill certain parasites.

A functional property related to the membrane receptors of the eosinophil is the cell’s ability to interact with the larval stages of some helminth parasites and damage them through oxidative mechanisms. Certain proteins released from eosinophilic granules damage antibody-coated Schistosoma parasites and may account for damage to endothelial cells in hypereosinophilic syndromes.

Basophils

Basophils (see Color Plate 6) have high concentrations of heparin and histamine in their granules, which play an important role in acute, systemic, hypersensitivity reactions (see Chapter 26). Degranulation occurs when an antigen such as pollen binds to two adjacent immunoglobulin E (IgE) antibody molecules located on the surface of mast cells. The events resulting from the release of the contents of these basophilic granules include increased vascular permeability, smooth muscle spasm, and vasodilation. If severe, this reaction can result in anaphylactic shock.

A class of compounds known as leukotrienes mediates the inflammatory functions of leukocytes. The observed systemic reactions related to leukotrienes were previously attributed to the slow-reacting substance of anaphylaxis.

Process of Phagocytosis

Phagocytosis can be divided into six stages—chemotaxis, adherence, engulfment, phagosome formation, fusion, and digestion and destruction (Fig. 3-2). The physical occurrence of damage to tissues, by trauma or microbial multiplication, releases substances such as activated complement components and products of infection to initiate phagocytosis.

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:

• Fc receptor—binds the Fc portion of antibody molecules, chiefly immunoglobulin G (IgG). The IgG attaches to the organism through its Fab site.

• Complement receptor—the third component of complement, C3, also binds to organisms and then attaches to the complement receptor.

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:

1. Antibody attached to the surface of a bacterium minimally binds the Fc phagocyte receptor.

2. Complement C3b is attached to the surface of the bacterium and binds loosely to the phagocyte C3b receptor.

3. Both antibody and C3b are attached to the surface of the bacterium and bound tightly to the phagocyte, allowing greater opportunity for the phagocyte to engulf the bacterium.

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.

Figure 3-3 Two phagocytic cells have engulfed numerous *Staphylococcus aureus* cells. (From Barrett JT: Textbook of immunology, ed 5, St Louis, 1988, Mosby.)

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.

Monocytes-Macrophages

In the past, the mononuclear monocyte-macrophage was known only as a scavenger cell. Only recently has its role as a complex cell of the immune system in the host defense against infection been recognized.

Mononuclear Phagocyte System

The macrophage (Fig. 3-4) and its precursors are widely distributed throughout the body. These cells constitute a physiologic system, the mononuclear phagocyte system (previously referred to as the reticuloendothelial system), which includes promonocytes and their precursors in the bone marrow, monocytes in the circulating blood, and macrophages in tissues. This collection of cells is considered to be a system because of the common origin, similar morphology, and shared functions, including rapid phagocytosis mediated by receptors for IgG and the major fragment of C3.

Figure 3-4 Electron micrograph of a macrophage. (From Barrett JT: Textbook of immunology, ed 5, St Louis, 1988, Mosby.)

Macrophages and monocytes (see Color Plate 7) migrate freely into the tissues from the blood to replenish and reinforce the macrophage population. Cells of the macrophage system originate in the bone marrow from the multipotential stem cell. This common committed progenitor cell can differentiate into the granulocyte or monocyte-macrophage pathway, depending on the microenvironment and chemical regulators. Maturation and differentiation of these cells may occur in various directions. Circulating monocytes may continue to be multipotential and give rise to different types of macrophages.

Macrophages exist as fixed or wandering cells. Specialized macrophages such as the pulmonary alveolar macrophages are the so-called dust phagocytes of the lung that function as the first line of defense against inhaled foreign particles and bacteria. Fixed macrophages line the endothelium of capillaries and the sinuses of organs such as the bone marrow, spleen, and lymph nodes. Macrophages, along with the network of reticular cells of the spleen, thymus, and other lymphoid tissues, are organized into the mononuclear phagocyte system (Fig. 3-5).

