Stimuli for Acute Inflammation

Acute inflammatory reactions may be triggered by a variety of stimuli:

Although each of these stimuli may induce reactions with some distinctive characteristics, in general, all inflammatory reactions have the same basic features.

In this section, we describe first how inflammatory stimuli are recognized by the host, then the typical reactions of acute inflammation and its morphologic features, and finally the chemical mediators responsible for these reactions.

Recognition of Microbes, Necrotic Cells, and Foreign Substances

A fundamental question relating to activation of the host response is how cells recognize the presence of potentially harmful agents such as microbes in the tissues. It was postulated that microbes and dead cells must elicit some sort of “danger signals” that distinguish them from normal tissues and mobilize the host response. It is now established that phagocytes, dendritic cells (cells in connective tissue and organs that capture microbes and initiate responses to them), and many other cells, such as epithelial cells, express receptors that are designed to sense the presence of infectious pathogens and substances released from dead cells. These receptors have been called “pattern recognition receptors” because they recognize structures (i.e., molecular patterns) that are common to many microbes or to dead cells. The two most important families of these receptors are the following:

Figure 2–3 Sensors of microbes and dead cells: Phagocytes, dendritic cells, and many types of epithelial cells express different classes of receptors that sense the presence of microbes and dead cells. A, Toll-like receptors (TLRs) located in the plasma membrane and endosomes and other cytoplasmic and plasma membrane receptors (members of families other than TLRs) recognize products of different classes of microbes. The proteins produced by TLR activation have numerous functions; only their role in inflammation is shown. B, The inflammasome is a protein complex that recognizes products of dead cells and some microbes and induces the secretion of biologically active interleukin-1 (IL-1). The inflammasome consists of a sensor protein (a leucine-rich protein called NLRP3), an adaptor, and the enzyme caspase-1, which is converted from an inactive to an active form. (Note that the inflammasome is distinct from phagolysosomes, which also are present in the cytoplasm but are vesicles that serve different functions in inflammation, as discussed later in the chapter.) CPP, calcium pyrophosphate; MSU, monosodium urate.

The functions of these sensors are referred to throughout the chapter. We now proceed with a discussion of the principal reactions of acute inflammation.

Vascular Changes

The main vascular reactions of acute inflammation are increased blood flow secondary to vasodilation and increased vascular permeability, both designed to bring blood cells and proteins to sites of infection or injury. While the initial encounter of an injurious stimulus, such as a microbe, is with macrophages and other cells in the connective tissue, the vascular reactions triggered by these interactions soon follow and dominate the early phase of the response.

Changes in Vascular Caliber and Flow

Changes in blood vessels are initiated rapidly after infection or injury but evolve at variable rates, depending on the nature and severity of the original inflammatory stimulus.

• After transient vasoconstriction (lasting only for seconds), arteriolar vasodilation occurs, resulting in locally increased blood flow and engorgement of the down-stream capillary beds (Fig. 2–2). This vascular expansion is the cause of the redness (erythema) and warmth characteristic of acute inflammation, and mentioned previously as two of the cardinal signs of inflammation.

• The microvasculature becomes more permeable, and protein-rich fluid moves into the extravascular tissues. This causes the red cells in the flowing blood to become more concentrated, thereby increasing blood viscosity and slowing the circulation. These changes are reflected microscopically by numerous dilated small vessels packed with red blood cells, called stasis.

• As stasis develops, leukocytes (principally neutrophils) begin to accumulate along the vascular endothelial surface—a process called margination. This is the first step in the journey of the leukocytes through the vascular wall into the interstitial tissue (described later).

Increased Vascular Permeability

Increasing vascular permeability leads to the movement of protein-rich fluid and even blood cells into the extravascular tissues (Fig. 2–4). This in turn increases the osmotic pressure of the interstitial fluid, leading to more outflow of water from the blood into the tissues. The resulting protein-rich fluid accumulation is called an exudate. Exudates must be distinguished from transudates, which are interstitial fluid accumulations caused by increased hydrostatic pressure, usually a consequence of reduced venous return. Transudates typically contain low concentrations of protein and few or no blood cells. Fluid accumulation in extravascular spaces, whether from an exudate or a transudate, produces tissue edema. Whereas exudates are typical of inflammation, transudates accumulate in various noninflammatory conditions, which are mentioned in Figure 2–4 and described in more detail in Chapter 3.

Figure 2–4 Formation of transudates and exudates. A, Normal hydrostatic pressure (blue arrows) is approximately 32 mm Hg at the arterial end of a capillary bed and 12 mm Hg at the venous end; the mean colloid osmotic pressure of tissues is approximately 25 mm Hg (green arrows), which is nearly equal to the mean capillary pressure. Therefore, the net flow of fluid across the vascular bed is almost nil. B, A transudate is formed when fluid leaks out because of increased hydrostatic pressure or decreased osmotic pressure. C, An exudate is formed in inflammation because vascular permeability increases as a result of the increase in interendothelial spaces.

Several mechanisms may contribute to increased vascular permeability in acute inflammatory reactions:

• Endothelial cell contraction leading to intercellular gaps in postcapillary venules is the most common cause of increased vascular permeability. Endothelial cell contraction occurs rapidly after binding of histamine, bradykinin, leukotrienes, and many other mediators to specific receptors, and is usually short-lived (15 to 30 minutes). A slower and more prolonged retraction of endothelial cells, resulting from changes in the cytoskeleton, may be induced by cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1). This reaction may take 4 to 6 hours to develop after the initial trigger and persist for 24 hours or more.

• Endothelial injury results in vascular leakage by causing endothelial cell necrosis and detachment. Endothelial cells are damaged after severe injury such as with burns and some infections. In most cases, leakage begins immediately after the injury and persists for several hours (or days) until the damaged vessels are thrombosed or repaired. Venules, capillaries, and arterioles can all be affected, depending on the site of the injury. Direct injury to endothelial cells may also induce a delayed prolonged leakage that begins after a delay of 2 to 12 hours, lasts for several hours or even days, and involves venules and capillaries. Examples are mild to moderate thermal injury, certain bacterial toxins, and x- or ultraviolet irradiation (i.e., the sunburn that has spoiled many an evening after a day in the sun). Endothelial cells may also be damaged as a consequence of leukocyte accumulation along the vessel wall. Activated leukocytes release many toxic mediators, discussed later, that may cause endothelial injury or detachment.

• Increased transcytosis of proteins by way of an intracellular vesicular pathway augments venular permeability, especially after exposure to certain mediators such as vascular endothelial growth factor (VEGF). Transcytosis occurs through channels formed by fusion of intracellular vesicles.

• Leakage from new blood vessels. As described later, tissue repair involves new blood vessel formation (angiogenesis). These vessel sprouts remain leaky until proliferating endothelial cells mature sufficiently to form intercellular junctions. New endothelial cells also have increased expression of receptors for vasoactive mediators, and some of the factors that stimulate angiogenesis (e.g., VEGF) also directly induce increased vascular permeability.

Although these mechanisms of vascular permeability are separable, all of them may participate in the response to a particular stimulus. For example, in a thermal burn, leakage results from chemically mediated endothelial contraction, as well as from direct injury and leukocyte-mediated endothelial damage.

Responses of Lymphatic Vessels

In addition to blood vessels, lymphatic vessels also participate in the inflammatory response. In inflammation, lymph flow is increased and helps drain edema fluid, leukocytes, and cell debris from the extravascular space. In severe inflammatory reactions, especially to microbes, the lymphatics may transport the offending agent, contributing to its dissemination. The lymphatics may become secondarily inflamed (lymphangitis), as may the draining lymph nodes (lymphadenitis). Inflamed lymph nodes are often enlarged because of hyperplasia of the lymphoid follicles and increased numbers of lymphocytes and phagocytic cells lining the sinuses of the lymph nodes. This constellation of pathologic changes is termed reactive, or inflammatory, lymphadenitis (Chapter 11). For clinicians, the presence of red streaks near a skin wound is a telltale sign of an infection in the wound. This streaking follows the course of the lymphatic channels and is diagnostic of lymphangitis; it may be accompanied by painful enlargement of the draining lymph nodes, indicating lymphadenitis.

  Summary

Vascular Reactions in Acute Inflammation

• Vasodilation is induced by chemical mediators such as histamine (described later) and is the cause of erythema and stasis of blood flow.

• Increased vascular permeability is induced by histamine, kinins, and other mediators that produce gaps between endothelial cells; by direct or leukocyte-induced endothelial injury; and by increased passage of fluids through the endothelium. This increased permeability allows plasma proteins and leukocytes to enter sites of infection or tissue damage; fluid leak through blood vessels results in edema.

Cellular Events: Leukocyte Recruitment and Activation

As mentioned earlier, an important function of the inflammatory response is to deliver leukocytes to the site of injury and to activate them. Leukocytes ingest offending agents, kill bacteria and other microbes, and eliminate necrotic tissue and foreign substances. A price that is paid for the defensive potency of leukocytes is that once activated, they may induce tissue damage and prolong inflammation, since the leukocyte products that destroy microbes can also injure normal host tissues. Therefore, host defense mechanisms include checks and balances that ensure that leukocytes are recruited and activated only when and where they are needed (i.e., in response to foreign invaders and dead tissues). Systemic activation of leukocytes can, in fact, have detrimental consequences, as in septic shock (Chapter 3).

Leukocyte Recruitment

Leukocytes normally flow rapidly in the blood, and in inflammation, they have to be stopped and brought to the offending agent or the site of tissue damage, which are typically outside the vessels. The sequence of events in the recruitment of leukocytes from the vascular lumen to the extravascular space consists of (1) margination and rolling along the vessel wall; (2) firm adhesion to the endothelium; (3) transmigration between endothelial cells; and (4) migration in interstitial tissues toward a chemotactic stimulus (Fig. 2–5). Rolling, adhesion, and transmigration are mediated by the interactions of adhesion molecules on leukocytes and endothelial surfaces (see later on). Chemical mediators—chemoattractants and certain cytokines—affect these processes by modulating the surface expression and binding affinity of the adhesion molecules and by stimulating directional movement of the leukocytes.

Figure 2–5 Mechanisms of leukocyte migration through blood vessels. The leukocytes (neutrophils shown here) first roll, then become activated and adhere to endothelium, then transmigrate across the endothelium, pierce the basement membrane, and migrate toward chemoattractants emanating from the source of injury. Different molecules play predominant roles in different steps of this process: selectins in rolling; chemokines (usually displayed bound to proteoglycans) in activating the neutrophils to increase avidity of integrins; integrins in firm adhesion; and CD31 (PECAM-1) in transmigration. ICAM-1, intercellular adhesion molecule-1; IL-1, interleukin-1; PECAM-1, platelet endothelial cell adhesion molecule-1; TNF, tumor necrosis factor.

Margination and Rolling

As blood flows from capillaries into postcapillary venules, circulating cells are swept by laminar flow against the vessel wall. Because the smaller red cells tend to move faster than the larger white cells, leukocytes are pushed out of the central axial column and thus have a better opportunity to interact with lining endothelial cells, especially as stasis sets in. This process of leukocyte accumulation at the periphery of vessels is called margination. If the endothelial cells are activated by cytokines and other mediators produced locally, they express adhesion molecules to which the leukocytes attach loosely. These cells bind and detach and thus begin to tumble on the endothelial surface, a process called rolling.

