Chapter 3: Inflammation and Repair

Changes in Vascular Flow and Caliber

Increased Vascular Permeability (Vascular Leakage)

Several mechanisms are responsible for the increased permeability of postcapillary venules, a hallmark of acute inflammation (Fig. 3.3).

• •  Contraction of endothelial cells resulting in opening of interendothelial gaps is the most common mechanism of vascular leakage. It is elicited by histamine, bradykinin, leukotrienes, and other chemical mediators. It is called the immediate transient response because it occurs rapidly after exposure to the mediator and is usually short-lived (15 to 30 minutes). In some forms of mild injury (e.g., after burns, irradiation or ultraviolet radiation, and exposure to certain bacterial toxins), vascular leakage begins after a delay of 2 to 12 hours and lasts for several hours or even days; this delayed prolonged leakage may be caused by contraction of endothelial cells or mild endothelial damage. Sunburn is a classic example of damage that results in late-appearing vascular leakage. Often the immediate and delayed responses occur along a continuum.
• •  Endothelial injury resulting in endothelial cell necrosis and detachment. Direct damage to the endothelium is encountered in severe physical injuries, for example, in thermal burns, or is induced by the actions of microbes and microbial toxins that damage endothelial cells. Neutrophils that adhere to the endothelium during inflammation may also injure endothelial cells and thus amplify the reaction. In most instances leakage starts immediately after injury and is sustained for several hours until the damaged vessels are thrombosed or repaired.

Although these mechanisms of increased vascular permeability are described separately, all probably contribute in varying degrees in responses to most stimuli. For example, at different stages of a thermal burn, leakage results from chemically mediated endothelial contraction and direct and leukocyte-dependent endothelial injury. The vascular leakage induced by these mechanisms can cause life-threatening loss of fluid in severely burned patients.

Responses of Lymphatic Vessels and Lymph Nodes

In addition to blood vessels, lymphatic vessels also participate in acute inflammation. The system of lymphatics and lymph nodes filters and polices the extravascular fluids. Lymphatics drain the small amount of extravascular fluid that seeps out of capillaries in the healthy state. In inflammation, lymph flow is increased and helps drain edema fluid that accumulates because of increased vascular permeability. In addition to fluid, leukocytes and cell debris, as well as microbes, may find their way into lymph. Lymphatic vessels, like blood vessels, proliferate during inflammatory reactions to handle the increased load. 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 macrophages. This constellation of pathologic changes is termed reactive, or inflammatory, lymphadenitis (Chapter 13). The presence of red streaks near a skin wound is a telltale sign of bacterial infection. The streaks represent inflamed lymphatic channels and are diagnostic of lymphangitis; it may be accompanied by painful enlargement of the draining lymph nodes, indicating lymphadenitis.

Leukocyte Adhesion to Endothelium

The attachment of leukocytes to endothelial cells is mediated by adhesion molecules whose expression is enhanced by cytokines, which are secreted by sentinel cells in tissues in response to microbes and other injurious agents, thus ensuring that leukocytes are recruited to the tissues where these stimuli are present. The two major families of proteins involved in leukocyte adhesion and migration are the selectins and integrins and their ligands (Table 3.3). They are expressed on leukocytes and endothelial cells.

• •  Selectins. The initial rolling interactions are mediated by selectins, of which there are three types: one expressed on leukocytes (L-selectin), one on endothelium (E-selectin), and one in platelets and on endothelium (P-selectin) (see Table 3.3). The ligands for selectins are sialylated oligosaccharides bound to mucin-like glycoproteins. The expression of selectins and their ligands is regulated by cytokines produced in response to infection and injury. Tissue macrophages, mast cells, and endothelial cells that encounter microbes and dead tissues respond by secreting several cytokines including tumor necrosis factor (TNF), IL-1, and chemokines (chemoattractant cytokines). (Cytokines are described in more detail later and in Chapter 6.) TNF and IL-1 act on the endothelial cells of postcapillary venules adjacent to the infection and induce the coordinate expression of numerous adhesion molecules. Within 1 to 2 hours the endothelial cells begin to express E-selectin and the ligands for L-selectin. Other mediators such as histamine and thrombin, described later, stimulate the redistribution of P-selectin from its normal intracellular stores in endothelial cell granules (called Weibel-Palade bodies) to the cell surface. Leukocytes express L-selectin at the tips of their microvilli and also express ligands for E-selectin and P-selectin, all of which bind to the complementary molecules on the endothelial cells. These are low-affinity interactions with a fast off-rate, so they are easily disrupted by the flowing blood. As a result, the bound leukocytes bind, detach, and bind again and thus begin to roll along the endothelial surface.
• •  Integrins. The weak rolling interactions slow down the leukocytes and give them the opportunity to bind more firmly to the endothelium. Firm adhesion is mediated by a family of heterodimeric leukocyte surface proteins called integrins (see Table 3.3). TNF and IL-1 induce endothelial expression of ligands for integrins, mainly vascular cell adhesion molecule 1 (VCAM-1), the ligand for the β1 integrin VLA-4, and intercellular adhesion molecule-1 (ICAM-1), the ligand for the β2 integrins LFA-1 and MAC-1. Leukocytes normally express integrins in a low-affinity state. Chemokines that were produced at the site of injury bind to endothelial cell proteoglycans and are displayed at high concentrations on the endothelial surface. These chemokines bind to and activate the rolling leukocytes. One of the consequences of activation is the conversion of VLA-4 and LFA-1 integrins on the leukocytes to a high-affinity state. The combination of cytokine-induced expression of integrin ligands on the endothelium and increased integrin affinity on the leukocytes results in firm integrin-mediated adhesion of the leukocytes to the endothelium at the site of inflammation. The leukocytes stop rolling, their cytoskeleton is reorganized, and they spread out on the endothelial surface.