Figure 3-5 Mononuclear phagocyte system. (Adapted from Roitt IM: Essential immunology, ed 5, Oxford, 1984, Blackwell Scientific.)

Functionally, the most important step in the maturation of macrophages is the cytokine-driven conversion of the normal resting macrophage to the activated macrophage. Macrophages can be activated during infection by the release of macrophage-activating cytokines such as interferon-gamma (IFN-γ) and granulocyte colony-stimulating factor (G-CSF) from T lymphocytes specifically sensitized to antigens from the infecting microorganisms. This interaction constitutes the basis of cell-mediated immunity. In addition, macrophages exposed to an endotoxin release a hormone, tumor necrosis factor α (TNF-α, cachectin), which can activate macrophages itself under certain in vitro conditions.

The terminal stage of development in the mononuclear phagocyte cell line is the multinucleated giant cell, which characterizes granulomatous inflammatory diseases such as tuberculosis. Both monocytes and macrophages can be shown in the lesions in these diseases before the formation of giant cells, thought to be precursors of the multinucleated cells.

Host Defense Functions

Functionally, monocytes-macrophages have phagocytosis as their major role, but these cells perform at least three distinct but interrelated functions in host defense. The categories of host defense functions of monocytes-macrophages include phagocytosis, antigen presentation and induction of the immune response, and secretion of biologically active molecules.

Phagocytosis

The principal functions of mononuclear phagocytes in body defenses result from the changes that take place in these functions when the macrophage is activated (Box 3-1). Macrophages carry out the fundamental function of ingesting and killing invading microorganisms such as intracellular parasites, M. tuberculosis, and some fungi. In addition, macrophages remove and eliminate such extracellular pathogens as pneumococci from the blood circulation. The macrophage also has the capacity to phagocytize particulate and aggregated soluble materials. This process is enhanced by the presence of receptors on the surface of the Fc portion of IgG and C3. The ability to internalize soluble substances supports the increased microbicidal and tumoricidal ability of activated macrophages. Activation of macrophages or monocytes can result in the release of parasiticidal mediators and in receptor-mediate phagocytosis during malaria infection. The most likely location for this innate immune response is within the spleen which is crucial for development of immunity to malaria.

Box 3-1 Functions of Mononuclear Phagocytes

Increased Activity in Activated Macrophages

Antigen presentation

Chemotaxis

Glucose transport and metabolism

Microbicidal activity

Phagocytosis (variable activity, depending on particle)

Phagocytosis-associated respiratory burst

Pinocytosis

Tumoricidal activity

Increased Constituents in Activated Macrophages

Complement components ^∗

Plasminogen activator

TNF-α (cachectin)^†

Decreased Constituents in Activated Macrophages

Apolipoprotein E and lipoprotein lipase

Elastase

Prostaglandins, leukotrienes

Constituent Demonstrating No Change in Activated Macrophages

Lysoenzyme

^∗Increased or no change.

^†When stimulated.

Adapted from Johnston RB: Current concepts: immunology. Monocytes and macrophages, N Engl J Med 318:747–752, 1988.

Another important phagocytic function of macrophages is their ability to dispose of damaged or dying cells. Macrophages lining the sinusoids of the spleen are particularly important in ingesting aging erythrocytes. They are also involved in removing tissue debris, repairing wounds, and removing debris as embryonic tissues replace one another.

Phagocytic activity increases when there is tissue damage and inflammation, which releases substances that attract macrophages. Activated macrophages migrate more vigorously in response to chemotactic factors and should enter sites of inflammation (e.g., locations of infection or cancer) more efficiently than resting macrophages. Migration of monocytes into different body tissues appears to be a random phenomenon in the absence of localized inflammation. An essential factor in the protective function of monocytes is the capacity of the cell to move through the endothelial wall of blood vessels (diapedesis) to the site of microbial invasion in tissues. The attracting forces for monocytes, chemotactic factors, include complement products and chemoattractants derived from neutrophils, lymphocytes, or cancer cells.

