Innate Immunity Inflammation and Wound Healing

The complement system intensifies or complements the capacity of cells of the innate and adaptive immune systems to clear pathogens and damaged cells and to activate inflammation.6 Complement consists of a large number of proteins (sometimes called complement factors), which, together, constitute about 10% of the total circulating serum protein. Activation of the complement system initiates a cascade of proteolytic steps that result in the formation of several substances that can destroy pathogens directly or can eradicate pathogens through enhancing the activity of other components of the immune response.

The activation of C3 and C5, two important components of the complement cascade, results in the creation of several potent molecules critical to the immune response:

Three major pathways or cascades control the activation of complement (see Fig. 7.3A): the classical pathway, the alternative pathway, and the lectin pathway. The classical pathway is activated by antibodies, which are components of the adaptive immune system. Antibodies bind to antigens, which are typically proteins or carbohydrates produced by infectious microorganisms. Such antibodies activate the first complement component, C1, which, in turn, leads to the sequential activation of other components of the complement cascade, specifically C3 and C5, triggering inflammation.

The alternative pathway is activated directly by substances found on the surface of infectious microorganisms. These substances would include lipopolysaccharides (endotoxins) found on bacterial membranes and carbohydrates (zymosan) found on yeast cell walls. This pathway uses unique proteins (factor B, factor D, and properdin) to form a complex that sequentially activates the complement proteins C3 and C5. At this point, the process converges with the classical pathway. The alternative pathway provides a mechanism whereby the complement system can be activated in the absence of antibodies.

The lectin pathway, like the alternative pathway, is independent of antibodies and is activated by several plasma proteins, particularly mannose-binding lectin (MBL). MBL binds to bacterial polysaccharides that contain the carbohydrate mannose and activates the complement cascade. Infectious agents that do not activate the alternative pathway may still be susceptible to the complement system through the lectin pathway.

In summary, the complement pathway or cascade is a sequential series of cellular events that serve as defense mechanisms for the body. It can be activated by at least three different pathways, resulting in one or more of four protective functions: opsonization (C3b), anaphylatoxic activity from mast cell degranulation (C3a, C5a), leukocyte chemotaxis (C5a), and cell lysis (C5b–C9; membrane attack complex). Because of this potent defense system, successful pathogens must develop mechanisms that resist complement activity in order to cause disease.⁷

While highly beneficial to host defense, the complement system can contribute to tissue damage in a wide range of acute and chronic inflammatory diseases (see Chapter 9). New therapies aimed at blocking complement are proving to be beneficial for a number of these conditions.⁸

The clotting system (also known as the coagulation system) is a group of plasma proteins, which, when activated sequentially, form a blood clot which is a meshwork of fibrin strands and platelets. The clotting system can be activated by a variety of substances released during tissue injury or infection, such as collagen, enzymes, and bacterial toxins. Clots serve to plug damaged vessels and stop bleeding (hemostasis), trap microorganisms to prevent their spread to adjacent tissues, and provide a framework for future repair and healing. When the wall of a blood vessel is injured, two converging pathways lead to clot formation (see Fig. 7.3B):

Both pathways converge at factor X and thrombin. Thrombin activates fibrinogen to form fibrin. Fibrin, in turn, comes together to form a fibrin clot. The purposes of the fibrin clot are to:

Platelets also play a crucial role in inflammation, including the secretion of immunoregulatory cytokines and the ability to bind infectious pathogens.¹⁰ Additional details concerning the clotting system are discussed in Chapter 28.

The third plasma protein system, the kinin system (see Fig. 7.3C), interacts closely with the clotting system. Both the clotting and kinin systems can be initiated through activation of Hageman factor (factor XII), which, in turn, results in the formation of factor XIIa, also known as prekallikrein activator. Factor XIIa activates prekallikrein, the first component of the kinin system. The next product in the cascade is kallikrein, followed by kininogen, a precursor molecule, and then bradykinin, the final product of the kinin system. Bradykinin causes dilation of blood vessels. Bradykinin also acts in concert with prostaglandins to induce pain, trigger smooth muscle cell contraction (i.e., bronchoconstriction), and increase vascular permeability.