The weak and transient interactions involved in rolling are mediated by the selectin family of adhesion molecules (Table 2–2). Selectins are receptors expressed on leukocytes and endothelium that contain an extracellular domain that binds sugars (hence the lectin part of the name). The three members of this family are E-selectin (also called CD62E), expressed on endothelial cells; P-selectin (CD62P), present on platelets and endothelium; and L-selectin (CD62L), on the surface of most leukocytes. Selectins bind sialylated oligosaccharides (e.g., sialyl–Lewis X on leukocytes) that are attached to mucin-like glycoproteins on various cells. The endothelial selectins are typically expressed at low levels or are not present at all on unactivated endothelium, and are up-regulated after stimulation by cytokines and other mediators. Therefore, binding of leukocytes is largely restricted to endothelium at sites of infection or tissue injury (where the mediators are produced). For example, in unactivated endothelial cells, P-selectin is found primarily in intracellular Weibel-Palade bodies; however, within minutes of exposure to mediators such as histamine or thrombin, P-selectin is distributed to the cell surface, where it can facilitate leukocyte binding. Similarly, E-selectin and the ligand for L-selectin, which are not expressed on normal endothelium, are induced after stimulation by the cytokines IL-1 and TNF.

Table 2–2 Endothelial and Leukocyte Adhesion Molecules

Endothelial Molecule	Leukocyte Molecule	Major Role(s)
Selectins and Selectin Ligands
P-selectin	Sialyl–Lewis X–modified proteins	Rolling
E-selectin	Sialyl–Lewis X–modified proteins	Rolling and adhesion
GlyCam-1, CD34	L-selectin*	Rolling (neutrophils, monocytes)
Integrins and Integrin Ligands
ICAM-1 (immunoglobulin family)	CD11/CD18 integrins (LFA-1, Mac-1)	Firm adhesion, arrest, transmigration
VCAM-1 (immunoglobulin family)	VLA-4 integrin	Adhesion
Others
CD31	CD31 (homotypic interaction)	Transmigration of leukocytes through endothelium

ICAM-1, intercellular adhesion molecule-1; LFA-1, leukocyte function–associated antigen-1; Mac-1, macrophage-1 antigen; VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late antigen-4.

* L-selectin is also involved in the binding of circulating lymphocytes to the high endothelial venules in lymph nodes and mucosal lymphoid tissues, and subsequent homing of lymphocytes to these tissues.

Adhesion

The rolling leukocytes are able to sense changes in the endothelium that initiate the next step in the reaction of leukocytes, which is firm adhesion to endothelial surfaces. This adhesion is mediated by integrins expressed on leukocyte cell surfaces interacting with their ligands on endothelial cells (Fig. 2–5 and Table 2–2). Integrins are transmembrane heterodimeric glycoproteins that mediate the adhesion of leukocytes to endothelium and of various cells to the extracellular matrix. They are normally expressed on leukocyte plasma membranes in a low-affinity form and do not adhere to their specific ligands until the leukocytes are activated by chemokines.

Chemokines are chemoattractant cytokines that are secreted by many cells at sites of inflammation and are displayed on the endothelial surface. (Cytokines are described later in the chapter.) When the adherent leukocytes encounter the displayed chemokines, the cells are activated, and their integrins undergo conformational changes and cluster together, thus converting to a high-affinity form. At the same time, other cytokines, notably TNF and IL-1 (also secreted at sites of infection and injury), activate endothelial cells to increase their expression of ligands for integrins. These ligands include intercellular adhesion molecule-1 (ICAM-1), which binds to the integrins leukocyte function–associated antigen-1 (LFA-1) (also called CD11a/CD18) and macrophage-1 antigen (Mac-1) (i.e., CD11b/CD18), and vascular cell adhesion molecule-1 (VCAM-1), which binds to the integrin very late antigen-4 (VLA-4) (Table 2–2). Engagement of integrins by their ligands delivers signals to the leukocytes that lead to cytoskeletal changes that mediate firm attachment to the substrate. Thus, the net result of cytokine-stimulated increased integrin affinity and increased expression of integrin ligands is stable attachment of leukocytes to endothelial cells at sites of inflammation.

Transmigration

After being arrested on the endothelial surface, leukocytes migrate through the vessel wall primarily by squeezing between cells at intercellular junctions. This extravasation of leukocytes, called diapedesis, occurs mainly in the venules of the systemic vasculature; it has also been noted in capillaries in the pulmonary circulation. Migration of leukocytes is driven by chemokines produced in extravascular tissues, which stimulate movement of the leukocytes toward their chemical gradient. In addition, platelet endothelial cell adhesion molecule-1 (PECAM-1) (also called CD31), a cellular adhesion molecule expressed on leukocytes and endothelial cells, mediates the binding events needed for leukocytes to traverse the endothelium. After passing through the endothelium, leukocytes secrete collagenases that enable them to pass through the vascular basement membrane.

Chemotaxis

After extravasating from the blood, leukocytes move toward sites of infection or injury along a chemical gradient by a process called chemotaxis. Both exogenous and endogenous substances can be chemotactic for leukocytes, including the following:

• Bacterial products, particularly peptides with N-formylmethionine termini

• Cytokines, especially those of the chemokine family

• Components of the complement system, particularly C5

• Products of the lipoxygenase pathway of arachidonic acid (AA) metabolism, particularly leukotriene B4 (LTB4)

These mediators, which are described in more detail later, are produced in response to infections and tissue damage and during immunologic reactions. Leukocyte infiltration in all of these situations results from the actions of various combinations of mediators.

Chemotactic molecules bind to specific cell surface receptors, which triggers the assembly of cytoskeletal contractile elements necessary for movement. Leukocytes move by extending pseudopods that anchor to the ECM and then pull the cell in the direction of the extension. The direction of such movement is specified by a higher density of chemokine receptors at the leading edge of the cell. Thus, leukocytes move to and are retained at the site where they are needed.

The type of emigrating leukocyte varies with the age of the inflammatory response and with the type of stimulus. In most forms of acute inflammation, neutrophils predominate in the inflammatory infiltrate during the first 6 to 24 hours and are replaced by monocytes in 24 to 48 hours (Fig. 2–6). Several factors account for this early abundance of neutrophils: These cells are the most numerous leukocytes in the blood, they respond more rapidly to chemokines, and they may attach more firmly to the adhesion molecules that are rapidly induced on endothelial cells, such as P- and E-selectins. In addition, after entering tissues, neutrophils are short-lived—they die by apoptosis and disappear within 24 to 48 hours—while monocytes survive longer. There are exceptions to this pattern of cellular infiltration, however. In certain infections (e.g., those caused by Pseudomonas organisms), the cellular infiltrate is dominated by continuously recruited neutrophils for several days; in viral infections, lymphocytes may be the first cells to arrive; and in some hypersensitivity reactions, eosinophils may be the main cell type.

Figure 2–6 Nature of leukocyte infiltrates in inflammatory reactions. The photomicrographs show an inflammatory reaction in the myocardium after ischemic necrosis (infarction). A, Early (neutrophilic) infiltrates and congested blood vessels. B, Later (mononuclear) cellular infiltrates. C, The approximate kinetics of edema and cellular infiltration. For sake of simplicity, edema is shown as an acute transient response, although secondary waves of delayed edema and neutrophil infiltration also can occur.

  Summary

Leukocyte Recruitment to Sites of Inflammation

• Leukocytes are recruited from the blood into the extravascular tissue, where infectious pathogens or damaged tissues may be located, and are activated to perform their functions.

• Leukocyte recruitment is a multi-step process consisting of loose attachment to and rolling on endothelium (mediated by selectins); firm attachment to endothelium (mediated by integrins); and migration through interendothelial spaces.

• Various cytokines promote expression of selectins and integrin ligands on endothelium (TNF, IL-1), increase the avidity of integrins for their ligands (chemokines), and promote directional migration of leukocytes (also chemokines); many of these cytokines are produced by tissue macrophages and other cells responding to pathogens or damaged tissues.

• Neutrophils predominate in the early inflammatory infiltrate and are later replaced by macrophages.

Leukocyte Activation

Once leukocytes have been recruited to the site of infection or tissue necrosis, they must be activated to perform their functions. Stimuli for activation include microbes, products of necrotic cells, and several mediators that are described later. As described earlier, leukocytes use various receptors to sense the presence of microbes, dead cells, and foreign substances. Engagement of these cellular receptors induces a number of responses in leukocytes that are part of their normal defensive functions and are grouped under the term leukocyte activation (Fig. 2–7). Leukocyte activation results in the enhancement of the following functions:

• Phagocytosis of particles

• Intracellular destruction of phagocytosed microbes and dead cells by substances produced in phagosomes, including reactive oxygen and nitrogen species and lysosomal enzymes

• Liberation of substances that destroy extracellular microbes and dead tissues, which are largely the same as the substances produced within phagocytic vesicles. A recently discovered mechanism by which neutrophils destroy extracellular microbes is the formation of extracellular “traps.”

• Production of mediators, including arachidonic acid metabolites and cytokines, that amplify the inflammatory reaction, by recruiting and activating more leukocytes

Figure 2–7 Leukocyte activation. Different classes of cell surface receptors of leukocytes recognize different stimuli. The receptors initiate responses that mediate the functions of the leukocytes. Only some receptors are depicted (see text for details). Lipopolysaccharide (LPS) first binds to a circulating LPS-binding protein (not shown). IFN-γ, interferon-γ.

Phagocytosis

Phagocytosis consists of three steps (Fig. 2–8): (1) recognition and attachment of the particle to the ingesting leukocyte; (2) engulfment, with subsequent formation of a phagocytic vacuole; and (3) killing and degradation of the ingested material.

Figure 2–8 Phagocytosis. Phagocytosis of a particle (e.g., a bacterium) involves (1) attachment and binding of the particle to receptors on the leukocyte surface, (2) engulfment and fusion of the phagocytic vacuole with granules (lysosomes), and (3) destruction of the ingested particle. iNOS, inducible nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species.

Leukocytes bind and ingest most microorganisms and dead cells by means of specific surface receptors. Some of these receptors recognize components of the microbes and dead cells and other receptors recognize host proteins, called opsonins, that coat microbes and target them for phagocytosis (the process called opsonization). The most important opsonins are antibodies of the immunoglobulin G (IgG) class that bind to microbial surface antigens, breakdown products of the complement protein C3 (described later), and plasma carbohydrate-binding lectins called collectins, which bind to microbial cell wall sugar groups. These opsonins either are present in the blood ready to coat microbes or are produced in response to the microbes. Leukocytes express receptors for opsonins that facilitate rapid phagocytosis of the coated microbes. These receptors include the Fc receptor for IgG (called FcγRI), complement receptors 1 and 3 (CR1 and CR3) for complement fragments, and C1q for the collectins.

Binding of opsonized particles to these receptors triggers engulfment and induces cellular activation that enhances degradation of ingested microbes. In engulfment, pseudopods are extended around the object, eventually forming a phagocytic vacuole. The membrane of the vacuole then fuses with the membrane of a lysosomal granule, resulting in discharge of the granule’s contents into the phagolysosome.