The activity of mononuclear phagocytes against cancer cells in humans is less well understood than the phagocytosis of microorganisms. Phagocytes are thought to suppress the growth of spontaneously arising tumors. The ability of these cells to control malignant cells may not involve phagocytosis but may be related to secreted cellular products such as lysosomal enzymes, oxygen metabolites (e.g., H₂O₂), proteinases, and TNF-α (cachectin). The proteolytic enzymes present on the surface membrane of monocytes also may play a role in tumor rejection.

Antigen Presentation and Induction of the Immune Response

The phagocytic property of the macrophage is particularly important in the processing of antigens as part of the immune response. Macrophages are believed to process antigens and physically present this biochemically modified and more reactive form of antigen to lymphocytes (particularly helper T cells) as an initial step in the immune response. Recognition of antigen on the macrophage surface by T lymphocytes, however, requires an additional match of the surface MHC class II gene product. This gene product is the Ia product in the mouse and D gene region product in humans. With proper recognition, the macrophage secretes a lymphocyte-activating factor (IL-1), lymphocyte proliferation ensues, and the immune response (T cell–B cell response) is facilitated.

Secretion of Biologically Active Molecules

Monocytes-macrophages release many factors associated with host defense and inflammation. These cells serve as supportive accessory cells to lymphocytes, at least partly by releasing soluble factors. In cellular immunity, monocytes assume a killer role in that they are activated by sensitized lymphocytes to phagocytize offending cells or antigen particles. This is important in fields such as tumor immunology.

In addition to their phagocytic properties, monocytes-macrophages are able to synthesize a number of biologically important compounds, including transferrin, complement, interferon, pyrogens, and certain growth factors. Approximately 100 distinct substances have been identified as being secreted by monocytes-macrophages.

Blood monocytes and tissue macrophages are primary sources of the polypeptide hormone called IL-1, which has a particularly potent effect on the inflammatory response. IL-1 also supports B lymphocyte proliferation and antibody production, as well as T lymphocyte production of lymphokines. The increased synthesis of IL-1 by activated macrophages could contribute to enhancement of the immune response. Endotoxin also induces the synthesis of IL-1. This effect is achieved at least partly by stimulation of the macrophages to release TNF-α, which then stimulates the production of IL-1 by endothelial cells and macrophages. Activated macrophages release much more TNF-α than resting macrophages exposed to endotoxin. Both TNF-α and IL-1 can induce the fever and synthesis of acute-phase reactants that characterize inflammation.

Acute Inflammation

Tissue damage results in inflammation, a series of biochemical and cellular changes that facilitate the phagocytosis of invading microorganisms or damaged cells (Fig. 3-6). If inflammation is sufficiently extensive, it is accompanied by an increase in the plasma concentration of acute-phase reactants (see Chapter 5). Leukocyte recruitment into inflamed tissue follows a well-defined cascade of events beginning with the capture of free-flowing WBCs to the vessel wall and subsequent leukocyte rolling along and adhesion to the inflamed endothelial layer. During rolling, WBCs come into close contact with the endothelial surface, which allows endothelium-bound chemokines to interact with their specific receptors on the leukocyte surface. This triggers the activation of integrins, which leads to firm leukocyte arrest on the endothelium. In addition, integrin-dependent signaling events induce cytoskeletal rearrangements and cell polarization, modifications necessary to help prepare the attached leukocyte to spread and crawl in search for a way out of the vasculature into tissue.