Multiple mechanisms are available to regulate the plasma protein systems, activating or inactivating the reactions to maintain balance, and contain the inflammatory response. Plasma entering the tissues during inflammation contains enzymes that destroy the mediators of inflammation and downregulate the inflammatory response. These enzymes include protease inhibitors (e.g., C1-inhibitor) and carboxypeptidase, which inhibit the complement system; and kininase and histaminase, which inhibit the kinin system. The formation of clots activates a fibrinolytic system. This system serves to limit the size of the clot and degrade the clot after bleeding has ceased. In this system thrombin activates plasminogen, forming the enzyme plasmin, which degrades the fibrin polymers in clots and has been implicated in a wide range of inflammatory diseases.11 Defects in these mechanisms can be genetic or acquired. For example, a genetic defect in the protease C1 inhibitor (C1-inh) results in hereditary angioedema, a self-limiting edema of cutaneous and mucosal layers. Acquired defects in plasminogen activation and control, which is seen in severe infection, can result in widespread clotting.¹²

Mast cells, macrophages, and dendritic cells have evolved a set of receptors referred to as pattern recognition receptors (PRRs) (Table 7.2). PRRs are generally expressed on cells in tissues near the body’s surfaces (skin, respiratory tract, GI tract, genitourinary tract), where they monitor the environment for products of cellular damage and infectious microorganisms. PRRs can also be found on mucosal epithelial cells, neutrophils, and some lymphocytes. PRRs recognize two types of molecular patterns:

An important group of PRRs are Toll-like receptors (TLRs), which recognize a large variety of PAMPs located on the surface of microorganisms (e.g., bacterial lipopolysaccharide, peptidoglycans, lipoproteins, yeast zymosan, flagellin, bacterial or viral nucleic acid, and viral coat proteins). TLRs are expressed on the surface of many cells that have direct and early contact with potential pathogens. Such cells include mucosal epithelial cells, mast cells, macrophages, dendritic cells, neutrophils, and lymphocytes. TLRs also are activated by oxidative stress, tissue injury, and damaged cellular contents (DAMPs). Activation of TLRs initiates a cascade of intracellular signaling pathways leading to the activation of nuclear factor-[kappa]B (NF-κB) in the cell nucleus.13 NF-κB activation results in increased transcription and subsequent release of numerous inflammatory cytokines. Among these are tumor necrosis factor (TNF), interleukins, and interferons (IFNs) which play key roles in the inflammatory process, activation of the adaptive immune response, and the destruction of many pathogenic microorganisms (these cytokines are discussed in detail later in this chapter). Pathophysiologic changes in TLR stimulation and NF-κB activation have been implicated in numerous conditions such as neuroinflammation, heart disease, and cancer¹³ (see Emerging Science Box: The Role of Toll-like Receptors [TLRs] and Nuclear Factor-kappaB [NF-κB] in Disease).¹³

Nucleotide-binding-like receptors (NLRs) (including nucleotide oligomerization domain–like [NOD-like] receptors) are cytoplasmic (intracellular) receptors in lymphocytes, macrophages, and dendritic cells. At least 23 NLRs have been identified in humans. They recognize intracellular microorganisms and damaged cells and initiate the production of proinflammatory mediators. NLRs also detect cells that are forming inflammasomes, which are large cytoplasmic complexes that activate cytokines. Inflammasomes are implicated in a wide variety of diseases including Coronavirus Disease 2019 (COVID-19) inflammatory lung damage.14

Scavenger receptors are membrane receptors primarily expressed on macrophages and are categorized into 12 classes.¹⁵ They have multiple functions including the recognition and subsequent phagocytosis of bacterial pathogens and damaged cells. They also recognize soluble lipoproteins, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and oxidized LDL, and have been implicated in atherosclerotic vascular diseases.¹⁶ C-type lectin receptors (CLRs) are a type of scavenger receptor that binds to both PAMPs and DAMPs. There are many types of CLRs, such as macrophage mannose, Dectin 1 and 2, and mannose-binding lectin receptors. They are particularly important in recognizing fungal antigens and subsequent activation of innate immune cells.

Complement and cytokine receptors are found on many cells involved in the immune response, as well as on some epithelial cells. These are not true PRRs but are essential to innate immune cell function. Complement receptors recognize several fragments produced through activation of the complement system, particularly C3a, C5a, and C3b. This results in chemotaxis and activation of innate immune cells. Cytokine receptors recognize both proinflammatory and anti-inflammatory cytokines (these cytokines are discussed later in this chapter).