Killing and Degradation of Phagocytosed Microbes

The culmination of the phagocytosis of microbes is killing and degradation of the ingested particles. The key steps in this reaction are the production of microbicidal substances within lysosomes and fusion of the lysosomes with phagosomes, thus exposing the ingested particles to the destructive mechanisms of the leukocytes (Fig. 2–8). The most important microbicidal substances are reactive oxygen species (ROS) and lysosomal enzymes. The production of ROS involves the following steps:

• Phagocytosis and the engagement of various cellular receptors stimulate an oxidative burst, also called the respiratory burst, which is characterized by a rapid increase in oxygen consumption, glycogen catabolism (glycogenolysis), increased glucose oxidation, and production of ROS. The generation of the oxygen metabolites is due to rapid activation of a leukocyte NADPH oxidase, called the phagocyte oxidase, which oxidizes NADPH (reduced nicotinamide adenine dinucleotide phosphate) and, in the process, converts oxygen to superoxide ion () (see Fig. 1–18, B, Chapter 1).

• Superoxide is then converted by spontaneous dismutation into hydrogen peroxide ( + 2H⁺ → H₂O₂). These ROS act as free radicals and destroy microbes by mechanisms that were described in Chapter 1.

• The quantities of H₂O₂ produced generally are insufficient to kill most bacteria (although superoxide and hydroxyl radical formation may be sufficient to do so). However, the lysosomes of neutrophils (called azurophilic granules) contain the enzyme myeloperoxidase (MPO), and in the presence of a halide such as Cl⁻, MPO converts H₂O₂ to HOCl^• (hypochlorous radical). HOCl^• is a powerful oxidant and antimicrobial agent (NaOCl is the active ingredient in chlorine bleach) that kills bacteria by halogenation, or by protein and lipid peroxidation.

Fortunately, the phagocyte oxidase is active only after its cytosolic subunit translocates to the membrane of the phagolysosome; thus, the reactive end products are generated mainly within the vesicles, and the phagocyte itself is not damaged. H₂O₂ is eventually broken down to water and O₂ by the actions of catalase, and the other ROS also are degraded (Chapter 1). Reactive nitrogen species, particularly nitric oxide (NO), act in the same way as that described for ROS.

The dead microorganisms are then degraded by the action of lysosomal acid hydrolases. Perhaps the most important lysosomal enzyme involved in bacterial killing is elastase.

Of note, in addition to ROS and enzymes, several other constituents of leukocyte granules are capable of killing infectious pathogens. These include bactericidal permeability-increasing protein (causing phospholipase activation and membrane phospholipid degradation), lysozyme (causing degradation of bacterial coat oligosaccharides), major basic protein (an important eosinophil granule constituent that is cytotoxic for parasites), and defensins (peptides that kill microbes by creating holes in their membranes).

Secretion of Microbicidal Substances

The microbicidal mechanisms of phagocytes are largely sequestered within phagolysosomes in order to protect the leukocytes from damaging themselves. Leukocytes also actively secrete granule components including enzymes such as elastase, which destroy and digest extracellular microbes and dead tissues, as well as antimicrobial peptides. The contents of lysosomal granules are secreted by leukocytes into the extracellular milieu by several mechanisms:

• The phagocytic vacuole may remain transiently open to the outside before complete closure of the phagolysosome (regurgitation during feeding).

• If cells encounter materials that cannot be easily ingested, such as immune complexes deposited on immovable surfaces (e.g., glomerular basement membrane), the attempt to phagocytose these substances (frustrated phagocytosis) triggers strong leukocyte activation, and lysosomal enzymes are released into the surrounding tissue or lumen.

• The membrane of the phagolysosome may be damaged if potentially injurious substances, such as silica particles, are phagocytosed.

Neutrophil Extracellular Traps (NETs)

These “traps” are extracellular fibrillar networks that are produced by neutrophils in response to infectious pathogens (mainly bacteria and fungi) and inflammatory mediators (such as chemokines, cytokines, complement proteins, and ROS). NETs contain a framework of nuclear chromatin with embedded granule proteins, such as antimicrobial peptides and enzymes (Fig. 2–9). The traps provide a high concentration of antimicrobial substances at sites of infection, and prevent the spread of the microbes by trapping them in the fibrils. In the process, the nuclei of the neutrophils are lost, leading to death of the cells. NETs also have been detected in blood neutrophils during sepsis. The nuclear chromatin in the NETs, which includes histones and associated DNA, has been postulated to be a source of nuclear antigens in systemic autoimmune diseases, particularly lupus, in which affected persons react against their own DNA and nucleoproteins (Chapter 4).

Figure 2–9 Neutrophil extracellular traps (NETs). A, Healthy neutrophils with nuclei stained red and cytoplasm green. B, Release of nuclear material from neutrophils (note that two have lost their nuclei), forming extracellular traps. C, An electron micrograph of bacteria (staphylococci) trapped in NETs.

(From Brinkmann V, Zychlinsky A: Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5:577, 2007, with the permission of the authors and publisher.)

Leukocyte-Induced Tissue Injury

Because leukocytes are capable of secreting potentially harmful substances such as enzymes and ROS, they are important causes of injury to normal cells and tissues under several circumstances:

In all of these situations, the mechanisms by which leukocytes damage normal tissues are the same as the mechanisms involved in the clearance of microbes and dead tissues, because once the leukocytes are activated, their effector mechanisms do not distinguish between offender and host. In fact, if unchecked or inappropriately directed against host tissues, leukocytes themselves become the main offenders. Leukocyte-dependent tissue injury underlies many acute and chronic human diseases (Table 2–3), as is evident in discussions of specific disorders throughout this book.

Table 2–3 Clinical Examples of Leukocyte-Induced Injury

IgE, immunoglobulin E.

* Listed are selected examples of diseases in which the host response plays a significant role in tissue injury. Some, such as asthma, can manifest with acute inflammation or a chronic illness with repeated bouts of acute exacerbation. These diseases and their pathogenesis are discussed in much more detail in relevant chapters.

Activated leukocytes, especially macrophages, also secrete many cytokines, which stimulate further inflammation and have important systemic effects, to be discussed later.

Defects in Leukocyte Function

Since leukocytes play a central role in host defense, it is not surprising that defects in leukocyte function, both acquired and inherited, lead to increased susceptibility to infections, which may be recurrent and life-threatening (Table 2–4). The most common causes of defective inflammation are bone marrow suppression caused by tumors or treatment with chemotherapy or radiation (resulting in decreased leukocyte numbers) and metabolic diseases such as diabetes (causing abnormal leukocyte functions). These are described elsewhere in the book.

Table 2–4 Defects in Leukocyte Functions

H₂O₂, hydrogen peroxide; MPO, myeloperoxidase.

Modified from Gallin JI: Disorders of phagocytic cells. In Gallin JI, et al (eds): Inflammation: Basic Principles and Clinical Correlates, 2nd ed. New York, Raven Press, 1992, pp 860, 861.

The genetic disorders, although individually rare, illustrate the importance of particular molecular pathways in the complex inflammatory response. Some of the better understood inherited diseases are the following:

Outcomes of Acute Inflammation

Although the consequences of acute inflammation are modified by the nature and intensity of the injury, the site and tissue affected, and the ability of the host to mount a response, acute inflammation generally has one of three outcomes (Fig. 2–10):

Figure 2–10 Outcomes of acute inflammation: resolution, healing by scarring (fibrosis), or chronic inflammation (see text).

Cell-Derived Mediators

Tissue macrophages, mast cells, and endothelial cells at the site of inflammation, as well as leukocytes that are recruited to the site from the blood, are all capable of producing different mediators of inflammation.

Vasoactive Amines

The two vasoactive amines, histamine and serotonin, are stored as preformed molecules in mast cells and other cells and are among the first mediators to be released in acute inflammatory reactions.

• Histamine is produced by many cell types, particularly mast cells adjacent to vessels, as well as circulating basophils and platelets. Preformed histamine is released from mast cell granules in response to a variety of stimuli: (1) physical injury such as trauma or heat; (2) immune reactions involving binding of IgE antibodies to Fc receptors on mast cells (Chapter 4); (3) C3a and C5a fragments of complement, the so-called anaphylatoxins (see later); (4) leukocyte-derived histamine-releasing proteins; (5) neuropeptides (e.g., substance P); and (6) certain cytokines (e.g., IL-1, IL-8). In humans, histamine causes arteriolar dilation and rapidly increases vascular permeability by inducing venular endothelial contraction and formation of interendothelial gaps. Soon after its release, histamine is inactivated by histaminase.

• Serotonin (5-hydroxytryptamine) is a preformed vasoactive mediator found within platelet granules that is released during platelet aggregation (Chapter 3). It induces vasoconstriction during clotting. It is produced mainly in some neurons and enterochromaffin cells, and is a neurotransmitter and regulates intestinal motility.

Arachidonic Acid Metabolites: Prostaglandins, Leukotrienes, and Lipoxins

Products derived from the metabolism of AA affect a variety of biologic processes, including inflammation and hemostasis. AA metabolites, also called eicosanoids (because they are derived from 20-carbon fatty acids—Greek eicosa, “twenty”), can mediate virtually every step of inflammation (Table 2–6); their synthesis is increased at sites of inflammatory response, and agents that inhibit their synthesis also diminish inflammation. Leukocytes, mast cells, endothelial cells, and platelets are the major sources of AA metabolites in inflammation. These AA-derived mediators act locally at the site of generation and then decay spontaneously or are enzymatically destroyed.

Table 2–6 Principal Inflammatory Actions of Arachidonic Acid Metabolites (Eicosanoids)

Action	Eicosanoid
Vasodilation	Prostaglandins PGI2 (prostacyclin), PGE1, PGE2, PGD2
Vasoconstriction	Thromboxane A2, leukotrienes C4, D4, E4
Increased vascular permeability	Leukotrienes C4, D4, E4
Chemotaxis, leukocyte adhesion	Leukotriene B4, HETE

HETE, hydroxyeicosatetraenoic acid.

AA is a 20-carbon polyunsaturated fatty acid (with four double bonds) produced primarily from dietary linoleic acid and present in the body mainly in its esterified form as a component of cell membrane phospholipids. It is released from these phospholipids through the action of cellular phospholipases that have been activated by mechanical, chemical, or physical stimuli, or by inflammatory mediators such as C5a. AA metabolism proceeds along one of two major enzymatic pathways: Cyclooxygenase stimulates the synthesis of prostaglandins and thromboxanes, and lipoxygenase is responsible for production of leukotrienes and lipoxins (Fig. 2–16).