Figure 3-6 Acute inflammatory response.
Neutrophils are among the first cells to arrive at the scene of an infection and are important contributors to the acute inflammatory response. As the neutrophil rolls along the blood vessel wall, the L-selectin on its surface binds to carbohydrate structures such as sialyl-Lewis X on the adhesion molecules on the vascular endothelium, and its progress is eventually halted. As the neutrophil becomes activated, it replaces L-selectin with other cell surface adhesion molecules, such as integrins. These molecules bind E-selectin, which is present on the blood vessel wall as a result of the influence of inflammatory mediators such as bacterial lipopolysaccharides and the cytokines IL-1 and TNF-α. The activated neutrophil then enters the tissues, where it is attracted to the infection site by a number of chemoattractants. The neutrophil can then phagocytose and destroy the C3b-coated bacteria. (Adapted from Delves PJ, Roitt IM: The immune system. First of two parts, N Engl J Med 343:37–49, 2000.)

Celsus, a practitioner of Greek medicine who was born in 25 bce, is credited with recording the cardinal signs of inflammation—rubor (redness), calor (heat), dolor (pain), and tumor (swelling). The primary objective of inflammation is to localize and eradicate the irritant and repair the surrounding tissue. The inflammatory response involves the following three major stages:

1. Dilation of capillaries to increase blood flow

2. Microvascular structural changes and escape of plasma proteins from the bloodstream

3. Leukocyte transmigration through endothelium and accumulation at the site of injury

Hypoxia can induce inflammation. Inflammation in response to hypoxia is clinically relevant. Ischemia in organ grafts increases the risk of inflammation and graft failure or rejection. Hypoxia has multiple effects on the innate and adaptive immune systems.

Once inflammation is triggered, it must be appropriately resolved or pathologic tissue damage will occur. In some diseases, the body’s defense system (immune system) inappropriately triggers an inflammatory response when no foreign substances are present. In these autoimmune disorders, the body’s normally protective immune system causes damage to its own tissues (see Chapter 28).

SEPSIS

If an inflammation overwhelms the whole body, systemic inflammatory response syndrome (SIRS) is diagnosed. Sepsis, severe sepsis, and septic shock are progressively severe stages of SIRS. The criteria for SIRS require two or more conditions: alteration of body temperature (>38°C or <36°C), increased heart rate, increased respiratory rate, and a total leukocyte count of >12.0 × 10(9)/L (or >10% immature forms). Sepsis is defined as SIRS + infection; severe sepsis is defined as sepsis + evidence of organ dysfunction. Patients with severe sepsis are considered to have defective adaptive immunity.

Sepsis begins when the innate immune system responds aggressively to the presence of bacteria. Toll-like receptors (TLR) cause the antigen presenting cell (APC) to produce proinflammatory cytokines. Biochemical markers associated with sepsis include tumor necrosis factor (TNF) and interleukins (ILs) IL-1 and IL-6, a proinflammatory cytokine. Other proteins produced in response infection and/or inflammation include procalcitonin and chemokine production. Another consequence of inflammation is that the liver is stimulated to produce C-reactive protein (C-RP) (see Chapter 5).

These cytokines produce systemic inflammation by activating circulating polymorphonuclear leukocytes (PMNs). APCs involve the adaptive immune system by presenting bacterial antigen to T-cell receptor using a Class II major histocompatibility complex (MHC) protein and co-stimulation of CD28.

Cell Surface Receptors

Cellular communication is essential to the development, tissue organization, and function of all multicellular organisms. Cells communicate with each other and their environment through soluble mediators and during direct contact (e.g., phagocytosis). An immunologic response is a result of the interactions of various leukocytes with each other and other cells in the body. These interactions occur through cell surface receptors that mediate cell-cell binding, or adhesion, of leukocytes.

The discovery of several cell surface receptors involved in cellular communication has been a key factor in understanding the mechanisms underlying inflammatory and immune phenomena. Three protein families—the immunoglobulin (Ig) family, integrin family, and the rather recently designated selectin family—form a network of cellular interactions in the immune system. Neutrophil tether to and roll on P- and E-selectin expressed on activated endothelial cells. Rolling neutrophils encounter immobilized chemokines. Chemokines activate integrins to their high-affinity states that enable interactions with intercellular adhesion molecule-1 (ICAM-1), which promote arrest, adhesion strengthening, intraluminal crawling, and transendothelial migration. E-selectin directly triggers signals in rolling PMN that cooperate with chemokine signals to minimize neutrophil recruitment during inflammation.