Mast cells are significant and potent activators of the inflammatory response. They have abundant granules containing biochemical mediators, which are released in instances of pathogen invasion and tissue injury (Fig. 7.4). Mast cells are activated by PAMPs and DAMPs binding to PRRs, as well as by direct physical, chemical, and thermal injury. They also can be activated by components of the complement cascade and by immunoglobulin E (IgE) antibodies produced in allergies (see Chapter 9). Located in connective tissue and close to vessels, they can be found near the body's surfaces (skin, GI, and respiratory tract linings). A variety of stimuli associated with tissue injury can induce inflammation by triggering the release of potent soluble substances from mast cells. These substances are released in two ways:

Panel A shows a colorized photomicrograph of a mast cell at the center surrounded densely by intracellular granules. Panel B shows flowchart tracking the degranulation and synthesis of a mast cell. Degranulation releases the following substances: 1. Chemotactic factors include neutrophil chemotactic factor that attracts neutrophils, and eosinophil chemotactic factor that attracts eosinophils. 2. Histamine causes vascular dilation and permeability. 3. Cytokines, for example, T N F alpha, I L 4, 5, 6, and 13, include inflammation and adaptive immunity. Synthesis releases the following substances: 1. Phospholipase A 2 includes arachidonic acid, and platelet-activating factor that causes vascular effects and platelet activation. Arachidonic acid, in turn, releases the following substances: 1. 5-lipoxygenase include leukotrienes that cause vascular dilation and permeability and bronchoconstriction, and cyclooxygenase include prostaglandins that cause vascular dilation and permeability, and pain. 2. Growth factors include endothelial cell, connective tissue, and smooth muscle proliferation.

Degranulation

Mast cells release biochemical mediators from their granules into the surrounding tissues within seconds of a stimulus. Substances within the granules include histamine, chemotactic factors, and cytokines. Their effects occur immediately.

Histamine is a small-molecular-weight molecule with potent effects on many other cells, particularly those that control the circulation. Histamine binds to histamine receptors (H1, H2, H3, and H4 receptors) on various cell surfaces. The binding of histamine to the H1 receptor is proinflammatory (Fig. 7.5). This causes a rapid, temporary constriction of smooth muscle, along with dilation of the postcapillary venules. The net result of these two effects is increased blood flow within the microvasculature. H1 receptor binding also causes increased vascular permeability secondary to the retraction of endothelial cells lining the capillaries (opening the tight junctions between them), and increased leukocyte adherence to the endothelial walls. The H1 receptor also is present on smooth muscle cells within the bronchi. H1 stimulation results in bronchial smooth muscle contraction and bronchoconstriction often seen in asthma. Antihistamines are drugs that block the binding of histamine to the H1 receptor, resulting in decreased vascular effects. H4 receptors are highly expressed on mast cells where their stimulation is proinflammatory by promoting histamine and cytokine generation, and H4 receptor blockers are being developed in an effort to better treat many allergic and inflammatory disorders.¹⁷ The role of histamine receptors and hypersensitivity is discussed in Chapter 9.

An illustrated table summarizes the effects of histamine through H 1 and H 2 receptors. The illustration of a cell shows histamine being attracted to H 1 receptor and H 2 receptor. When bound to H 1 receptor, G T P is converted to c G M P, increasing its level and leading to cell activation. When bound to H 2 receptor, A T P is converted to c A M P, increasing its level and leading to cell inactivation. The table at the top identifies the target cell and lists the effect of histamine on the target cell. The data from the table for cell activation are as follows. • Smooth muscle cell: Contraction. • Endothelial cell: Contraction (retraction at endothelial junctions) • Neutrophil: Increased chemotaxis. • Mast cell: Prostaglandin synthesis. The data from the table for cell inactivation are as follows. • Parietal cell of stomach mucosa: secretion of gastric acid. • Lymphocyte: Decreased activity. • Eosinophil: Decreased activity. • Neutrophil: Decreased chemotaxis. • Mast cell: Decreased degranulation.