• Prostaglandins and thromboxanes. Products of the cyclooxygenase pathway include prostaglandin E₂ (PGE₂), PGD₂, PGF_2α, PGI₂ (prostacyclin), and thromboxane A₂ (TXA₂), each derived by the action of a specific enzyme on an intermediate. Some of these enzymes have a restricted tissue distribution. For example, platelets contain the enzyme thromboxane synthase, and hence TXA₂, a potent platelet-aggregating agent and vasoconstrictor, is the major prostaglandin produced in these cells. Endothelial cells, on the other hand, lack thromboxane synthase but contain prostacyclin synthase, which is responsible for the formation of PGI₂, a vasodilator and a potent inhibitor of platelet aggregation. The opposing roles of TXA₂ and PGI₂ in hemostasis are discussed further in Chapter 3. PGD₂ is the major metabolite of the cyclooxygenase pathway in mast cells; along with PGE₂ and PGF_2α (which are more widely distributed), it causes vasodilation and potentiates edema formation. The prostaglandins also contribute to the pain and fever that accompany inflammation; PGE₂ augments pain sensitivity to a variety of other stimuli and interacts with cytokines to cause fever.

• Leukotrienes. Leukotrienes are produced by the action of 5-lipoxygenase, the major AA-metabolizing enzyme in neutrophils. The synthesis of leukotrienes involves multiple steps (Fig. 2–16). The first step generates leukotriene A₄ (LTA₄), which in turn gives rise to LTB₄ or LTC₄. LTB₄ is produced by neutrophils and some macrophages and is a potent chemotactic agent for neutrophils. LTC₄ and its subsequent metabolites, LTD₄ and LTE₄, are produced mainly in mast cells and cause bronchoconstriction and increased vascular permeability.

• Lipoxins. Once leukocytes enter tissues, they gradually change their major lipoxygenase-derived AA products from leukotrienes to anti-inflammatory mediators called lipoxins, which inhibit neutrophil chemotaxis and adhesion to endothelium and thus serve as endogenous antagonists of leukotrienes. Platelets that are activated and adherent to leukocytes also are important sources of lipoxins. Platelets alone cannot synthesize lipoxins A₄ and B₄ (LXA₄ and LXB₄), but they can form these mediators from an intermediate derived from adjacent neutrophils, by a transcellular biosynthetic pathway. By this mechanism, AA products can pass from one cell type to another.

Figure 2–16 Production of arachidonic acid metabolites and their roles in inflammation. Note the enzymatic activities whose inhibition through pharmacologic intervention blocks major pathways (denoted with a red X). COX-1, COX-2, cyclooxygenases 1 and 2; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid.

Anti-inflammatory Drugs That Block Prostaglandin Production

The central role of eicosanoids in inflammatory processes is emphasized by the clinical utility of agents that block eicosanoid synthesis. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, inhibit cyclooxygenase activity, thereby blocking all prostaglandin synthesis (hence their efficacy in treating pain and fever). There are two forms of the cyclooxygenase enzyme, COX-1 and COX-2. COX-1 is produced in response to inflammatory stimuli and also is constitutively expressed in most tissues, where it stimulates the production of prostaglandins that serve a homeostatic function (e.g., fluid and electrolyte balance in the kidneys, cytoprotection in the gastrointestinal tract). By contrast, COX-2 is induced by inflammatory stimuli but it is absent from most normal tissues. Therefore, COX-2 inhibitors have been developed with the expectation that they will inhibit harmful inflammation but will not block the protective effects of constitutively produced prostaglandins. These distinctions between the roles of the two cyclooxygenases are not absolute, however. Furthermore, COX-2 inhibitors may increase the risk for cardiovascular and cerebrovascular events, possibly because they impair endothelial cell production of prostacyclin (PGI₂), an inhibitor of platelet aggregation, but leave intact the COX-1–mediated production by platelets of TXA₂, a mediator of platelet aggregation. Glucocorticoids, which are powerful anti-inflammatory agents, act in part by inhibiting the activity of phospholipase A₂ and thus the release of AA from membrane lipids.

Platelet-Activating Factor

Originally named for its ability to aggregate platelets and cause their degranulation, platelet-activating factor (PAF) is another phospholipid-derived mediator with a broad spectrum of inflammatory effects. PAF is acetyl glycerol ether phosphocholine; it is generated from the membrane phospholipids of neutrophils, monocytes, basophils, endothelial cells, and platelets (and other cells) by the action of phospholipase A₂. PAF acts directly on target cells through the effects of a specific G protein–coupled receptor. In addition to stimulating platelets, PAF causes bronchoconstriction and is 100 to 1000 times more potent than histamine in inducing vasodilation and increased vascular permeability. It also stimulates the synthesis of other mediators, such as eicosanoids and cytokines, from platelets and other cells. Thus, PAF can elicit many of the reactions of inflammation, including enhanced leukocyte adhesion, chemotaxis, leukocyte degranulation, and the respiratory burst.

Cytokines

Cytokines are polypeptide products of many cell types that function as mediators of inflammation and immune responses (Chapter 4). Different cytokines are involved in the earliest immune and inflammatory reactions to noxious stimuli and in the later adaptive (specific) immune responses to microbes. Some cytokines stimulate bone marrow precursors to produce more leukocytes, thus replacing the ones that are consumed during inflammation and immune responses. Molecularly characterized cytokines are called interleukins (abbreviated IL and numbered), referring to their ability to mediate communications between leukocytes. However, the nomenclature is imperfect—many interleukins act on cells other than leukocytes, and many cytokines that do act on leukocytes are not called interleukins, for historical reasons.

The major cytokines in acute inflammation are TNF, IL-1, IL-6, and a group of chemoattractant cytokines called chemokines. Other cytokines that are more important in chronic inflammation include interferon-γ (IFN-γ) and IL-12. A cytokine called IL-17, produced by T lymphocytes and other cells, plays an important role in recruiting neutrophils and is involved in host defense against infections and in inflammatory diseases.

Tumor Necrosis Factor and Interleukin-1

TNF and IL-1 are produced by activated macrophages, as well as mast cells, endothelial cells, and some other cell types (Fig. 2–17). Their secretion is stimulated by microbial products, such as bacterial endotoxin, immune complexes, and products of T lymphocytes generated during adaptive immune responses. As mentioned earlier, IL-1 is also the cytokine induced by activation of the inflammasome. The principal role of these cytokines in inflammation is in endothelial activation. Both TNF and IL-1 stimulate the expression of adhesion molecules on endothelial cells, resulting in increased leukocyte binding and recruitment, and enhance the production of additional cytokines (notably chemokines) and eicosanoids. TNF also increases the thrombogenicity of endothelium. IL-1 activates tissue fibroblasts, resulting in increased proliferation and production of ECM.

Figure 2–17 The roles of cytokines in acute inflammation. The cytokines TNF, IL-1, and IL-6 are key mediators of leukocyte recruitment in local inflammatory responses and also play important roles in the systemic reactions of inflammation.

Although TNF and IL-1 are secreted by macrophages and other cells at sites of inflammation, they may enter the circulation and act at distant sites to induce the systemic acute-phase reaction that is often associated with infection and inflammatory diseases. Components of this reaction include fever, lethargy, hepatic synthesis of various acute-phase proteins (also stimulated by IL-6), metabolic wasting (cachexia), neutrophil release into the circulation, and fall in blood pressure. These systemic manifestations of inflammation are described later in the chapter.

Chemokines

The chemokines are a family of small (8 to 10 kDa), structurally related proteins that act primarily as chemoattractants for different subsets of leukocytes. The two main functions of chemokines are to recruit leukocytes to the site of inflammation and to control the normal anatomic organization of cells in lymphoid and other tissues. Combinations of chemokines that are produced transiently in response to inflammatory stimuli recruit particular cell populations (e.g., neutrophils, lymphocytes or eosinophils) to sites of inflammation. Chemokines also activate leukocytes; one consequence of such activation, as mentioned earlier, is increased affinity of leukocyte integrins for their ligands on endothelial cells. Some chemokines are produced constitutively in tissues and are responsible for the anatomic segregation of different cell populations in tissues (e.g., the segregation of T and B lymphocytes in different areas of lymph nodes and spleen). Chemokines mediate their activities by binding to specific G protein–coupled receptors on target cells; two of these chemokine receptors (called CXCR4 and CCR5) are important coreceptors for the binding and entry of the human immunodeficiency virus into lymphocytes (Chapter 4).

Chemokines are classified into four groups based on the arrangement of conserved cysteine residues. The two major groups are the CXC and CC chemokines:

• CXC chemokines have one amino acid separating the conserved cysteines and act primarily on neutrophils. IL-8 is typical of this group; it is produced by activated macrophages, endothelial cells, mast cells, and fibroblasts, mainly in response to microbial products and other cytokines such as IL-1 and TNF.

• CC chemokines have adjacent cysteine residues and include monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) (both chemotactic predominantly for monocytes), RANTES (regulated on activation, normal T cell–expressed and secreted) (chemotactic for memory CD4+ T cells and monocytes), and eotaxin (chemotactic for eosinophils).

Reactive Oxygen Species

ROS are synthesized via the NADPH oxidase (phagocyte oxidase) pathway and are released from neutrophils and macrophages that are activated by microbes, immune complexes, cytokines, and a variety of other inflammatory stimuli. The synthesis and regulation of these oxygen-derived free radicals have been described in Chapter 1, in the context of cell injury, and earlier in this chapter in the discussion of leukocyte activation. When the ROS are produced within lysosomes they function to destroy phagocytosed microbes and necrotic cells. When secreted at low levels, ROS can increase chemokine, cytokine, and adhesion molecule expression, thus amplifying the cascade of inflammatory mediators. At higher levels, these mediators are responsible for tissue injury by several mechanisms, including (1) endothelial damage, with thrombosis and increased permeability; (2) protease activation and antiprotease inactivation, with a net increase in breakdown of the ECM; and (3) direct injury to other cell types (e.g., tumor cells, red cells, parenchymal cells). Fortunately, various antioxidant protective mechanisms (e.g., mediated by catalase, superoxide dismutase, and glutathione) present in tissues and blood minimize the toxicity of the oxygen metabolites (Chapter 1).

Nitric Oxide

NO is a short-lived, soluble, free radical gas produced by many cell types and capable of mediating a variety of functions. In the central nervous system it regulates neurotransmitter release as well as blood flow. Macrophages use it as a cytotoxic agent for killing microbes and tumor cells. When produced by endothelial cells it relaxes vascular smooth muscle and causes vasodilation.

NO is synthesized de novo from l-arginine, molecular oxygen, and NADPH by the enzyme nitric oxide synthase (NOS). There are three isoforms of NOS, with different tissue distributions.

• Type I, neuronal NOS (nNOS), is constitutively expressed in neurons, and does not play a significant role in inflammation.

• Type II, inducible NOS (iNOS), is induced in macrophages and endothelial cells by a number of inflammatory cytokines and mediators, most notably by IL-1, TNF, and IFN-γ, and by bacterial endotoxin, and is responsible for production of NO in inflammatory reactions. This inducible form is also present in many other cell types, including hepatocytes, cardiac myocytes, and respiratory epithelial cells.

• Type III, endothelial NOS, (eNOS), is constitutively synthesized primarily (but not exclusively) in endothelium.

An important function of NO is as a microbicidal (cytotoxic) agent in activated macrophages. NO plays other roles in inflammation, including vasodilation, antagonism of all stages of platelet activation (adhesion, aggregation, and degranulation), and reduction of leukocyte recruitment at inflammatory sites.