Members of the Ig superfamily include antigen-specific receptors (e.g., T cell receptor [TCR] and surface immunoglobulin [sIg]), as well as antigen-independent receptors and their counterreceptors, such as CD2 and lymphocyte function–associated antigen-3. Ig superfamily members function in cell activation, differentiation, and cell-cell interaction. In some cases, both an adhesion receptor and the counterreceptor to which it binds are members of the Ig superfamily.

Three selectin family molecules—endothelial cell adhesion molecule-1, leukocyte adhesion molecule (LAM-1, Mel-14), and CD62, also known as platelet activation–dependent granule–external membrane protein and granule membrane protein of 140 kDa (GMP-140)—have been implicated in a number of leukocyte adhesion phenomena, including leukocyte homing to lymphoid tissue. Selectins are expressed on leukocytes and endothelial cells. Mel-14 functions early in neutrophil-endothelium adhesion.

The integrin family consists of at least 14 alpha-beta heterodimers divided into subfamilies with distinct structural and functional characteristics. The subfamily of leukocyte integrins contains three members—LFA-1, Mac-1, and p150,95. These molecules are glycoproteins composed of noncovalently associated alpha and beta subunits. LFA-1 is expressed on all leukocytes, whereas Mac-1 and p150,95 are found primarily on granulocytes and monocytes.

The integrin family is phylogenetically ancient. Integrin family members engage in interactions with cell surface ligands and extracellular matrix (ECM) components. ECM components, including fibronectin, collagen, and laminin, have been shown to be ligands for members of the beta-1 and beta-3 subfamilies. Members of these subfamilies are of great significance in embryogenesis, growth and repair, and hemostasis. The leukocyte integrins, or beta-2 subfamily, have been shown to be involved in a diverse number of leukocyte adhesion–dependent phenomena, giving them a critical role in inflammatory and immune responses. The term integrin was initially used to emphasize that these receptors integrate signals from the extracellular environment with the intracellular cytoskeleton. A signal is transduced from outside to inside the cell.

In addition to the involvement of these receptors in a variety of immune functions, integrin molecules play a role in the spread of malignant cells. The major cause of death in malignant disease is not the primary tumor but rather the metastasis of tumor cells to distant sites within the body. Metastasis is a complex multistep process that begins with the detachment of a few tumor cells from the primary tumor. The tumor cells then move into the circulatory system, where they can be transported to other organs. While in the circulatory system, tumor cells must survive the natural defense system of the body before attaching to and invading the tissues of another organ. A better understanding of the metastatic process could provide the basis for diagnostic and therapeutic strategies.

Disorders of Neutrophils

Noninfectious Neutrophil-Mediated Inflammatory Disease

Although neutrophils provide the major means of defense against bacterial and fungal infections, they can also be destructive to host tissues. The same oxidative and nonoxidative processes that destroy microorganisms can affect adjacent host tissues. A number of disease states correspond to inappropriate phagocytosis (Box 3-2), as with prolonged activation of NADPH oxidase. This process occurs when phagocytes attempt to engulf particles that are too large. The phagocyte releases oxygen radicals and granule contents onto the particle, but these escape into the surrounding tissues, generating tissue damage. This is often observed in response to dust inhalation and smoking (e.g., nicotine) and in persistent infections such as cystic fibrosis. In addition, many autoimmune diseases are thought to be caused by inappropriate activation of the process of phagocytosis, whereby the body attacks its own cells and tissues. Examples include rheumatoid arthritis, multiple sclerosis, and Graves’ disease.

Abnormal Neutrophil Function

Patients with quantitative or qualitative defects of neutrophils have a high rate of infection, which illustrates the importance of the neutrophil to body defenses. Individuals with a marked decrease of neutrophils (neutropenia) or severe defects in neutrophil function frequently have recurrent systemic bacterial infections (e.g., pneumonia), disseminated cutaneous pyogenic lesions, and other types of life-threatening bacterial and fungal infections.