In contrast, histamine binding to the H2 receptor is generally anti-inflammatory because it results in the suppression of leukocyte function. Both H1 and H2 receptors are often present on the same immune cells where they may act in an antagonistic fashion. For example, stimulation of H1 receptors on neutrophils results in an augmentation of neutrophil chemotaxis, whereas stimulation of the H2 receptor results in its inhibition. The H2 receptor also is abundant on parietal cells of the stomach mucosa, where stimulation induces the secretion of gastric acid as part of the normal physiology of the stomach. H3 receptors inhibit the release of histamine and other neurotransmitters on neurons in the central nervous system. They are involved in blood–brain barrier function, and non-inflammatory processes including cognition, sleep, and regulation of food intake.18 Research into the role of H3 receptors in health and disease are revealing potential uses treating neurodegeneration (Alzheimer and Parkinson disease), migraine, narcolepsy, and control of eating in obesity.¹⁹

Mast cell granules also contain chemotactic factors (chemokines). Chemotaxis is directional movement of cells along a chemical gradient formed by a chemotactic factor. Two important factors are neutrophil chemotactic factor (NCF) and eosinophil chemotactic factor of anaphylaxis (ECF-A). Neutrophils are the predominant cells that destroy bacteria in inflammation. Eosinophils help regulate the inflammatory response. Both cells are discussed in more detail later in this chapter.

Synthesis of mediators

Activated mast cells initiate synthesis of other mediators of inflammation. These mediators include leukotrienes, prostaglandins, platelet-activating factor and growth factors (see Fig. 7.4B).

1. 1. Leukotrienes (slow-reacting substances of anaphylaxis [SRS-A]) are products of arachidonic acid. Arachidonic acid is released from mast cell membranes by intracellular phospholipase A2 which acts on membrane phospholipids. Leukotrienes are produced from arachidonic acid by the action of lipoxygenase and consist of several different subtypes (Fig. 7.6) Leukotrienes induce smooth muscle contraction (especially bronchoconstriction) and increased vascular permeability. Leukotrienes appear to be important in the later stages of the inflammatory response because they stimulate slower and more prolonged inflammatory responses than does the rapid-acting histamine.
2. 2. Prostaglandins also are products of arachidonic acid. They cause increased vascular permeability, neutrophil chemotaxis, and pain. Pain results from the direct effects on nerves. Prostaglandins are long-chain, unsaturated fatty acids produced by the action of cyclooxygenase (COX). COX exists in two different forms: COX-1, which produces prostaglandins that activate platelets and protect the stomach lining, and COX-2, which activates prostaglandins associated with inflammation (see Fig. 7.6). Aspirin, acetaminophen, and nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX-1 and/or COX-2. They suppress inflammation and improve symptoms but have associated side effects. NSAIDs can cause GI tract bleeding, hypertension, renal dysfunction, and cardiovascular disease especially in older individuals who take them regularly for underlying conditions such as osteoarthritis.²⁰
3. 3. Platelet-activating factor (PAF) molecules are produced by neutrophils, monocytes, endothelial cells, mast cells, and platelets. The biologic activity of PAF is similar to that of leukotrienes. It causes increased vascular permeability, leukocyte adhesion to endothelial cells, and platelet activation (see Fig. 7.6).
4. 4. Growth factors synthesized and released by mast cells include vascular endothelial growth factor (VEGF) which promotes endothelial cell proliferation, and platelet-derived growth factor (PDGF) which promotes connective tissue and smooth muscle proliferation (see Fig. 7.4B). These growth factors contribute to wound healing.

A flowchart tracks the production of lipid vasoactive substances by mast cells. The data from the flow chart is as follows. • Cell membrane phosphatidylcholine: phospholipase A 2. • Phospholipase A 2: P A F and arachidonic acid. • P A F: Enhanced inflammation. Eosinophil chemotaxis, vasoconstriction, bronchoconstriction, platelet activation (vasodilation, increased capillary permeability at low PAF concentrations). • Arachidonic acid: Cyclooxygenase and 5-Lipoxygenase. • Cyclooxygenase (C O X-1, C O X 2): P G D 2. Enhanced inflammation. Smooth muscle contraction (bronchoconstriction), increased capillary permeability, increased vasodilation. • 5-lipoxygenase: L T A 4. • L T A 4: L T C 4 and L T B 4. • L T C 4: L T D 4. L T E 4. Enhanced inflammation. Smooth muscle contraction (bronchoconstriction), increased capillary permeability, increased vasodilation. • L T B 4: Enhanced inflammation. Neutrophil chemotaxis.

Macrophages are derived from circulating monocytes. As with other blood cell types, monocytes are produced in bone marrow and enter into the circulation. They migrate to tissues throughout the body where they transform to macrophages. These tissue macrophages have many different names depending on where they are located, including Kupffer cells (liver), alveolar macrophages (lungs), and microglia (brain) (see Table 28.3). Tissue macrophages are important initial mediators of the inflammatory response.