Leukocyte Migration Through Endothelium

The most telling proof of the importance of leukocyte adhesion molecules in the host inflammatory response are genetic deficiencies in these molecules, which result in increased susceptibility to bacterial infections. These leukocyte adhesion deficiencies are described in Chapter 6.

Chemotaxis of Leukocytes

After exiting the circulation, leukocytes move in the tissues toward the site of injury by a process called chemotaxis, which is defined as locomotion along a chemical gradient. Both exogenous and endogenous substances act as chemoattractants. The most common exogenous factors are bacterial products, including peptides with N-formylmethionine terminal amino acids and some lipids. Endogenous chemoattractants include several chemical mediators (described later): (1) cytokines, particularly those of the chemokine family (e.g., IL-8); (2) components of the complement system, particularly C5a; and (3) arachidonic acid (AA) metabolites, mainly leukotriene B₄ (LTB₄). All these chemotactic agents bind to specific seven-transmembrane G protein–coupled receptors on the surface of leukocytes. Signals initiated from these receptors result in activation of second messengers that induce polymerization of actin at the leading edge of the cell and localization of myosin filaments at the back. This reorganization of the cytoskeleton allows the leading edge of the leukocyte to extend filopodia that pull the back of the cell in the direction of extension, much as an automobile with front-wheel drive is pulled by the wheels in front (Fig. 3.5). The net result is that leukocytes migrate in the direction of locally produced chemoattractants emanating from the site of the inflammatory stimulus.

The nature of the leukocyte infiltrate varies with the age of the inflammatory response and 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. 3.6). There are several reasons for the early preponderance of neutrophils: they are more numerous than are other leukocytes, respond more rapidly to chemokines, and may attach more firmly to the adhesion molecules that are rapidly induced on endothelial cells such as P-selectin and E-selectin. After entering tissues, neutrophils are short-lived; most neutrophils in extravascular tissues undergo apoptosis within a few days. Monocytes not only survive longer but may also proliferate in the tissues, and thus they become the dominant population in prolonged inflammatory reactions. There are, however, exceptions to this stereotypic pattern of cellular infiltration. In certain infections—for example, those produced by Pseudomonas bacteria—the cellular infiltrate is dominated by continuously recruited neutrophils for several days; in viral infections, lymphocytes may be the first cells to arrive; some hypersensitivity reactions are dominated by activated lymphocytes, macrophages, and plasma cells (reflecting the immune response); and in helminthic infections and allergic reactions, eosinophils may be the main cell type.

The molecular understanding of leukocyte recruitment and migration has led to development of a large number of drugs for controlling harmful inflammation, including agents that block TNF (discussed later), and antagonists of leukocyte integrins that are approved for inflammatory diseases or are being tested in clinical trials. Predictably, these antagonists not only have the desired effect of controlling the inflammation but can also compromise the ability of treated patients to defend themselves against microbes, which, of course, is the physiologic function of the inflammatory response.

Phagocytosis

Phagocytosis involves sequential steps (Fig. 3.8):

• • Recognition and attachment of the particle to be ingested by the leukocyte;
• • Engulfment, with subsequent formation of a phagocytic vacuole; and
• • Killing of the microbe and degradation of the ingested material.

Intracellular Destruction of Microbes and Debris

Killing of microbes is accomplished by reactive oxygen species (ROS), also called reactive oxygen intermediates, and reactive nitrogen species, mainly derived from nitric oxide (NO), and these as well as lysosomal enzymes destroy phagocytosed materials (see Fig. 3.8). This is the final step in the elimination of infectious agents and necrotic cells. The killing and degradation of microbes and dead cell debris within neutrophils and macrophages occur most efficiently after activation of the phagocytes. All these killing mechanisms are normally sequestered in lysosomes, to which phagocytosed materials are brought. Thus, potentially harmful substances are segregated from the cell's cytoplasm and nucleus to avoid damage to the phagocyte while it is performing its normal function.

Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are extracellular fibrillar networks that concentrate antimicrobial substances at sites of infection and trap microbes, helping to prevent their spread. They are produced by neutrophils in response to infectious pathogens (mainly bacteria and fungi) and inflammatory mediators (e.g., chemokines, cytokines [mainly interferons], complement proteins, and ROS). The extracellular traps consist of a viscous meshwork of nuclear chromatin that binds and concentrates granule proteins such as antimicrobial peptides and enzymes (Fig. 3.9). NET formation starts with ROS-dependent activation of an arginine deaminase that converts arginines to citrulline, leading to chromatin decondensation. Other enzymes that are produced in activated neutrophils, such as MPO and elastase, enter the nucleus and cause further chromatin decondensation, culminating in rupture of the nuclear envelope and release of chromatin. In this process, the nuclei of the neutrophils are lost, leading to death of the cells. NETs have also been detected in the blood 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 individuals react against their own DNA and nucleoproteins (Chapter 6).