Leukocyte mobility may be impaired in some diseases (e.g., rheumatoid arthritis, cirrhosis, CGD). Defective locomotion or leukocyte immobility can also be seen in patients receiving steroids and in those with lazy leukocyte syndrome. A marked defect in the cellular response to chemotaxis, an important step in phagocytosis, can be seen in patients with diabetes mellitus, Chédiak-Higashi anomaly (syndrome), or sepsis, as well as in those with high levels of antibody immunoglobulin E (IgE), as in Job’s syndrome.

Congenital Neutrophil Abnormalities

A small number of patients have congenital abnormalities of neutrophil structure and function (Box 3-3).

Box 3-3 Congenital Neutrophil Abnormalities

Chédiak-Higashi syndrome (anomaly)

Chronic granulomatous disease (CGD)

Complement receptor 3 (CR3) deficiency

Myeloperoxidase deficiency

Specific granule deficiency

Chédiak-Higashi Syndrome

The Chédiak-Higashi syndrome represents a qualitative disorder of neutrophils. It is a rare familial disorder inherited as an autosomal recessive trait and expressed as an abnormal granulation of neutrophils. Neutrophils having giant granules display impaired chemotaxis and delayed killing of ingested bacteria.

Chronic Granulomatous Disease

The chronic granulomatous diseases (CGDs) are a genetically heterogeneous group of disorders of oxidative metabolism affecting the cascade of events required for H₂O₂ production by phagocytes. Patients with X-linked CGD (X-CGD) have a mutation in CYBB encoding the transmembrane gp91^phox subunit of phagocyte NADPH oxidase required for microbicidal ROS production by neutrophils and monocytes. As a result, patients have life-threatening infections and granulomatous complications. If a suitable hematopoietic stem cell donor is available, it can cure X-CGD, but graft-versus-host disease (see Chapter 31) is a significant risk.

A number of types of inheritance of the disorder have been described, including sex-linked (X chromosome–linked) in 66%, autosomal recessive in 34%, and autosomal dominant in less than 1% of cases. Patients with the autosomal recessive form may have a less severe clinical course than patients with the X-linked form. CGD is a defect of neutrophil microbicidal ROS generation resulting from gp91phox deficiency. CGD is caused by a missense, nonsense, frameshift, splice, or deletion mutation in the genes for p22 phox, p40 phox, p47 phox, p67 phox (autosomal CGD), or gy91phox (X-linked CGD), which results in variable production of neutrophil-derived ROIs.

The onset of CGD is during infancy, with one third of patients dying before the age of 7 years because of infections. It was observed that in the presence of normal or elevated leukocyte counts, the neutrophilic granulocytes in vitro ingested and destroyed only streptococci, not staphylococci. Subsequent testing revealed that cells from patients with CGD can phagocytize non–H₂O₂-producing bacteria such as Staphylococcus aureus and gram-negative rods (e.g., Enterobacteriaceae), but cannot destroy them. In the X-linked form, the defective leukocytes fail to exhibit increased anaerobic metabolism during phagocytosis because of a cytochrome b558 deficiency (which expresses itself as a defect in the 91,000-Da glycoprotein membrane anchor of the cytochrome complex), or these defective leukocytes produce H₂O₂ because of a myeloperoxidase deficiency.

Patients with CGD have infections with catalase-positive bacteria and fungi affecting the skin, lungs, liver, and bones. They also develop granulomas, resulting from a lack of resolution of inflammatory foci, even after the infection has been eliminated. This leads to extensive granuloma formation and, in some circumstances, impairment of physiologic processes (e.g., obstruction of the esophagus or urinary tract).