Lysosomal Enzymes of Leukocytes

The lysosomal granules of neutrophils and monocytes contain many enzymes that destroy phagocytosed substances and are capable of causing tissue damage. Lysosomal granule contents also may be released from activated leukocytes, as described earlier. Acid proteases generally are active only in the low-pH environment of phagolysosomes, whereas neutral proteases, including elastase, collagenase, and cathepsin, are active in extracellular locations and cause tissue injury by degrading elastin, collagen, basement membrane, and other matrix proteins. Neutral proteases also can cleave the complement proteins C3 and C5 directly to generate the vasoactive mediators C3a and C5a and can generate bradykinin-like peptides from kininogen.

The potentially damaging effects of lysosomal enzymes are limited by antiproteases present in the plasma and tissue fluids. These include α₁-antitrypsin, the major inhibitor of neutrophil elastase, and α₂-macroglobulin. Deficiencies of these inhibitors may result in sustained activation of leukocyte proteases, resulting in tissue destruction at sites of leukocyte accumulation. For instance, α₁-antitrypsin deficiency in the lung can cause a severe panacinar emphysema (Chapter 12).

Neuropeptides

Like the vasoactive amines, neuropeptides can initiate inflammatory responses; these are small proteins, such as substance P, that transmit pain signals, regulate vessel tone, and modulate vascular permeability. Nerve fibers that secrete neuropeptides are especially prominent in the lung and gastrointestinal tract.

  Summary

Major Cell-Derived Mediators of Inflammation

• Vasoactive amines—histamine, serotonin: Their main effects are vasodilation and increased vascular permeability.

• Arachidonic acid metabolites—prostaglandins and leukotrienes: Several forms exist and are involved in vascular reactions, leukocyte chemotaxis, and other reactions of inflammation; they are antagonized by lipoxins.

• Cytokines: These proteins, produced by many cell types, usually act at short range; they mediate multiple effects, mainly in leukocyte recruitment and migration; principal ones in acute inflammation are TNF, IL-1, IL-6, and chemokines.

• ROS: Roles include microbial killing and tissue injury.

• NO: Effects are vasodilation and microbial killing.

• Lysosomal enzymes: Roles include microbial killing and tissue injury.

Plasma Protein–Derived Mediators

Circulating proteins of three interrelated systems—the complement, kinin, and coagulation systems—are involved in several aspects of the inflammatory reaction.

Complement

The complement system consists of plasma proteins that play an important role in host defense (immunity) and inflammation. Upon activation, different complement proteins coat (opsonize) particles, such as microbes, for phagocytosis and destruction, and contribute to the inflammatory response by increasing vascular permeability and leukocyte chemotaxis. Complement activation ultimately generates a porelike membrane attack complex (MAC) that punches holes in the membranes of invading microbes. Here we summarize the role of the complement system in inflammation.

• Complement components, numbered C1 to C9, are present in plasma in inactive forms, and many of them are activated by proteolysis to acquire their own proteolytic activity, thus setting up an enzymatic cascade.

• The critical step in the generation of biologically active complement products is the activation of the third component, C3 (Fig. 2–18). C3 cleavage occurs by three pathways: (1) the classical pathway, triggered by fixation of the first complement component C1 to antigen-antibody complexes; (2) the alternative pathway, triggered by bacterial polysaccharides (e.g., endotoxin) and other microbial cell wall components, and involving a distinct set of plasma proteins including properdin and factors B and D; and (3) the lectin pathway, in which a plasma lectin binds to mannose residues on microbes and activates an early component of the classical pathway (but in the absence of antibodies).

• All three pathways lead to the formation of a C3 convertase that cleaves C3 to C3a and C3b. C3b deposits on the cell or microbial surface where complement was activated and then binds to the C3 convertase complex to form C5 convertase; this complex cleaves C5 to generate C5a and C5b and initiate the final stages of assembly of C6 to C9.

Figure 2–18 The activation and functions of the complement system. Activation of complement by different pathways leads to cleavage of C3. The functions of the complement system are mediated by breakdown products of C3 and other complement proteins, and by the membrane attack complex (MAC).

The complement-derived factors that are produced along the way contribute to a variety of phenomena in acute inflammation:

• Vascular effects. C3a and C5a increase vascular permeability and cause vasodilation by inducing mast cells to release histamine. These complement products are also called anaphylatoxins because their actions mimic those of mast cells, which are the main cellular effectors of the severe allergic reaction called anaphylaxis (Chapter 4). C5a also activates the lipoxygenase pathway of AA metabolism in neutrophils and macrophages, causing release of more inflammatory mediators.

• Leukocyte activation, adhesion, and chemotaxis. C5a, and to lesser extent, C3a and C4a, activate leukocytes, increasing their adhesion to endothelium, and is a potent chemotactic agent for neutrophils, monocytes, eosinophils, and basophils.

• Phagocytosis. When fixed to a microbial surface, C3b and its inactive proteolytic product iC3b act as opsonins, augmenting phagocytosis by neutrophils and macrophages, which express receptors for these complement products.

• The MAC, which is made up of multiple copies of the final component C9, kills some bacteria (especially thin-walled Neisseria) by creating pores that disrupt osmotic balance.

The activation of complement is tightly controlled by cell-associated and circulating regulatory proteins. The presence of these inhibitors in host cell membranes protects normal cells from inappropriate damage during protective reactions against microbes. Inherited deficiencies of these regulatory proteins lead to spontaneous complement activation:

• A protein called C1 inhibitor blocks activation of C1, and its inherited deficiency causes a disease called hereditary angioedema, in which excessive production of kinins secondary to complement activation results in edema in multiple tissues, including the larynx.

• Another protein called decay-accelerating factor (DAF) normally limits the formation of C3 and C5 convertases. In a disease called paroxysmal nocturnal hemoglobinuria, there is an acquired deficiency of DAF that results in complement-mediated lysis of red cells (which are more sensitive to lysis than most nucleated cells) (Chapter 11).

• Factor H is a plasma protein that also limits convertase formation; its deficiency is associated with a kidney disease called the hemolytic uremic syndrome (Chapter 13), as well as spontaneous vascular permeability in macular degeneration of the eye.

Even in the presence of regulatory proteins, inappropriate or excessive complement activation (e.g., in antibody-mediated diseases) can overwhelm the regulatory mechanisms; this is why complement activation is responsible for serious tissue injury in a variety of immunologic disorders (Chapter 4).

Coagulation and Kinin Systems

Some of the molecules activated during blood clotting are capable of triggering multiple aspects of the inflammatory response. Hageman factor (also known as factor XII of the intrinsic coagulation cascade) (Fig. 2–19) is a protein synthesized by the liver that circulates in an inactive form until it encounters collagen, basement membrane, or activated platelets (e.g., at a site of endothelial injury). Activated Hageman factor (factor XIIa) initiates four systems that may contribute to the inflammatory response: (1) the kinin system, producing vasoactive kinins; (2) the clotting system, inducing the activation of thrombin, fibrinopeptides, and factor X, all with inflammatory properties; (3) the fibrinolytic system, producing plasmin and inactivating thrombin; and (4) the complement system, producing the anaphylatoxins C3a and C5a. These are described below.

• Kinin system activation leads ultimately to the formation of bradykinin from its circulating precursor, high-molecular-weight kininogen (HMWK) (Fig. 2–19). Like histamine, bradykinin causes increased vascular permeability, arteriolar dilation, and bronchial smooth muscle contraction. It also causes pain when injected into the skin. The actions of bradykinin are short-lived because it is rapidly degraded by kininases present in plasma and tissues. Of note, kallikrein, an intermediate in the kinin cascade with chemotactic activity, also is a potent activator of Hageman factor and thus constitutes another link between the kinin and clotting systems.

• In the clotting system (Chapter 3), the proteolytic cascade leads to activation of thrombin, which then cleaves circulating soluble fibrinogen to generate an insoluble fibrin clot. Factor Xa, an intermediate in the clotting cascade, causes increased vascular permeability and leukocyte emigration. Thrombin participates in inflammation by binding to protease-activated receptors that are expressed on platelets, endothelial cells, and many other cell types. Binding of thrombin to these receptors on endothelial cells leads to their activation and enhanced leukocyte adhesion. In addition, thrombin generates fibrinopeptides (during fibrinogen cleavage) that increase vascular permeability and are chemotactic for leukocytes. Thrombin also cleaves C5 to generate C5a, thus linking coagulation with complement activation.

• As a rule, whenever clotting is initiated (e.g., by activated Hageman factor), the fibrinolytic system is also activated concurrently. This mechanism serves to limit clotting by cleaving fibrin, thereby solubilizing the fibrin clot (Chapter 3). Plasminogen activator (released from endothelium, leukocytes, and other tissues) and kallikrein cleave plasminogen, a plasma protein bound up in the evolving fibrin clot. The resulting product, plasmin, is a multifunctional protease that cleaves fibrin and is therefore important in lysing clots. However, fibrinolysis also participates in multiple steps in the vascular phenomena of inflammation. For example, fibrin degradation products increase vascular permeability, and plasmin cleaves the C3 complement protein, resulting in production of C3a and vasodilation and increased vascular permeability. Plasmin can also activate Hageman factor, thereby amplifying the entire set of responses.

Figure 2–19 Interrelationships among the four plasma mediator systems triggered by activation of factor XII (Hageman factor). See text for details.

As is evident from the preceding discussion, many molecules are involved in different aspects of the inflammatory reaction, and these molecules often interact with, amplify, and antagonize one another. From this almost bewildering potpourri of chemical mediators, it is possible to identify the major contributors to various components of acute inflammation (Table 2–7). The relative contributions of individual mediators to inflammatory reactions to different stimuli have yet to be fully elucidated. Such knowledge would have obvious therapeutic implications since it might allow one to “custom design” antagonists for various inflammatory diseases.

Table 2–7 Role of Mediators in Different Reactions of Inflammation

Inflammatory Component	Mediators
Vasodilation	Prostaglandins Nitric oxide Histamine
Increased vascular permeability	Histamine and serotonin C3a and C5a (by liberating vasoactive amines from mast cells, other cells) Bradykinin Leukotrienes C4, D4, E4 PAF Substance P
Chemotaxis, leukocyte recruitment and activation	TNF, IL-1 Chemokines C3a, C5a Leukotriene B4 Bacterial products (e.g., N-formyl methyl peptides)
Fever	IL-1, TNF Prostaglandins
Pain	Prostaglandins Bradykinin
Tissue damage	Lysosomal enzymes of leukocytes Reactive oxygen species Nitric oxide

IL-1, interleukin-1; PAF, platelet-activating factor; TNF, tumor necrosis factor.

  Summary

Plasma Protein–Derived Mediators of Inflammation

• Complement proteins: Activation of the complement system by microbes or antibodies leads to the generation of multiple breakdown products, which are responsible for leukocyte chemotaxis, opsonization and phagocytosis of microbes and other particles, and cell killing.

• Coagulation proteins: Activated factor XII triggers the clotting, kinin, and complement cascades and activates the fibrinolytic system.

• Kinins: Produced by proteolytic cleavage of precursors, this group mediates vascular reaction and pain.