Leukocyte-Mediated Tissue Injury

In all these situations, the mechanisms by which leukocytes damage normal tissues are the same as the mechanisms involved in antimicrobial defense because once the leukocytes are activated, their effector mechanisms do not distinguish between offender and host. During activation and phagocytosis, neutrophils and macrophages produce microbicidal substances (ROS, NO, and lysosomal enzymes) within the phagolysosome; under some circumstances, these substances are also released into the extracellular space. These released substances are capable of damaging host cells such as vascular endothelium and may thus amplify the effects of the initial injurious agent. If unchecked or inappropriately directed against host tissues, the leukocyte infiltrate itself becomes the offender, and indeed leukocyte-dependent inflammation and tissue injury underlie many acute and chronic human diseases (see Table 3.1). This fact becomes evident in the discussion of specific disorders throughout this book.

The contents of lysosomal granules are secreted by leukocytes into the extracellular milieu by several mechanisms. Controlled secretion of granule contents is a normal response of activated leukocytes. If phagocytes encounter materials that cannot be easily ingested, such as immune complexes deposited on large surfaces (e.g., glomerular basement membrane), the inability of the leukocytes to surround and ingest these substances (frustrated phagocytosis) triggers strong activation and the release of lysosomal enzymes into the extracellular environment. Some phagocytosed substances, such as urate crystals, may damage the membrane of the phagolysosome, also leading to the release of lysosomal granule contents.

Other Functional Responses of Activated Leukocytes

In this discussion of acute inflammation, we emphasize the importance of neutrophils and macrophages. However, it has recently become clear that some T lymphocytes, which are cells of adaptive immunity, also contribute to acute inflammation. The most important of these cells are those that produce the cytokine IL-17 (so-called Th17 cells), discussed in more detail in Chapter 6. IL-17 induces the secretion of chemokines that recruit other leukocytes. In the absence of effective Th17 responses, individuals are susceptible to fungal and bacterial infections and tend to develop “cold abscesses,” particularly in the skin, that lack the classic features of acute inflammation, such as warmth and redness.

Vasoactive Amines: Histamine and Serotonin

The richest source of histamine are mast cells that are normally present in the connective tissue adjacent to blood vessels. It is also found in basophils and platelets. Histamine is stored in mast cell granules and is released by mast cell degranulation in response to a variety of stimuli, including (1) physical injury (such as trauma), cold, and heat, all by unknown mechanisms; (2) binding of antigen to IgE antibodies displayed on the surfaces of mast cells, which underlies immediate hypersensitivity (allergic) reactions (Chapter 6); and (3) products of complement called anaphylatoxins (C3a and C5a), described later. Antibodies and complement products bind to specific receptors on mast cells and trigger signaling pathways that induce rapid degranulation. Neuropeptides (e.g., substance P) and cytokines (IL-1, IL-8) may also trigger release of histamine.

Histamine causes dilation of arterioles and increases the permeability of venules. Histamine is considered to be the principal mediator of the immediate transient phase of increased vascular permeability, producing interendothelial gaps in venules, as discussed earlier. Its vasoactive effects are mediated mainly via binding to receptors on microvascular endothelial cells. The antihistamine drugs that are commonly used to treat some inflammatory reactions, such as allergies, are histamine receptor antagonists that bind to and block the receptor. Histamine also causes contraction of some smooth muscles.

Serotonin (5-hydroxytryptamine) is a preformed vasoactive mediator present in platelets and certain neuroendocrine cells, such as in the gastrointestinal tract. Its primary function is as a neurotransmitter in the gastrointestinal tract and the central nervous system. It is also a vasoconstrictor, but the importance of this action in inflammation is unclear.

Arachidonic Acid Metabolites

The lipid mediators prostaglandins and leukotrienes are produced from arachidonic acid (AA) present in membrane phospholipids and stimulate vascular and cellular reactions in acute inflammation. AA is a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid) that is derived from dietary sources or by synthesis from a precursor molecule, the essential fatty acid linoleic acid. Active AAs are derived from an esterified precursor found in membrane phospholipids. Mechanical, chemical, and physical stimuli or other mediators (e.g., C5a) release AA from membrane phospholipids through the action of cellular phospholipases, mainly phospholipase A₂. AA-derived mediators, also called eicosanoids (because they are derived from 20-carbon fatty acids; Greek eicosa = 20), are synthesized by two major classes of enzymes: cyclooxygenases (which generate prostaglandins) and lipoxygenases (which produce leukotrienes and lipoxins) (Fig. 3.10). Eicosanoids bind to G protein–coupled receptors on many cell types and can mediate virtually every step of inflammation (Table 3.6).

Cytokines and Chemokines

Cytokines are proteins produced by many cell types (principally activated lymphocytes, macrophages, and dendritic cells, but also endothelial, epithelial, and connective tissue cells) that mediate and regulate immune and inflammatory reactions. By convention, growth factors that act on epithelial and mesenchymal cells are not grouped under cytokines. The general properties and functions of cytokines are discussed in Chapter 6. The cytokines involved in acute inflammation are reviewed here (Table 3.7).

Tumor Necrosis Factor and Interleukin-1

TNF and IL-1 serve critical roles in leukocyte recruitment by promoting adhesion of leukocytes to endothelium and their migration through blood vessels. These cytokines are produced mainly by activated macrophages and dendritic cells; TNF is also produced by T lymphocytes and mast cells, and IL-1 is produced by some epithelial cells as well. The secretion of TNF and IL-1 can be stimulated by microbial products, dead cells, immune complexes, foreign bodies, physical injury, and a variety of other inflammatory stimuli. The production of TNF is induced by signals through TLRs and other microbial sensors. The synthesis of IL-1 is stimulated by the same signals, but the generation of the biologically active form of this cytokine is dependent on the inflammasome (described earlier).