Laboratory evaluation of CGD begins with non-specific testing to rule out other disorders. These assays include serum quantitative immunoglobulin, complement activity enzyme immunoassay, CBC with differential, myeloperoxidase stain and a neutrophil receptor profile. The evaluation of neutrophil phagocytic function is best determined by the neutrophil oxidative burst assay (DHR) via flow cytometry (see Appendix C) that can indicate CGD by the absence or significant alteration of activity. Other, less-reliable tests include measurement of superoxide production, ferrocytochrome reduction, and the classic nitroblue tetrazolium test (NBT).

Complement Receptor 3 Deficiency

The complement receptor 3 (CR3) deficiency is a rare condition inherited as an autosomal recessive trait. A deficiency of CR3 on phagocytic cells presents as a leukocyte adhesion deficiency. Leukocyte adhesion deficiency type 1 (LAD-1) is caused by a deficiency of CD18. LAD-2 is caused by the absence of sialyl–Lewis X (CD15s) blood group antigen.

A CR3 deficiency in neutrophils is associated with marked abnormalities of adherence-related functions, including decreased aggregation of neutrophils to each other after activation, decreased adherence of neutrophils to endothelial cells, poor adherence and phagocytosis of opsonized microorganisms, defective spreading, and decreased diapedesis and chemotaxis. Patients may also lack an intravascular marginating pool of neutrophils. Defects in T lymphocytes are characterized by faulty lymphocyte-mediated cytotoxicity, with poor adherence to target cells. Abnormalities of B lymphocytes have also been observed.

Clinically, a deficiency can manifest as delayed separation of the umbilical cord. Other signs and symptoms include early onset of bacterial infections, including skin infections, mucositis, otitis, gingivitis, and periodontitis. A depressed inflammatory response and neutrophilia can be observed.

Myeloperoxidase Deficiency

A deficiency of myeloperoxidase is inherited as an autosomal recessive trait on chromosome 17. Myeloperoxidase is an iron-containing heme protein responsible for the peroxidase activity characteristic of azurophilic granules; it accounts for the greenish color of pus. Human neutrophils contain many granules of various sizes that are morphologically, biochemically, and functionally distinct. The azurophilic granules normally contain myeloperoxidase. In this disorder, azurophilic granules are present, but myeloperoxidase is decreased or absent. If phagocytes are deficient in myeloperoxidase, the patient’s phagocytes manifest a mild to moderate defect in bacterial killing and a marked defect in fungal killing in vitro.

Persons with a myeloperoxidase deficiency are generally healthy and do not have an increased frequency of infection, probably because of other microbicidal mechanisms compensating for the deficiency. Patients with diabetes and myeloperoxidase deficiency, however, may have deep fungal infections caused by Candida spp.

Specific Granule Deficiency

Specific granule deficiency is believed to be an autosomal recessive disease. It is caused by a failure to synthesize specific granules and some contents of other granules during differentiation of neutrophils in the bone marrow. Patients with specific granule deficiency have recurrent, severe bacterial infections of the skin and deep tissues, with a depressed inflammatory response.

Monocyte-Macrophage Disorders

Monocytes-macrophages have been shown to be abnormal in a variety of diseases (Table 3-2). The abnormality is partial and no related association with increased susceptibility to infection has been established. In cases of severely depressed migration of monocytes, however, it is likely that this dysfunction predisposes a patient to infection because other defects of host defense coexist in these disorders.

Table 3-2

Primary and Secondary Abnormalities of Monocyte-Macrophage Function

Abnormality	Condition/Group
Defect in phagocyte killing	Chronic granulomatous disease, corticosteroid therapy, newborn infants, viral infections
Defective monocyte cytotoxicity	Cancer, Wiskott-Aldrich syndrome
Defective release of macrophage-activating factors	Acquired immunodeficiency syndrome (AIDS), intracellular infections (e.g., lepromatous leprosy, tuberculosis, visceral leishmaniasis)
Depressed migration	AIDS, burns, diabetes, immunosuppressive therapy, newborn infants
Impaired phagocytosis	Congenital deficiency of CD11 to CD18, monocytic leukemia, systemic lupus erythematosus