Anti-inflammatory Mechanisms

Inflammatory reactions subside because many of the mediators are short-lived and are destroyed by degradative enzymes. In addition, there are several mechanisms that counteract inflammatory mediators and function to limit or terminate the inflammatory response. Some of these, such as lipoxins, and complement regulatory proteins, have been mentioned earlier. Activated macrophages and other cells secrete a cytokine, IL-10, whose major function is to down-regulate the responses of activated macrophages, thus providing a negative feedback loop. In a rare inherited disease in which IL-10 receptors are mutated, affected patients develop severe colitis in infancy. Other anti-inflammatory cytokines include TGF-β, which is also a mediator of fibrosis in tissue repair after inflammation. Cells also express a number of intracellular proteins, such as tyrosine phosphatases, that inhibit pro-inflammatory signals triggered by receptors that recognize microbes and cytokines.

Chronic Inflammatory Cells and Mediators

The combination of prolonged and repeated inflammation, tissue destruction and fibrosis that characterizes chronic inflammation involves complex interactions between several cell populations and their secreted mediators. Understanding the pathogenesis of chronic inflammatory reactions requires an appreciation of these cells and their biologic responses and functions.

Macrophages

Macrophages, the dominant cells of chronic inflammation, are tissue cells derived from circulating blood monocytes after their emigration from the bloodstream. Macrophages are normally diffusely scattered in most connective tissues and are also found in organs such as the liver (where they are called Kupffer cells), spleen and lymph nodes (where they are called sinus histiocytes), central nervous system (microglial cells), and lungs (alveolar macrophages). Together these cells constitute the so-called mononuclear phagocyte system, also known by the older name of reticuloendothelial system. In all tissues, macrophages act as filters for particulate matter, microbes, and senescent cells, as well as the effector cells that eliminate microbes in cellular and humoral immune responses (Chapter 4).

Monocytes arise from precursors in the bone marrow and circulate in the blood for only about a day. Under the influence of adhesion molecules and chemokines, they migrate to a site of injury within 24 to 48 hours after the onset of acute inflammation, as described earlier. When monocytes reach the extravascular tissue, they undergo transformation into macrophages, which are somewhat larger and have a longer lifespan and a greater capacity for phagocytosis than do blood monocytes.

Tissue macrophages are activated by diverse stimuli to perform a range of functions. Two major pathways of macrophage activation, classical and alternative, have been described (Fig. 2–21):

• Classical macrophage activation is induced by microbial products such as endotoxin, by T cell–derived signals, importantly the cytokine IFN-γ, and by foreign substances including crystals and particulate matter. Classically activated macrophages produce lysosomal enzymes, NO, and ROS, all of which enhance their ability to kill ingested organisms, and secrete cytokines that stimulate inflammation. These macrophages are important in host defense against ingested microbes and in many chronic inflammatory reactions.

• Alternative macrophage activation is induced by cytokines other than IFN-γ, such as IL-4 and IL-13, produced by T lymphocytes and other cells, including mast cells and eosinophils. Alternatively activated macrophages are not actively microbicidal; instead, their principal role is in tissue repair. They secrete growth factors that promote angiogenesis, activate fibroblasts and stimulate collagen synthesis. It may be that in response to most injurious stimuli, macrophages are initially activated by the classical pathway, designed to destroy the offending agents, and this is followed by alternative activation, which initiates tissue repair. However, such a precise sequence is not well documented in most inflammatory reactions.

Figure 2–21 Pathways of macrophage activation. Different stimuli activate monocytes/macrophages to develop into functionally distinct populations. Classically activated macrophages are induced by microbial products and cytokines, particularly IFN-γ, and are microbicidal and involved in potentially harmful inflammation. Alternatively activated macrophages are induced by IL-4 and IL-13, produced by T_H2 cells (a helper T cell subset) and other leukocytes, and are important in tissue repair and fibrosis. IFN-γ, interferon-γ; IL-4, IL-13, interkeukin-4, -13.

Macrophages have several critical roles in host defense and the inflammatory response.

• Macrophages, like the other type of phagocyte, the neutrophils, ingest and eliminate microbes and dead tissues. Because macrophages respond to activating signals from T lymphocytes, they are the most important phagocytes in the cell-mediated arm of adaptive immune responses (Chapter 4).

• Macrophages initiate the process of tissue repair and are involved in scar formation and fibrosis.

• Macrophages secrete mediators of inflammation, such as cytokines (TNF, IL-1, chemokines, and others) and eicosanoids. These cells are therefore central to the initiation and propagation of all inflammatory reactions.

• Macrophages display antigens to T lymphocytes and respond to signals from T cells, thus setting up a feedback loop that is essential for defense against many microbes by cell-mediated immune responses. The same bidirectional interactions are central to the development of chronic inflammatory diseases. The roles of cytokines in these interactions are discussed later.

After the initiating stimulus is eliminated and the inflammatory reaction abates, macrophages eventually die or wander off into lymphatics. In chronic inflammatory sites, however, macrophage accumulation persists, because of continued recruitment from the blood and local proliferation. IFN-γ can also induce macrophages to fuse into large, multinucleate giant cells.

Lymphocytes

Lymphocytes are mobilized in the setting of any specific immune stimulus (i.e., infections) as well as non–immune-mediated inflammation (e.g., due to ischemic necrosis or trauma), and are the major drivers of inflammation in many autoimmune and other chronic inflammatory diseases. The activation of T and B lymphocytes is part of the adaptive immune response in infections and immunologic diseases (Chapter 4). Both classes of lymphocytes migrate into inflammatory sites using some of the same adhesion molecule pairs and chemokines that recruit other leukocytes. In the tissues, B lymphocytes may develop into plasma cells, which secrete antibodies, and CD4+ T lymphocytes are activated to secrete cytokines.

By virtue of cytokine secretion, CD4+ T lymphocytes promote inflammation and influence the nature of the inflammatory reaction. There are three subsets of CD4+ helper T cells that secrete different sets of cytokines and elicit different types of inflammation:

• T_H1 cells produce the cytokine IFN-γ, which activates macrophages in the classical pathway.

• T_H2 cells secrete IL-4, IL-5, and IL-13, which recruit and activate eosinophils and are responsible for the alternative pathway of macrophage activation.

• T_H17 cells secrete IL-17 and other cytokines that induce the secretion of chemokines responsible for recruiting neutrophils and monocytes into the reaction.

Both T_H1 and T_H17 cells are involved in defense against many types of bacteria and viruses and in autoimmune diseases. T_H2 cells are important in defense against helminthic parasites and in allergic inflammation. These T cell subsets and their functions are described in more detail in Chapter 4.

Lymphocytes and macrophages interact in a bidirectional way, and these interactions play an important role in propagating chronic inflammation (Fig. 2–22). Macrophages display antigens to T cells, express membrane molecules (called costimulators), and produce cytokines (IL-12 and others) that stimulate T cell responses (Chapter 4). Activated T lymphocytes, in turn, produce cytokines, described earlier, which recruit and activate macrophages and thus promote more antigen presentation and cytokine secretion. The result is a cycle of cellular reactions that fuel and sustain chronic inflammation. In some strong and prolonged inflammatory reactions, the accumulation of lymphocytes, antigen-presenting cells, and plasma cells may assume the morphologic features of lymphoid organs, and akin to lymph nodes, may even contain well-formed germinal centers. This pattern of lymphoid organogenesis is often seen in the synovium of patients with long-standing rheumatoid arthritis and the thyroid of patients with autoimmune thyroiditis.

Figure 2–22 Macrophage–lymphocyte interactions in chronic inflammation. Activated lymphocytes and macrophages stimulate each other, and both cell types release inflammatory mediators that affect other cells. IFN-γ, interferon-γ; IL-1, interleukin-1; TNF, tumor necrosis factor.

Granulomatous Inflammation

Granulomatous inflammation is a distinctive pattern of chronic inflammation characterized by aggregates of activated macrophages with scattered lymphocytes. Granulomas are characteristic of certain specific pathologic states; consequently, recognition of the granulomatous pattern is important because of the limited number of conditions (some life-threatening) that cause it (Table 2–8). Granulomas can form under three settings:

Table 2–8 Examples of Diseases with Granulomatous Inflammation

The formation of a granuloma effectively “walls off” the offending agent and is therefore a useful defense mechanism. However, granuloma formation does not always lead to eradication of the causal agent, which is frequently resistant to killing or degradation, and granulomatous inflammation with subsequent fibrosis may even be the major cause of organ dysfunction in some diseases, such as tuberculosis.

  Morphology

In the usual H&E preparations (Fig. 2–23), some of the activated macrophages in granulomas have pink, granular cytoplasm with indistinct cell boundaries; these are called epithelioid cells because of their resemblance to epithelia. Typically, the aggregates of epithelioid macrophages are surrounded by a collar of lymphocytes. Older granulomas may have a rim of fibroblasts and connective tissue. Frequently, but not invariably, multinucleate giant cells 40 to 50 μm in diameter are found in granulomas. Such cells consist of a large mass of cytoplasm and many nuclei, and they derive from the fusion of multiple activated macrophages. In granulomas associated with certain infectious organisms (most classically the tubercle bacillus), a combination of hypoxia and free radical injury leads to a central zone of necrosis. On gross examination, this has a granular, cheesy appearance and is therefore called caseous necrosis (Chapters 1 and 13). On microscopic examination, this necrotic material appears as eosinophilic amorphous, structureless, granular debris, with complete loss of cellular details. The granulomas associated with Crohn disease, sarcoidosis, and foreign body reactions tend to not have necrotic centers and are said to be “noncaseating.” Healing of granulomas is accompanied by fibrosis that may be quite extensive.

Figure 2–23 A typical granuloma resulting from infection with Mycobacterium tuberculosis showing central area of caseous necrosis, activated epithelioid macrophages, giant cells, and a peripheral accumulation of lymphocytes.

The Control of Cell Proliferation

Several cell types proliferate during tissue repair. These include the remnants of the injured tissue (which attempt to restore normal structure), vascular endothelial cells (to create new vessels that provide the nutrients needed for the repair process), and fibroblasts (the source of the fibrous tissue that forms the scar to fill defects that cannot be corrected by regeneration). The proliferation of these cell types is driven by proteins called growth factors. The production of polypeptide growth factors and the ability of cells to divide in response to these factors are important determinants of the adequacy of the repair process.

The normal size of cell populations is determined by a balance among cell proliferation, cell death by apoptosis, and emergence of new differentiated cells from stem cells (Fig. 2–25). The key processes in the proliferation of cells are DNA replication and mitosis. The sequence of events that control these two processes is known as the cell cycle, described in detail in Chapter 5 in the context of cancer. At this stage, it is sufficient to note that nondividing cells are in cell cycle arrest in the G₁ phase or have exited the cycle and are in the G₀ phase. Growth factors stimulate cells to transition from G₀ into the G₁ phase and beyond into DNA synthesis (S), G₂, and mitosis (M) phases. Progression is regulated by cyclins, whose activity is controlled by cyclin-dependent kinases. Once cells enter the S phase, their DNA is replicated and they progress through G₂ and mitosis.

Figure 2–25 Mechanisms regulating cell populations. Cell numbers can be altered by increased or decreased rates of stem cell input, cell death by apoptosis, or changes in the rates of proliferation or differentiation.