The actions of TNF and IL-1 contribute to the local and systemic reactions of inflammation (Fig. 3.11). The most important roles of these cytokines in inflammation are the following.

• •  Endothelial activation. Both TNF and IL-1 act on endothelium to induce a spectrum of changes referred to as endothelial activation. These changes include increased expression of endothelial adhesion molecules, mostly E-selectin and P-selectin and ligands for leukocyte integrins; increased production of various mediators, including other cytokines and chemokines, growth factors, and eicosanoids; and increased procoagulant activity of the endothelium.
• •  Activation of leukocytes and other cells. TNF augments responses of neutrophils to other stimuli such as bacterial endotoxin and stimulates the microbicidal activity of macrophages, in part by inducing production of NO. IL-1 activates fibroblasts to synthesize collagen and stimulates proliferation of synovial and other mesenchymal cells. IL-1 also stimulates Th17 responses, which in turn induce acute inflammation.
• •  Systemic acute-phase response. IL-1 and TNF (as well as IL-6) induce the systemic acute-phase responses associated with infection or injury, discussed later. TNF regulates energy balance by promoting lipid and protein mobilization and by suppressing appetite. Therefore, sustained production of TNF contributes to cachexia, a pathologic state characterized by weight loss and anorexia that is seen in some chronic infections and neoplastic diseases.

TNF antagonists have been remarkably effective in the treatment of chronic inflammatory diseases, particularly rheumatoid arthritis, psoriasis, and some types of inflammatory bowel disease. One of the complications of this therapy is that patients become susceptible to mycobacterial infection, reflecting the reduced ability of macrophages to kill intracellular microbes. Although many of the actions of TNF and IL-1 seem overlapping, IL-1 antagonists are not as effective for reasons that remain unclear. Also, blocking either cytokine has no effect on the outcome of sepsis, perhaps because other cytokines contribute to this systemic inflammatory reaction.

Complement System

The complement system is a collection of plasma proteins that function mainly in host defense against microbes and in pathologic inflammatory reactions. The system consists of more than 20 proteins, some of which are numbered C1 through C9. The complement proteins function in both innate and adaptive immunity for defense against microbial pathogens. In the process of complement activation, several cleavage products of complement proteins are elaborated that cause increased vascular permeability, chemotaxis, and opsonization. The activation and functions of complement are outlined in Fig. 3.12.

Complement proteins are present in inactive forms in the plasma, and many of them are activated to become proteolytic enzymes that degrade other complement proteins, thus forming an enzymatic cascade capable of tremendous amplification. The critical step in complement activation is the proteolysis of the third (and most abundant) component, C3. Cleavage of C3 can occur by one of three pathways:

• • The classical pathway, which is triggered by binding of C1 to antibody (IgM or IgG) that has combined with antigen.
• • The alternative pathway, which can be triggered by microbial surface molecules (e.g., endotoxin, or lipopolysaccharide [LPS]), complex polysaccharides, cobra venom, and other substances, in the absence of antibody.
• • The lectin pathway, in which plasma mannose-binding lectin binds to carbohydrates on microbes and directly activates C1.

All three pathways of complement activation lead to the formation of an active enzyme called C3 convertase, which splits C3 into two functionally distinct fragments, C3a and C3b. C3a is released, and C3b becomes covalently attached to the cell or molecule where complement is being activated. More C3b then binds to the previously generated fragments to form C5 convertase, which cleaves C5 to release C5a and leave C5b attached to the cell surface. C5b binds the late components (C6–C9), culminating in the formation of the membrane attack complex (composed of multiple C9 molecules).

The complement system has three main functions (see Fig. 3.12).

• •  Inflammation. C5a, C3a, and, to a lesser extent, C4a are cleavage products of the corresponding complement components that stimulate histamine release from mast cells and thereby increase vascular permeability and cause vasodilation. They are called anaphylatoxins because they have effects similar to those of mast cell mediators that are involved in the reaction called anaphylaxis (Chapter 6). C5a is also a chemotactic agent for neutrophils, monocytes, eosinophils, and basophils. In addition, C5a activates the lipoxygenase pathway of AA metabolism in neutrophils and monocytes, causing further release of inflammatory mediators.
• •  Opsonization and phagocytosis. C3b and its cleavage product inactive C3b (iC3b), when fixed to a microbial cell wall, act as opsonins and promote phagocytosis by neutrophils and macrophages, which bear cell surface receptors for the complement fragments.
• •  Cell lysis. The deposition of the membrane attack complex on cells makes these cells permeable to water and ions and results in osmotic lysis of the cells. This role of complement is particularly important for killing microbes with thin cell walls, such as Neisseria bacteria, and deficiency of the terminal components of complement predisposes to Neisseria infections.