Macrophages and dendritic cells have an abundance of PRRs on their surface that can recognize a wide range of pathogens and molecules released from damaged tissues. PRR receptor binding to PAMPs and DAMPs results in intracellular communication leading to activation of NF-κB and the subsequent release of proinflammatory chemokines and cytokines. Chemokines (a type of cytokine) are members of a family of low-molecular-weight peptides that function primarily to induce leukocyte chemotaxis. They facilitate the movement of phagocytes from the blood stream through the vessel wall into the area of injury. Cytokines constitute a family of intercellular signaling molecules that are secreted, bind to specific membrane receptors, and regulate innate and adaptive immunity. Macrophages release both proinflammatory and antiinflammatory cytokines (Fig. 7.7). The most important proinflammatory cytokines secreted by macrophages are tumor necrosis factor-alpha (TNF-α), interleukins, and IFNs.

An illustrated flowchart summarizes the tissue macrophage-derived cytokines. Tissue macrophage is a result of S Rs and N L Rs from tissues injury D A M Ps and T L Rs from microbial P A M Ps, and it causes: • Proinflammatory: T N F-alpha, interleukins (I L-1, I L-6), interferons (I F N-alpha, I N F-beta, I N F-gamma). • Anti-inflammatory: Interleukins (I L-10), Interferons (T G F-beta). Local effects from proinflammation: • Chemotaxis of leukocytes. • Leukocyte adhesion and migration. • Activate phagocytes. • Immune cell proliferation. • Promote adaptive immunity. Systemic effects from proinflammation: • Fever. • Increase in circulating white blood cells. • Increase in acute phase proteins.

Dendritic cells can be considered a specialized type of macrophage derived from a common precursor stem cell in the bone marrow. Some, like macrophages, differentiate from circulating monocytes. They provide one of the major links between the innate and adaptive immune responses. They are located in tissues that have contact with the outside environment, e.g. the gut, respiratory tract, and skin. Like tissue macrophages, they recognize invaders with PRRs and phagocytose them. They are different from tissue macrophages in that they migrate from the tissues through lymphatic vessels to lymph nodes, where they present antigens from the phagocytosed invaders to T lymphocytes resulting in an adaptive immune response (see Chapter 8). Dendritic cells produce many of the same cytokines as macrophages.

In addition to phagocytosis, neutrophils can form neutrophil extracellular traps (NETs) which are crucial in attacking pathogens. NETs are formed of material from cell nucleus breakdown (chromatin and histones) and neutrophil granules which contribute numerous enzymes that can degrade microorganisms. These enzymes include neutrophil elastase, myeloperoxidase, matrix metalloproteinases, and others.24 NET formation usually leads to the destruction of the neutrophil. NETs may also contribute to tissue damage and autoimmune diseases.²⁵

Monocyte-derived macrophages are also essential phagocytes in inflammation. They begin appearing at the inflammatory site as soon as 24 hours after the initial neutrophil infiltration but do not arrive in large numbers until 3 to 7 days later. They are attracted by chemotactic factors released by the neutrophils that are already present at the site. Macrophage activation results in two subpopulations, M1 and M2 macrophages, each with specialized functions. M1 macrophages are proinflammatory and have bactericidal activity, promote adaptive immunity, and can attack cancer cells.26M2 macrophages are primarily involved in tissue healing and repair. Macrophages can survive and divide in the acidic environment of the inflammatory site and so are capable of conducting prolonged phagocytosis of microorganisms and damaged cells. They play an essential role in removing debris and promoting the formation of new blood vessels (angiogenesis) and secretion of growth factors that prepare the tissue for healing.²⁷

Phagocytosis is the ingestion of microbes, foreign particles, or cell fragments. The two most important cells for phagocytizing pathogens or damaged cells are neutrophils and macrophages. Inflammatory cytokines and chemokines serve to activate these cells and through chemotactic factors cause them to move toward the area of injury. These cytokines also open the tight junctions between endothelial cells, creating spaces between them. Additionally, inflammation induces endothelial nitric oxide synthase (iNOS), increasing the amount of nitric oxide (NO) production. Effects of NO in inflammation include vasodilation and changes in inflammatory cell function.28