(Modified from McCarthy NJ, et al: Apoptosis in the development of the immune system: growth factors, clonal selection and bcl-2. Cancer Metastasis Rev 11:157, 1992.)

Proliferative Capacities of Tissues

The ability of tissues to repair themselves is critically influenced by their intrinsic proliferative capacity. On the basis of this criterion, the tissues of the body are divided into three groups.

With the exception of tissues composed primarily of nondividing permanent cells (e.g., cardiac muscle, nerve), most mature tissues contain variable proportions of three cell types: continuously dividing cells, quiescent cells that can return to the cell cycle, and cells that have lost replicative ability.

Stem Cells

In most dividing tissues the mature cells are terminally differentiated and short-lived. As mature cells die, the tissue is replenished by the differentiation of cells generated from stem cells. Thus, in these tissues there is a homeostatic equilibrium between the replication, self-renewal, and differentiation of stem cells and the death of the mature, fully differentiated cells. Such relationships are particularly evident in the continuously dividing epithelium of the skin and the gastrointestinal tract, in which stem cells live near the basal layer of the epithelium, and cells differentiate as they migrate to the upper layers of the epithelium before they die and are shed from the surface.

Stem cells are characterized by two important properties: self-renewal capacity and asymmetric replication. Asymmetric replication means that when a stem cell divides, one daughter cell enters a differentiation pathway and gives rise to mature cells, while the other remains an undifferentiated stem cell that retains its self-renewal capacity. Self-renewal enables stem cells to maintain a functional population of precursors for long periods of time. Although the scientific literature is replete with descriptions of various types of stem cells, fundamentally there are two kinds:

Whereas the normal function of ES cells is to give rise to all cells of the body, adult stem cells are involved in tissue homeostasis. They maintain the compartment size both in tissues with high turnover, such as skin, bone marrow, and gut epithelium, and in those with low cell turnover, such as heart and blood vessels. Although there is much interest in isolation and infusion of tissue stem cells for replenishment of specialized cells in organs such as the heart (after a myocardial infarct) and brain (after a stroke), tissue stem cells are rare and very difficult to isolate to purity. Furthermore, they occur in specialized microenvironments within the organ called stem cell niches. Apparently, signals from other cells in such niches keep the stem cells quiescent and undifferentiated. Stem cell niches have been identified in many organs. In the brain, neural stem cells occur in the subventricular zone and dentate gyrus; in the skin, tissue stem cells are found in the bulge region of the hair follicle; and in the cornea, they are found at the limbus.

Perhaps the most extensively studied tissue stem cells are hematopoietic stem cells found in the bone marrow. Although rare, they can be purified to virtual purity based on cell surface markers. Hematopoietic stem cells can be isolated from bone marrow as well as from the peripheral blood after mobilization by administration of certain cytokines such as granulocyte colony-stimulating factor (G-CSF). As is well known, they can give rise to all blood cell lineages and continuously replenish the formed elements of the blood as these are consumed in the periphery. In clinical practice, marrow stem cells are used for treatment of diseases such as leukemia and lymphomas (Chapter 11). In addition to hematopoietic stem cells, the bone marrow also contains a somewhat distinctive population of tissue stem cells, often called mesenchymal stem cells. These cells can give rise to a variety of mesenchymal cells, such as chondroblasts, osteoblasts, and myoblasts. Hence, there is great interest in their therapeutic potential.

The ability to identify and isolate stem cells has given rise to the new field of regenerative medicine, which has as its main goal the repopulation of damaged organs by using differentiated progeny of ES cells or adult stem cells. Since ES cells have extensive self-renewal capacity and can give rise to all cell lineages, they often are considered ideal for developing specialized cells for therapeutic purposes. However, since ES cells are derived from blastocysts (typically produced from in vitro fertilization), their progeny carry histocompatibility molecules (human leukocyte antigen [HLA] in people) (Chapter 4) of the donors of the egg and sperm. Thus, they are likely to evoke immunologically mediated rejection by the host, just as organs transplanted from genetically disparate hosts do. Hence, much effort has gone into producing cells with the potential of ES cells from patient tissues. To accomplish this goal, the expressed genes in ES cells and differentiated cells have been compared and a handful of genes that are critical for the “stem-cell-ness” of ES cells have been identified. Introduction of such genes into fully differentiated cells, such as fibroblasts or skin epithelial cells, leads, quite remarkably, to reprogramming of the somatic cell nucleus, such that the cells acquire many of the properties of ES cells. These cells are called induced pluripotent stem cells (iPS cells) (Fig. 2–26). Since iPS cells can be derived from each patient, their differentiated progeny should engraft successfully and restore or replace damaged or deficient cells in the patient—for example, insulin-secreting β cells in a patient with diabetes. Although iPS cells hold considerable promise, their clinical usefulness remains to be proved.

Figure 2–26 The production of induced pluripotent stem cells (iPS cells). Genes that confer stem cell properties are introduced into a patient’s differentiated cells, giving rise to stem cells, which can be induced to differentiate into various lineages.

Growth Factors

Most growth factors are proteins that stimulate the survival and proliferation of particular cells, and may also promote migration, differentiation, and other cellular responses. They induce cell proliferation by binding to specific receptors and affecting the expression of genes whose products typically have several functions: They promote entry of cells into the cell cycle, they relieve blocks on cell cycle progression (thus promoting replication), they prevent apoptosis, and they enhance the synthesis of cellular proteins in preparation for mitosis. A major activity of growth factors is to stimulate the function of growth control genes, many of which are called proto-oncogenes because mutations in them lead to unrestrained cell proliferation characteristic of cancer (oncogenesis) (Chapter 5).

There is a huge (and ever-increasing) list of known growth factors. In the following discussion, rather than attempting an exhaustive cataloguing, we highlight only selected molecules that contribute to tissue repair (Table 2–9). Many of the growth factors that are involved in repair are produced by macrophages and lymphocytes that are recruited to the site of injury or are activated at this site, as part of the inflammatory process. Other growth factors are produced by parenchymal cells or stromal (connective tissue) cells in response to cell injury. We start the discussion by describing general principles of growth factor actions. We return to the roles of individual growth factors in the repair process later in the chapter.

Table 2–9 Growth Factors Involved in Regeneration and Repair

ECM, extracellular membrane.

Role of the Extracellular Matrix in Tissue Repair

Tissue repair depends not only on growth factor activity but also on interactions between cells and ECM components. The ECM is a complex of several proteins that assembles into a network that surrounds cells and constitutes a significant proportion of any tissue. ECM sequesters water, providing turgor to soft tissues, and minerals, giving rigidity to bone. It also regulates the proliferation, movement, and differentiation of the cells living within it, by supplying a substrate for cell adhesion and migration and serving as a reservoir for growth factors. The ECM is constantly being remodeled; its synthesis and degradation accompany morphogenesis, wound healing, chronic fibrosis, and tumor invasion and metastasis.

ECM occurs in two basic forms: interstitial matrix and basement membrane (Fig. 2–27).

Figure 2–27 The major components of the extracellular matrix (ECM), including collagens, proteoglycans, and adhesive glycoproteins. Note that although there is some overlap in their constituents, basement membrane and interstitial ECM differ in general composition and architecture. Both epithelial and mesenchymal cells (e.g., fibroblasts) interact with ECM through integrins. For simplification, many ECM components have been left out (e.g., elastin, fibrillin, hyaluronan, syndecan).

Functions of the Extracellular Matrix

The ECM is much more than a space filler around cells. Its various functions include

• Mechanical support for cell anchorage and cell migration, and maintenance of cell polarity

• Control of cell proliferation by binding and displaying growth factors and by signaling through cellular receptors of the integrin family. The type of ECM proteins can affect the degree of differentiation of the cells in the tissue, again acting largely through cell surface integrins.

• Scaffolding for tissue renewal. Because maintenance of normal tissue structure requires a basement membrane or stromal scaffold, the integrity of the basement membrane or the stroma of parenchymal cells is critical for the organized regeneration of tissues. Thus, although labile and stable cells are capable of regeneration, disruption of the ECM results in a failure of the tissues to regenerate and repair by scar formation (Fig. 2–24).

• Establishment of tissue microenvironments. Basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney.

  Summary

Extracellular Matrix and Tissue Repair

• The ECM consists of the interstitial matrix between cells, made up of collagens and several glycoproteins, and basement membranes underlying epithelia and surrounding vessels, made up of nonfibrillar collagen and laminin.

• The ECM serves several important functions:

It provides mechanical support to tissues; this is the role of collagens and elastin.

It acts as a substrate for cell growth and the formation of tissue microenvironments.

It regulates cell proliferation and differentiation; proteoglycans bind growth factors and display them at high concentration, and fibronectin and laminin stimulate cells through cellular integrin receptors.

• An intact ECM is required for tissue regeneration, and if the ECM is damaged, repair can be accomplished only by scar formation.

Having described the basic components of tissue repair, we now proceed to a discussion of repair by regeneration and by scar formation.

Role of Regeneration in Tissue Repair

The importance of regeneration in the replacement of injured tissues varies in different types of tissues and with the severity of injury.

Figure 2–28 Regeneration of the liver. Computed tomography scans show a donor liver in living-donor liver transplantation. A, The donor liver before the operation. Note the right lobe (outline), which will be resected and used as a transplant. B, Scan of the same liver 1 week after resection of the right lobe; note the enlargement of the left lobe (outline) without regrowth of the right lobe.

(Courtesy of R. Troisi, MD, Ghent University, Flanders, Belgium.)

A point worthy of emphasis is that extensive regeneration or compensatory hyperplasia can occur only if the residual connective tissue framework is structurally intact, as after partial surgical resection. By contrast, if the entire tissue is damaged by infection or inflammation, regeneration is incomplete and is accompanied by scarring. For example, extensive destruction of the liver with collapse of the reticulin framework, as occurs in a liver abscess, leads to scar formation even though the remaining liver cells have the capacity to regenerate.

Steps in Scar Formation

Repair by connective tissue deposition consists of sequential processes that follow the inflammatory response (Fig. 2–29):

Figure 2–29 Steps in repair by scar formation. Injury to a tissue that has limited regenerative capacity first induces inflammation, which clears dead cells and microbes, if any. This is followed by formation of vascularized granulation tissue and then deposition of ECM to form the scar. ECM, extracellular matrix.

Repair begins within 24 hours of injury by the emigration of fibroblasts and the induction of fibroblast and endothelial cell proliferation. By 3 to 5 days, the specialized granulation tissue that is characteristic of healing is apparent. The term granulation tissue derives from the gross appearance, such as that beneath the scab of a skin wound. Its histologic appearance is characterized by proliferation of fibroblasts and new thin-walled, delicate capillaries (angiogenesis) in a loose ECM, often with admixed inflammatory cells, mainly macrophages (Fig. 2–30, A). Granulation tissue progressively accumulates more fibroblasts, which lay down collagen, eventually resulting in the formation of a scar (Fig. 2–30, B). Scars remodel over time. We next describe each of the steps in this process.