The activation of complement is tightly controlled by cell-associated and circulating regulatory proteins. Different regulatory proteins inhibit the production of active complement fragments or remove fragments that deposit on cells. These regulators are expressed on normal host cells and are thus designed to prevent healthy tissues from being injured at sites of complement activation. Regulatory proteins can be overwhelmed when large amounts of complement are deposited on host cells and in tissues, as happens in autoimmune diseases, in which individuals produce complement-fixing antibodies against their own cell and tissue antigens (Chapter 6). The most important of these regulatory proteins are the following:

• •  C1 inhibitor (C1 INH) blocks the activation of C1, the first protein of the classical complement pathway. Inherited deficiency of this inhibitor is the cause of hereditary angioedema.
• •  Decay accelerating factor (DAF) and CD59 are two proteins that are linked to plasma membranes by a glycophosphatidylinositol (GPI) anchor. DAF prevents formation of C3 convertases, and CD59 inhibits formation of the membrane attack complex. An acquired deficiency of the enzyme that creates GPI anchors leads to deficiency of these regulators and excessive complement activation and lysis of red cells (which are sensitive to complement-mediated cell lysis) in the disease called paroxysmal nocturnal hemoglobinuria (PNH) (Chapter 14).
• •  Complement Factor H is a circulating glycoprotein that inhibits the alternative pathway of complement activation by promoting the cleavage and destruction of C3b and the turnover of the C3 convertases. Inherited defects in Factor H and several other regulatory proteins that interact with Factor H cause an atypical form of hemolytic uremic syndrome (Chapter 20), in which complement deposits in glomerular vessels, leading to endothelial damage and formation of platelet-rich thrombi. Polymorphisms in the Factor H gene have also been linked to age-related macular degeneration (Chapter 29), an important cause of vision loss in older adults.

The complement system contributes to disease in several ways. The activation of complement by antibodies or antigen-antibody complexes deposited on host cells and tissues is an important mechanism of cell and tissue injury (Chapter 6). Inherited deficiencies of complement proteins cause increased susceptibility to infections (Chapter 6), and, as mentioned earlier, deficiencies of regulatory proteins cause a variety of disorders resulting from excessive complement activation.

Serous Inflammation

Serous inflammation is marked by the exudation of cell-poor fluid into spaces created by cell injury or into body cavities lined by the peritoneum, pleura, or pericardium. Typically, the fluid in serous inflammation does not contain microbes or large numbers of leukocytes (which tend to produce purulent inflammation, described later). In body cavities the fluid may be derived from the plasma (as a result of increased vascular permeability) or from the secretions of mesothelial cells (as a result of local irritation); accumulation of fluid in these cavities is called an effusion. (Effusions also occur in noninflammatory conditions, such as reduced blood outflow in heart failure or reduced plasma protein levels in some kidney and liver diseases.) The skin blister resulting from a burn or viral infection represents accumulation of serous fluid within or immediately beneath the damaged epidermis of the skin (Fig. 3.13).

Fibrinous Inflammation

With greater increase in vascular permeability, large molecules such as fibrinogen pass out of the blood, and fibrin is formed and deposited in the extracellular space. A fibrinous exudate develops when the vascular leaks are large or there is a local procoagulant stimulus (e.g., caused by cancer cells). A fibrinous exudate is characteristic of inflammation in the lining of body cavities, such as the meninges, pericardium (Fig. 3.14A), and pleura. Histologically, fibrin appears as an eosinophilic meshwork of threads or sometimes as an amorphous coagulum (Fig. 3.14B). Fibrinous exudates may be dissolved by fibrinolysis and cleared by macrophages. If the fibrin is not removed, over time it may stimulate the ingrowth of fibroblasts and blood vessels and thus lead to scarring. Conversion of the fibrinous exudate to scar tissue (organization) within the pericardial sac leads to opaque fibrous thickening of the pericardium and epicardium in the area of exudation and, if the fibrosis is extensive, obliteration of the pericardial space.

Purulent (Suppurative) Inflammation and Abscess

Abscesses are localized collections of pus caused by suppuration buried in a tissue, an organ, or a confined space. They are produced by seeding of pyogenic bacteria into a tissue (Fig. 3.15). Abscesses have a central liquefied region composed of necrotic leukocytes and tissue cells. There is usually a zone of preserved neutrophils around this necrotic focus, and outside this region there may be vascular dilation and parenchymal and fibroblastic proliferation, indicating chronic inflammation and repair. In time the abscess may become walled off and ultimately replaced by connective tissue.

Ulcers

An ulcer is a local defect, or excavation, of the surface of an organ or tissue that is produced by the sloughing (shedding) of inflamed necrotic tissue (Fig. 3.16). Ulceration can occur only when tissue necrosis and resultant inflammation exist on or near a surface. It is most common in (1) the mucosa of the mouth, stomach, intestines, or genitourinary tract, and (2) the skin and subcutaneous tissue of the lower extremities in individuals with disorders that predispose to vascular insufficiency, such as diabetes, sickle cell anemia, and peripheral vascular disease.

Ulcerations are best exemplified by peptic ulcer of the stomach or duodenum, in which acute and chronic inflammation coexist. During the acute stage there is intense polymorphonuclear infiltration and vascular dilation in the margins of the defect. With chronicity, the margins and base of the ulcer develop fibroblastic proliferation, scarring, as well as the accumulation of lymphocytes, macrophages, and plasma cells.