The first step in the migration of phagocytes to the area of invasion or injury starts when endothelial cells, monocytes, and neutrophils begin expressing new CAMs on their surfaces. The most important CAMs related to vascular inflammation are selectins, integrins, and intracellular adhesion molecules (ICAMs). These CAMs increase the adhesion, or stickiness, between phagocytes and endothelial cells in the walls of the capillaries. At first, the phagocytes roll down the endothelial surface. This is followed by tight binding to the endothelium in a process called margination, or pavementing (Fig. 7.8A). Other adhesion molecules such as platelet endothelial cell adhesion molecules (PECAMs) then are expressed leading to diapedesis or emigration of the cells through the openings between endothelial cells. The leukocytes digest the basement membrane and migrate into the surrounding tissues.²⁹ Monoclonal antibodies that block adhesion molecules are used to reduce inflammation in a variety of conditions including cancer (see Chapter 9).³⁰

Three panels, A, B, and C, illustrate tissue damage, recognition and attachment, and phagocytosis, respectively. Top panel, tissue damage. An illustration shows a splinter punctured through the epithelium, causing a wound. A capillary with normal endothelium and a basement membrane contains white blood cells. Adherence margination and diapedesis of the blood cells is illustrated inside the capillary. 1. Neutrophils in the capillary. 2. Margination: cell moves closer to the endothelium. 3. Retracted intercellular junction: an open space between two endothelium cells. 4. Endothelial cell retraction: cell passe through the junction, chemotaxis. Bacterium from the site of the wound enters the connective tissue, which is combated by the white blood cells as a result of chemotactic gradient. Bottom left panel, recognition and attachment. An illustration shows a bacterium. Ligands and receptors binding to the ligands around the bacterium are as follows: • A g: A b R through A b. • C 3 b: C 3 b R. • P A M P: P R R. Bottom right panel, phagocytosis. An illustration shows bacterium being engulfed into the cell. The following structures in the cell are identified. • Bacteria opsonized by A b and C 3 b. • Phagosome. • Phagolysosome. • Phagocyte. • Lysosome.

Once inside the connective tissue in the perivascular space, phagocytes migrate to the inflammatory site by means of chemotaxis. They detect chemotactic factors (chemokines) in the environment through chemoreceptors on their plasma membranes and migrate in the direction of highest concentration (see Fig. 7.8A). The most important chemotactic factors include bacterial products, complement fragments C3a and C5a, and chemokines. Neutrophils also respond to chemotactic factors released from mast cells (NCF). Monocyte-derived macrophages are attracted to monocyte chemotactic factor (MCF) that has already been released by neutrophils at the site of injury.

Phagocytosis at the inflammatory site involves four steps (see Fig. 7.8C):

Most phagocytes can trap and engulf pathogens using their PRRs which recognize PAMPs on the microorganisms (see Fig. 7.8B). However, some pathogens such as encapsulated bacteria (see Chapter 10) are more difficult to engulf. Opsonization greatly enhances adherence of the phagocyte to the target microorganism or cell. Opsonins that coat the target bacteria or cell act as a “glue” tightening the affinity between the phagocyte and the target, making phagocytosis more effective. The most efficient opsonins are C3b of the complement system and antibodies produced during the adaptive immune response.

Engulfment (endocytosis) is a process whereby the microorganism is drawn into the interior of a phagocytic cell. Engulfment is carried out by small pseudopods that extend from the plasma membrane of the phagocyte to surround the target, forming an intracellular phagocytic vacuole, or phagosome. Upon formation of a phagosome, the lysosomes (containing destructive enzymes) converge, fuse with the phagosome, and discharge their contents to create a phagolysosome. Destruction of the target takes place within the phagolysosome. Destruction is accomplished by both oxygen-dependent and oxygen-independent mechanisms.

Oxygen-dependent killing mechanisms result from the production of toxic oxygen species (e.g., hydrogen peroxide, singlet oxygen, or hydroxyl radicals). Phagocytosis is accompanied by a burst of oxygen uptake by the phagocyte. Chemical mediators and other reactive oxygen species are highly damaging to bacteria.

Oxygen-independent mechanisms of microbial killing include (1) the acidic pH (3.5 to 4.0) of the phagolysosome, (2) cationic proteins that bind to and damage target cell membranes, (3) enzymatic attack of the microorganism's cell wall by lysozyme and other enzymes, and (4) inhibition of bacterial growth by lactoferrin binding of iron (iron is essential for bacterial growth). Throughout the process, these toxic substances are isolated within membrane-bound vesicles. This isolation protects the phagocyte from harm secondary to the phagocyte's own enzymes.