Figure 2–30 A, Granulation tissue showing numerous blood vessels, edema, and a loose ECM containing occasional inflammatory cells. Collagen is stained blue by the trichrome stain; minimal mature collagen can be seen at this point. B, Trichrome stain of mature scar, showing dense collagen with only scattered vascular channels. ECM, extracellular matrix.

Angiogenesis

Angiogenesis is the process of new blood vessel development from existing vessels, primarily venules. It is critical in healing at sites of injury, in the development of collateral circulations at sites of ischemia, and in allowing tumors to increase in size beyond the constraints of their original blood supply. Much work has been done to understand the mechanisms underlying angiogenesis, and therapies to either augment the process (e.g., to improve blood flow to a heart ravaged by coronary atherosclerosis) or inhibit it (e.g., to frustrate tumor growth or block pathologic vessel growth such as in diabetic retinopathy) are being developed.

Angiogenesis involves sprouting of new vessels from existing ones and consists of the following steps (Fig. 2–31):

Figure 2–31 Mechanism of angiogenesis. In tissue repair, angiogenesis occurs mainly by growth factor–driven outgrowth of residual endothelium, sprouting of new vessels, and recruitment of pericytes to form new vessels.

The process of angiogenesis involves a variety of growth factors, cell–cell interactions, interactions with ECM proteins, and tissue enzymes.

Activation of Fibroblasts and Deposition of Connective Tissue

The laying down of connective tissue in the scar occurs in two steps: (1) migration and proliferation of fibroblasts into the site of injury and (2) deposition of ECM proteins produced by these cells. The recruitment and activation of fibroblasts to synthesize connective tissue proteins are driven by many growth factors, including PDGF, FGF-2 (described earlier), and TGF-β. The major source of these factors is inflammatory cells, particularly macrophages, which are present at sites of injury and in granulation tissue. Sites of inflammation are also rich in mast cells, and in the appropriate chemotactic milieu, lymphocytes may be present as well. Each of these cell types can secrete cytokines and growth factors that contribute to fibroblast proliferation and activation.

As healing progresses, the number of proliferating fibroblasts and new vessels decreases; however, the fibroblasts progressively assume a more synthetic phenotype, so there is increased deposition of ECM. Collagen synthesis, in particular, is critical to the development of strength in a healing wound site. As described later, collagen synthesis by fibroblasts begins early in wound healing (days 3 to 5) and continues for several weeks, depending on the size of the wound. Net collagen accumulation, however, depends not only on increased synthesis but also on diminished collagen degradation (discussed later). Ultimately, the granulation tissue evolves into a scar composed of largely inactive, spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, and other ECM components (Fig. 2–30, B). As the scar matures, there is progressive vascular regression, which eventually transforms the highly vascularized granulation tissue into a pale, largely avascular scar.

Growth Factors Involved in ECM Deposition and Scar Formation

Many growth factors are involved in these processes, including TGF-β, PDGF, and FGF. Because FGF also is involved in angiogenesis, it was described earlier. Here we briefly describe the major properties of TGF-β and PDGF.

• Transforming growth factor-β (TGF-β) belongs to a family of homologous polypeptides (TGF-β1, -β2, and -β3) that includes other cytokines such as bone morphogenetic proteins. The TGF-β1 isoform is widely distributed and is usually referred to as TGF-β. The active factor binds to two cell surface receptors with serine-threonine kinase activity, triggering the phosphorylation of transcription factors called Smads. TGF-β has many and often opposite effects, depending on the cell type and the metabolic state of the tissue. In the context of inflammation and repair, TGF-β has two main functions:

TGF-β stimulates the production of collagen, fibronectin, and proteoglycans, and it inhibits collagen degradation by both decreasing proteinase activity and increasing the activity of tissue inhibitors of proteinases known as TIMPs (discussed later on). TGF-β is involved not only in scar formation after injury but also in the development of fibrosis in lung, liver, and kidneys that follows chronic inflammation.

TGF-β is an anti-inflammatory cytokine that serves to limit and terminate inflammatory responses. It does so by inhibiting lymphocyte proliferation and the activity of other leukocytes. Mice lacking TGF-β exhibit widespread inflammation and abundant lymphocyte proliferation.

• Platelet-derived growth factor (PDGF) belongs to a family of closely related proteins, each consisting of two chains, designated A and B. There are five main PDGF isoforms, of which the BB isoform is the prototype; it is often referred to simply as PDGF. PDGFs bind to receptors designated as PDGFRα and PDGFRβ. PDGF is stored in platelets and released on platelet activation and is also produced by endothelial cells, activated macrophages, smooth muscle cells, and many tumor cells. PDGF causes migration and proliferation of fibroblasts and smooth muscle cells and may contribute to the migration of macrophages.

• Cytokines (discussed earlier as mediators of inflammation, and in Chapter 4 in the context of immune responses) may also function as growth factors and participate in ECM deposition and scar formation. IL-1 and IL-13, for example, act on fibroblasts to stimulate collagen synthesis, and can also enhance the proliferation and migration of fibroblasts.

Remodeling of Connective Tissue

After its synthesis and deposition, the connective tissue in the scar continues to be modified and remodeled. Thus, the outcome of the repair process is a balance between synthesis and degradation of ECM proteins. We have already discussed the cells and factors that regulate ECM synthesis. The degradation of collagens and other ECM components is accomplished by a family of matrix metalloproteinases (MMPs), which are dependent on zinc ions for their activity. MMPs should be distinguished from neutrophil elastase, cathepsin G, plasmin, and other serine proteinases that can also degrade ECM but are not metalloenzymes. MMPs include interstitial collagenases, which cleave fibrillar collagen (MMP-1, -2, and -3); gelatinases (MMP-2 and -9), which degrade amorphous collagen and fibronectin; and stromelysins (MMP-3, -10, and -11), which degrade a variety of ECM constituents, including proteoglycans, laminin, fibronectin, and amorphous collagen.

MMPs are produced by a variety of cell types (fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells), and their synthesis and secretion are regulated by growth factors, cytokines, and other agents. The activity of the MMPs is tightly controlled. They are produced as inactive precursors (zymogens) that must be first activated; this is accomplished by proteases (e.g., plasmin) likely to be present only at sites of injury. In addition, activated MMPs can be rapidly inhibited by specific tissue inhibitors of metalloproteinases (TIMPs), produced by most mesenchymal cells. Thus, during scarring, MMPs are activated to remodel the deposited ECM, and then their activity is shut down by the TIMPs.

Healing of Skin Wounds

Cutaneous wound healing is a process that involves both epithelial regeneration and the formation of connective tissue scar and is thus illustrative of the general principles that apply to healing in all tissues.

Depending on the nature and size of the wound, the healing of skin wounds is said to occur by first or second intention.

Healing by First Intention

One of the simplest examples of wound repair is the healing of a clean, uninfected surgical incision approximated by surgical sutures (Fig. 2–33). This is referred to as primary union, or healing by first intention. The incision causes only focal disruption of epithelial basement membrane continuity and death of relatively few epithelial and connective tissue cells. As a result, epithelial regeneration is the principal mechanism of repair. A small scar is formed, but there is minimal wound contraction. The narrow incisional space first fills with fibrin-clotted blood, which then is rapidly invaded by granulation tissue and covered by new epithelium. The steps in the process are well defined:

• Within 24 hours, neutrophils are seen at the incision margin, migrating toward the fibrin clot. Basal cells at the cut edge of the epidermis begin to show increased mitotic activity. Within 24 to 48 hours, epithelial cells from both edges have begun to migrate and proliferate along the dermis, depositing basement membrane components as they progress. The cells meet in the midline beneath the surface scab, yielding a thin but continuous epithelial layer.

• By day 3, neutrophils have been largely replaced by macrophages, and granulation tissue progressively invades the incision space. Collagen fibers are now evident at the incision margins, but these are vertically oriented and do not bridge the incision. Epithelial cell proliferation continues, yielding a thickened epidermal covering layer.

• By day 5, neovascularization reaches its peak as granulation tissue fills the incisional space. Collagen fibrils become more abundant and begin to bridge the incision. The epidermis recovers its normal thickness as differentiation of surface cells yields a mature epidermal architecture with surface keratinization.

• During the second week, there is continued collagen accumulation and fibroblast proliferation. The leukocyte infiltrate, edema, and increased vascularity are substantially diminished. The long process of “blanching” begins, accomplished by increasing collagen deposition within the incisional scar and the regression of vascular channels.

• By the end of the first month, the scar consists of a cellular connective tissue, largely devoid of inflammatory cells, covered by an essentially normal epidermis. However, the dermal appendages destroyed in the line of the incision are permanently lost. The tensile strength of the wound increases with time, as described later.

Figure 2–33 Steps in wound healing by first intention (left) and second intention (right). In the latter case, note the large amount of granulation tissue and wound contraction.

Healing by Second Intention

When cell or tissue loss is more extensive, such as in large wounds, at sites of abscess formation, ulceration, and ischemic necrosis (infarction) in parenchymal organs, the repair process is more complex and involves a combination of regeneration and scarring. In second intention healing of skin wounds, also known as healing by secondary union (Fig. 2–34; see also Fig. 2–33), the inflammatory reaction is more intense, and there is development of abundant granulation tissue, with accumulation of ECM and formation of a large scar, followed by wound contraction mediated by the action of myofibroblasts.

Figure 2–34 Healing of skin ulcers. A, Pressure ulcer of the skin, commonly found in diabetic patients. B, A skin ulcer with a large gap between the edges of the lesion. C, A thin layer of epidermal re-epithelialization, and extensive granulation tissue formation in the dermis. D, Continuing re-epithelialization of the epidermis and wound contraction.

(Courtesy of Z. Argenyi, MD, University of Washington, Seattle, Wash.)

Secondary healing differs from primary healing in several respects:

• A larger clot or scab rich in fibrin and fibronectin forms at the surface of the wound.

• Inflammation is more intense because large tissue defects have a greater volume of necrotic debris, exudate, and fibrin that must be removed. Consequently, large defects have a greater potential for secondary, inflammation-mediated, injury.

• Larger defects require a greater volume of granulation tissue to fill in the gaps and provide the underlying framework for the regrowth of tissue epithelium. A greater volume of granulation tissue generally results in a greater mass of scar tissue.

• Secondary healing involves wound contraction. Within 6 weeks, for example, large skin defects may be reduced to 5% to 10% of their original size, largely by contraction. This process has been ascribed to the presence of myofibroblasts, which are modified fibroblasts exhibiting many of the ultrastructural and functional features of contractile smooth muscle cells.

Fibrosis in Parenchymal Organs

Deposition of collagen is part of normal wound healing. The term fibrosis is used to denote the excessive deposition of collagen and other ECM components in a tissue. As already mentioned, the terms scar and fibrosis are used interchangeably, but fibrosis most often refers to the deposition of collagen in chronic diseases.

The basic mechanisms of fibrosis are the same as those of scar formation during tissue repair. However, tissue repair typically occurs after a short-lived injurious stimulus and follows an orderly sequence of steps, whereas fibrosis is induced by persistent injurious stimuli such as infections, immunologic reactions, and other types of tissue injury. The fibrosis seen in chronic diseases such as pulmonary fibrosis is often responsible for organ dysfunction and even organ failure.