Role of Macrophages

Macrophages are tissue cells derived from hematopoietic stem cells in the bone marrow in postnatal life and from progenitors in the embryonic yolk sac and fetal liver during early development (Fig. 3.19). Circulating cells of this lineage are known as monocytes. Macrophages are normally diffusely scattered in most connective tissues. In addition, they are present in specific locations in organs such as the liver (where they are called Kupffer cells), spleen and lymph nodes (called sinus histiocytes), central nervous system (microglial cells), and lungs (alveolar macrophages). Together, these cells comprise the mononuclear phagocyte system, also known by the older (and inaccurate) name reticuloendothelial system.

Committed progenitors in the bone marrow give rise to monocytes, which enter the blood, migrate into various tissues, and differentiate into macrophages. This is typical of macrophages at sites of inflammation and in some tissues such as the skin and intestinal tract. The half-life of blood monocytes is about 1 day, whereas the lifespan of tissue macrophages may be several months or years. Other specialized types of macrophages, such as microglia, Kupffer cells, and alveolar macrophages, arise from progenitors in the yolk sac or fetal liver very early in embryogenesis and migrate to the developing brain, liver, and lung, where they persist throughout life as a stable population of resident cells. As discussed earlier, in inflammatory reactions, monocytes begin to emigrate into extravascular tissues quite early, and within 48 hours they may constitute the predominant cell type. Extravasation of monocytes is governed by the same factors that are involved in neutrophil emigration, that is, adhesion molecules and chemotactic factors.

The products of activated macrophages eliminate injurious agents such as microbes and initiate the process of repair, but are also responsible for much of the tissue injury in chronic inflammation. Several functions of macrophages are central to the development and persistence of chronic inflammation and the accompanying tissue injury.

• • Macrophages, like the other type of phagocytes, the neutrophils, ingest and eliminate microbes and dead tissues.
• • Macrophages initiate the process of tissue repair and are involved in scar formation and fibrosis. These processes are discussed later in the chapter.
• • Macrophages secrete mediators of inflammation, such as cytokines (TNF, IL-1, chemokines, and others) and eicosanoids. Thus, macrophages contribute to the initiation and propagation of 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. These interactions are described further in the subsequent discussion of the role of lymphocytes in chronic inflammation and in more detail in Chapter 6 where cell-mediated immunity is considered.

There are two major pathways of macrophage activation, called classical and alternative, that endow macrophages with different functional activities (Fig. 3.20).

• •  Classical macrophage activation may be induced by microbial products such as endotoxin, which engage TLRs and other sensors; by T cell–derived signals, importantly the cytokine IFN-γ, in immune responses; or by foreign substances, including crystals and particulate matter. Classically activated (also called M1) macrophages produce NO and lysosomal enzymes, which enhance their ability to kill ingested organisms, and secrete cytokines that stimulate inflammation. The main role of these macrophages in host defense is to destroy microbes and promote the inflammatory response.
• •  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. These macrophages are not actively microbicidal; instead, their principal functions are to terminate inflammation and promote tissue repair.

It seems plausible that in response to most injurious stimuli the first activation pathway is the classical one, designed to destroy the offending agents, and this is followed by alternative activation, which initiates tissue repair. However, such a precise sequence is not documented in most inflammatory reactions, and most reactions contain varying numbers of both types. Also, the M1 and M2 phenotypes are the extreme forms, and there may be many intermediate types that are difficult to characterize as typical M1 or M2.

Their impressive arsenal of mediators makes macrophages powerful allies in the body's defense against unwanted invaders, but the same weaponry can also induce considerable tissue destruction when macrophages are inappropriately or excessively activated. It is largely because of these activities of macrophages that tissue destruction is one of the hallmarks of chronic inflammation.

In some instances, if the irritant is eliminated, macrophages eventually disappear (either dying off or making their way into the lymphatics and lymph nodes). In others, macrophage accumulation persists, as a result of continuous recruitment from the circulation and local proliferation at the site of inflammation.

Role of Lymphocytes

Both Th1 and Th17 cells are involved in defense against many types of bacteria and viruses and in autoimmune diseases in which tissue injury is caused by chronic inflammation. Th2 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 6.

Lymphocytes and macrophages interact in a bidirectional way, and these interactions play an important role in propagating chronic inflammation (Fig. 3.21). Macrophages display antigens to T cells, express membrane molecules called costimulators, and produce cytokines (IL-12 and others) that stimulate T-cell responses (Chapter 6). Activated T lymphocytes, in turn, produce cytokines, described earlier, which recruit and activate macrophages, promoting more antigen presentation and cytokine secretion. The result is a cycle of cellular reactions that fuel and sustain chronic inflammation. A typical consequence of such a chronic T cell–macrophage reaction is the formation of granulomas, described later.

Activated B lymphocytes and antibody-producing plasma cells are also often present at sites of chronic inflammation. The antibodies produced may be specific for persistent foreign antigens or self antigens in the inflammatory site or against altered tissue components. However, the contribution of antibodies to most chronic inflammatory disorders is unclear.

In some chronic inflammatory reactions, the accumulated lymphocytes, antigen-presenting cells, and plasma cells cluster together to form organized lymphoid follicles resembling those seen in lymph nodes. These are called tertiary lymphoid organs; this type of lymphoid organogenesis is often seen in the synovium of patients with long-standing rheumatoid arthritis and in the thyroid in Hashimoto thyroiditis. It has been postulated that the local formation of lymphoid follicles may perpetuate the immune reaction, but the significance of these structures is not established.