Several bacteria are resistant to destruction by phagocytes and can survive inside macrophages. Microorganisms, such as Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy), Salmonella typhi (typhoid fever), Brucella abortus (brucellosis), and Listeria monocytogenes (listeriosis), can remain dormant or multiply inside the phagolysosomes of macrophages.³¹

In addition to removing pathogens and damaged cells, phagocytes (especially macrophages) are responsible for removing cells that have completed their normal life cycle. For example, white blood cells and erythrocytes are programmed to live for only weeks or months. These dead cells number in the billions per day and must be recognized and removed from the body via phagocytosis.³² Phagocytosis of a red blood cell is illustrated in Fig. 7.9.

Dying phagocytes can cause tissue damage at the site of inflammation. When a phagocyte dies at an inflammatory site, it frequently lyses (breaks open), releasing its enzymatic contents into the tissue. These enzymes can cause inflammation-associated tissue destruction. The destructive effects of enzymes released by dying phagocytes are minimized by natural inhibitors found in blood. Examples of such inhibitors would include catalase, which breaks down hydrogen peroxide, and alpha1-antitrypsin (α1-antitrypsin), a protease inhibitor produced by the liver. An inherited deficiency of α₁-antitrypsin, a disease known as alpha-1 antitrypsin deficiency, results in pulmonary emphysema secondary to chronic lung inflammation, even in nonsmokers (see Chapter 35).³³

Another type of innate immune cell is the eosinophil. Although eosinophils are only mildly phagocytic, they serve as the body's primary defense against parasites and regulate vascular mediators released from mast cells. Lysosomal granules within the eosinophil contain enzymes that degrade vasoactive substances from mast cells, thus limiting and controlling the vascular effects of inflammation. Eosinophils also are important components of allergic conditions and can contribute to tissue damage; these effects are discussed in Chapter 9.

The basophil is the least prevalent granulocyte in the blood. It is similar to mast cells with respect to the contents of its granules. Basophilic granules also contain heparin, a naturally occurring anticoagulation product. Basophils release histamine which, as discussed previously, has potent vasoactive properties. Basophils are an important source of cytokines involved in the adaptive immune response, particularly responses associated with allergies and asthma.

Lymphocytes, another type of leukocyte, initiate specific, protective immune responses against pathogens and cancer. B lymphocytes produce antibodies, and T lymphocytes regulate other immune cells and kill viruses and cancer cells (see Chapter 8). Natural killer (NK) cells, a type of lymphocyte, eliminate virally infected and cancerous cells. NK cells have inhibitory and activating receptors that allow differentiation between infected or tumor cells and normal cells. If the NK cell binds to a target cell through activating receptors, it produces several cytokines and toxic molecules, which, in turn, kill the target cell. Innate lymphoid cells (ILCs) also are derived from lymphocyte precursors that reside near mucosal surfaces. There are three major groups of ILCs (ILC1s, ILC2s, and ILC3s) which contribute in different ways to preventing pathogen invasion and activating both the innate and adaptive immune responses (see Chapter 8).³⁴

The classic or cardinal signs of the acute local inflammatory response were described in the first century by a Roman named Celsus. They include the following:

Inflammatory exudates result from increased vascular permeability and the leakage of fluid into tissues. Exudates vary in composition, depending on the stage of inflammation, and, to a lesser extent, the triggering event. In early or mild inflammation, the exudate may be a serous exudate (watery) with very few plasma proteins or leukocytes (e.g., fluid in a blister). In more severe or advanced inflammation, the exudate may be a fibrinous exudate (thick and clotted) (e.g., the fluid exudates in the lungs of an individual with pericarditis). A purulent exudate (pus) is the accumulation of a large number of leukocytes, as occurs in bacterial infections (e.g., sputum in persons with pneumonia). A purulent exudate can also occur within walled-off lesions, known as cysts or abscesses. When bleeding occurs, the exudate is filled with erythrocytes and is described as a hemorrhagic exudate.

Leukocytosis is an increase in the number of circulating white blood cells beyond the upper limit of normal (11,000/mL³ in adults) (see Chapter 29). Inflammation stimulates the proliferation and release of granulocyte and monocyte precursors in bone marrow. The cells migrate into the systemic circulation resulting in both an increase in the total number of circulating leukocytes and an increased ratio of immature to mature cell forms. In particular, increased numbers of immature neutrophil forms such as band cells, metamyelocytes, and (rarely) myelocytes are released from the marrow into blood causing an abnormal complete blood count (CBC), a common laboratory analysis of blood cells. The differential count is a measure of the ratio or proportion of each of the various blood cell types circulating in the blood. An increase in the total number of white blood cells in combination with an abnormal differential count is a clue that there may be an ongoing infection.