Other Cells in Chronic Inflammation

• •  Eosinophils are abundant in immune reactions mediated by IgE and in parasitic infections (Fig. 3.22). Their recruitment is driven by adhesion molecules similar to those used by neutrophils and by specific chemokines (e.g., eotaxin) derived from leukocytes and epithelial cells. Eosinophils have granules that contain major basic protein, a highly cationic protein that is toxic to helminths but also may injure host epithelial cells. This is why eosinophils are of benefit in controlling helminth infections, yet they also contribute to tissue damage in immune reactions such as allergies (Chapter 6).

  
  

• •  Mast cells are widely distributed in connective tissues and participate in both acute and chronic inflammatory reactions. Mast cells express on their surface the receptor (FcεRI) that binds the Fc portion of IgE antibody. In immediate hypersensitivity reactions, IgE antibodies bound to the cells’ Fc receptors specifically recognize antigen, and the cells degranulate and release mediators such as histamine and prostaglandins (Chapter 6). This type of response occurs during allergic reactions to foods, insect venom, or drugs, sometimes with catastrophic results (e.g., anaphylactic shock). Mast cells are also present in chronic inflammatory reactions, and because they secrete a plethora of cytokines, they may promote inflammatory reactions in different situations.
• • Although neutrophils are characteristic of acute inflammation, many forms of chronic inflammation continue to show large numbers of neutrophils, induced either by persistent microbes or by mediators produced by activated macrophages and T lymphocytes. In chronic bacterial infection of bone (osteomyelitis), a neutrophilic exudate can persist for many months. Neutrophils are also important in the chronic damage induced in lungs by smoking and other irritant stimuli (Chapter 15). This pattern of inflammation has been called acute on chronic.

Cell Proliferation: Signals and Control Mechanisms

Cell proliferation is driven by signals provided by growth factors and from the ECM. Many different growth factors have been described; some act on multiple cell types, and others are cell-type specific (see Table 1.1 in Chapter 1). Growth factors are typically produced by cells near the site of damage. The most important sources of these growth factors are macrophages that are activated by the tissue injury, but epithelial and stromal cells also produce some of these factors. Several growth factors are displayed at high concentrations bound to ECM proteins. All growth factors activate signaling pathways that stimulate DNA replication (Chapter 1), while also fostering changes in metabolism that promote the biosynthesis of other cellular components (membranes, organelles, proteins) that are needed for a “mother” cell to produce two daughter cells. In addition to responding to growth factors, cells use integrins to bind to ECM proteins, and signals from the integrins can also stimulate cell proliferation.

Mechanisms of Tissue Regeneration

Steps in Scar Formation

Repair by connective tissue deposition consists of sequential processes that follow tissue injury. Within minutes after injury, a hemostatic plug composed of platelets (Chapter 4) is formed, which stops bleeding and provides a scaffold for the deposition of fibrin. The subsequent steps are summarized here (Fig. 3.26):

• •  Inflammation. Breakdown products of complement activation, chemokines released from activated platelets, and other mediators produced at the site of injury function as chemotactic agents to recruit neutrophils and then monocytes over the next 6 to 48 hours. These inflammatory cells eliminate the offending agents, such as microbes that may have entered through the wound, and clear the debris. As the injurious agents and necrotic cells are cleared, the inflammation resolves.
• •  Cell proliferation. In the next stage, which takes up to 10 days, several cell types, including epithelial cells, endothelial and other vascular cells, and fibroblasts, proliferate and migrate to close the now clean wound. Each cell type serves unique functions.
  • •  Epithelial cells respond to locally produced growth factors and migrate over the wound to cover it up.
  • •  Endothelial cells and pericytes proliferate to form new blood vessels, a process known as angiogenesis. Because of the importance of this process in physiologic host responses and in many pathologic conditions, it is described in more detail subsequently.
  • •  Fibroblasts proliferate and migrate into the site of injury and lay down collagen fibers that form the scar.
• •  Formation of granulation tissue. Migration and proliferation of fibroblasts and deposition of loose connective tissue, together with the vessels and interspersed mononuclear leukocytes, form granulation tissue. The term granulation tissue derives from the pink, soft, granular gross appearance, such as that seen 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. 3.27A). Granulation tissue progressively fills the site of injury; the amount of granulation tissue that is formed depends on the size of the tissue defect created by the wound and the intensity of inflammation.

  
  

• •  Deposition of connective tissue. Granulation tissue is progressively replaced by deposition of collagen. The amount of connective tissue increases in the granulation tissue, eventually resulting in the formation of a stable fibrous scar (Fig. 3.27B).

Macrophages play a central role in repair by clearing offending agents and dead tissue, providing growth factors for the proliferation of various cells, and secreting cytokines that stimulate fibroblast proliferation and connective tissue synthesis and deposition. The macrophages that are involved in repair are mostly of the alternatively activated (M2) type. It is not clear how the classically activated macrophages that dominate during inflammation, and are involved in getting rid of microbes and dead tissues, are gradually replaced by alternatively activated macrophages that serve to terminate inflammation and induce repair.

We next describe the steps in the formation of granulation tissue and the scar.

Angiogenesis

Angiogenesis involves sprouting of new vessels from existing ones and consists of the following steps (Fig. 3.28):

• •  Vasodilation in response to nitric oxide and increased permeability induced by vascular endothelial growth factor (VEGF).
• •  Separation of pericytes from the abluminal surface and breakdown of the basement membrane to allow formation of a vessel sprout.
• •  Migration of endothelial cells toward the area of tissue injury.
• •  Proliferation of endothelial cells just behind the leading front (“tip”) of migrating cells.
• • Remodeling into capillary tubes.
• •  Recruitment of periendothelial cells (pericytes for small capillaries and smooth muscle cells for larger vessels) to form the mature vessel.
• •  Suppression of endothelial proliferation and migration and deposition of the basement membrane.

Deposition of Connective Tissue

Remodeling of Connective Tissue

Healing of Skin Wounds

Healing by First Intention

When the injury involves only the epithelial layer, the principal mechanism of repair is epithelial regeneration, also called primary union or healing by first intention. One of the simplest examples of this type of wound repair is the healing of a clean, uninfected surgical incision approximated by surgical sutures (Fig. 3.29). The incision causes only focal disruption of epithelial basement membrane continuity and death of relatively few epithelial and connective tissue cells. The repair consists of the same three connected processes that we have described previously: inflammation, proliferation of epithelial and other cells, and maturation of the connective tissue scar.

• • Wounding causes the rapid activation of coagulation pathways, which results in the formation of a blood clot on the wound surface (Chapter 4). The clot serves to stop bleeding and supports migrating cells, which are attracted by growth factors, cytokines, and chemokines released into the area.
• • Within 24 hours, neutrophils are seen at the incision margin, migrating toward the fibrin clot. Basal cells at the edge of the incision 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 yielding a thin but continuous epithelial layer that closes the wound.
• • By day 3, neutrophils have been largely replaced by macrophages, and granulation tissue progressively invades the incision space. The macrophages clear extracellular debris, fibrin, and other foreign material and promote angiogenesis and ECM deposition.
• • By day 5, neovascularization reaches its peak as granulation tissue fills the incisional space. The new vessels are leaky, allowing the passage of plasma proteins and fluid into the extravascular space. Thus, new granulation tissue is often edematous. Fibroblasts progressively migrate into the granulation tissue, where they proliferate and lay down collagen and ECM.
• • During the second week, there is continued collagen accumulation and fibroblast proliferation. The leukocyte infiltrate, edema, and increased vascularity are substantially diminished.
• • By the end of the first month, the scar comprises a cellular connective tissue largely devoid of inflammatory cells and covered by an essentially normal epidermis. The tensile strength of the wound increases with time, as described later.

Fibrosis in Parenchymal Organs

Defects in Healing: Chronic Wounds

• •  Venous leg ulcers (Fig. 3.30A) develop most often in elderly people as a result of chronic venous hypertension, which may be caused by severe varicose veins or congestive heart failure. Deposits of iron pigment (hemosiderin) are common, resulting from red cell breakdown, and there may be accompanying chronic inflammation. These ulcers fail to heal because of poor delivery of oxygen to the site of the ulcer.

  
  

• •  Arterial ulcers (Fig. 3.30B) develop in individuals with atherosclerosis of peripheral arteries, especially associated with diabetes. The ischemia results in atrophy and then necrosis of the skin and underlying tissues. These lesions can be quite painful.
• •  Diabetic ulcers (Fig. 3.30C) affect the lower extremities, particularly the feet. There is tissue necrosis and failure to heal as a result of vascular disease causing ischemia, neuropathy, systemic metabolic abnormalities, and secondary infections. Histologically, these lesions are characterized by epithelial ulceration (Fig. 3.30E) and extensive granulation tissue in the underlying dermis (Fig. 3.30F).
• •  Pressure sores (Fig. 3.30D) are areas of skin ulceration and necrosis of underlying tissues caused by prolonged compression of tissues against a bone, e.g., in elderly patients with numerous morbidities lying in bed without moving. The lesions are caused by mechanical pressure and local ischemia.

When a surgical incision reopens internally or externally it is called wound dehiscence. The risk factors for such an occurrence are obesity, malnutrition, infections, and vascular insufficiency. In abdominal wounds it can be precipitated by vomiting and coughing.

Excessive Scarring

Excessive formation of the components of the repair process can give rise to hypertrophic scars and keloids. The accumulation of excessive amounts of collagen may give rise to a raised scar known as a hypertrophic scar. These often grow rapidly and contain abundant myofibroblasts, but they tend to regress over several months (Fig. 3.31A). If the scar tissue grows beyond the boundaries of the original wound and does not regress, it is called a keloid (Fig. 3.31B, C). Keloid formation seems to be an individual predisposition, and for unknown reasons it is somewhat more common in African Americans. Hypertrophic scars generally develop after thermal or traumatic injury that involves the deep layers of the dermis.

Exuberant granulation is another deviation in wound healing consisting of the formation of excessive amounts of granulation tissue, which protrudes above the level of the surrounding skin and blocks reepithelialization (this process has been called, with more literary fervor, proud flesh). Excessive granulation must be removed by cautery or surgical excision to permit restoration of the continuity of the epithelium. Rarely, incisional scars or traumatic injuries may be followed by exuberant proliferation of fibroblasts and other connective tissue elements that may recur after excision. Called desmoids, or aggressive fibromatoses, these neoplasms lie in the interface between benign and malignant (though low-grade) tumors.

Contraction in the size of a wound is an important part of the normal healing process. An exaggeration of this process gives rise to contracture and results in deformities of the wound and the surrounding tissues. Contractures are particularly prone to develop on the palms, the soles, and the anterior aspect of the thorax. Contractures are commonly seen after serious burns and can compromise the movement of joints.