Laboratory tests that measure levels of acute-phase reactants are available. For example, an increase in blood levels of the acute-phase reactants fibrinogen is associated with an increased adhesiveness of erythrocytes. Erythrocytes adhere to one another under these circumstances, forming large clumps that are buoyant and slow to settle to the bottom of a test tube of blood. This results in an increased value for the laboratory test referred to as erythrocyte sedimentation rate (ESR) or, as it is more commonly known, the “sed rate.” Although this increased ESR is a nonspecific reaction, persons found to have an elevated ESR will likely have an inflammatory process going on somewhere within the body. Another common laboratory measure of inflammation is the C-reactive protein (CRP), a laboratory measurement which also is increased during an inflammatory response. CRP is used to look for subclinical inflammation in individuals at risk for heart disease and to estimate the severity of certain infectious and autoimmune diseases.35

In its simplest terms, the difference between acute and chronic inflammation is duration. Chronic inflammation is slow, long-term inflammation lasting for prolonged periods of several months to years. It is characterized by low-grade persistent inflammatory changes that result in collateral damage to tissues.36 Virtually all chronic diseases are characterized by a component of chronic inflammation. Although the cause of defective resolution and a persistent inflammatory response is often unknown, some common conditions associated with chronic inflammation include increasing age, cardiac and neurologic disorders, malignancy, autoimmune disorders, and chronic infection.

Chronic inflammation is most often thought to be preceded by an unsuccessful acute inflammatory response, although that initial insult may not have been recognized (see Algorithm 7.3). For example, if bacterial contamination or foreign objects (dirt, wood splinter, silica, glass, etc.) persist in a wound, the inflammatory response will be prolonged. Some microorganisms are resistant to killing by the acute inflammatory response or may produce toxins which damage tissue and cause persistent inflammation, even after the original microorganism is killed. Chronic inflammation also can occur as a result of defective resolution of acute inflammation, due to an inability to appropriately dampen the innate immune response.³⁷ Trained immunity in which innate immune cells remain hyperreactive to inflammatory stimuli may also play a role (see Emerging Science Box: Understanding the Causes of Chronic Inflammatory Disease).³⁸

There are two major types of chronic inflammation (Fig. 7.10). Nonspecific proliferative chronic inflammation is characterized by the infiltration of lymphocytes and macrophages followed by a proliferation of fibroblasts, connective tissue, and epithelial cells. This often leads to scar or polyp formation. Granulomatous chronic inflammation occurs when macrophages and eosinophils attempt to wall off and isolate a foreign body or an infected area by forming a granuloma (Fig. 7.11). Infections caused by certain bacteria (tuberculosis, listeriosis, brucellosis), fungi (histoplasmosis, coccidioidomycosis), and parasites (leishmaniasis, schistosomiasis, toxoplasmosis) commonly result in granuloma formation. Many autoimmune conditions also are associated with granulomatous tissue changes.³⁹

An illustration identifies the types of chronic inflammation. Persistent inflammatory responses: • Proinflammatory cytokine release. • Neutrophil degranulation and death. • Lymphocyte activation. • Fibroblast activation. All responses lead to nonspecific proliferative chronic inflammation (repair or scar) and granulomatous chronic inflammation. A closeup of a hypertrophic scar shows a thin line of raised fibrous tissue running around the upper throat of a person. A photomicrograph of cells affected by tuberculous granuloma shows a smooth central area (amorphous caseous necrosis) and some pits indicating langhans giant cells, surrounded by epithelioid macrophage cells and lymphocytes.

A photomicrograph of cells affected by tuberculous granuloma shows a smooth central area (amorphous caseous necrosis) and some pits indicating langhans giant cells, surrounded by epithelioid macrophage cells and lymphocytes.

Granuloma formation is mediated by TNF-α and other proinflammatory cytokines. Macrophages are called to the area of persistent inflammation, where some differentiate into large epithelioid cells, which specialize in taking up debris and other small particles. Other macrophages fuse into multinucleated giant cells, which are active phagocytes, which can engulf particles larger than those that can be engulfed by a single

macrophage. These two types of specialized cells form the center of the granuloma and are surrounded by lymphocytes. The granuloma itself is often encapsulated by fibrous deposits of collagen. It may become cartilaginous or even calcified by deposits of calcium carbonate and calcium phosphate.