Inflammation and Repair

Vascular Reactions in Acute Inflammation

Vasodilation is one of the earliest reactions of acute inflammation and is responsible for the externally visible redness (erythema) and warmth that accompany most acute inflammatory reactions. The most important chemical mediator of vasodilation is histamine, discussed later.

Vasodilation is quickly followed by increased permeability of the microvasculature and the outpouring of protein-rich fluid into the extravascular tissues. The escape of fluid, proteins, and blood cells from the vascular system into the interstitial tissue or body cavities is known as exudation ( Fig. 2.2 ). An exudate is an extravascular fluid that has a high protein concentration and contains cellular debris. Its presence implies that there is an increase in the permeability of small blood vessels, typically during an inflammatory reaction. By contrast, a transudate is a fluid with low protein content (most of which is albumin), little or no cellular material, and low specific gravity. A transudate is essentially an ultrafiltrate of blood plasma that is produced as a result of osmotic or hydrostatic imbalance across the vessel wall without an increase in vascular permeability and is usually not associated with inflammation ( Chapter 3 ). Edema denotes an excess of fluid in the interstitial tissue or serous cavities; it can be either an exudate or a transudate. Pus , a purulent exudate, is an inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of dead cells, and, in many cases, microbes.

The principal mechanism of increased vascular permeability is the contraction of endothelial cells, which creates interendothelial openings. It is elicited by histamine, bradykinin, leukotrienes, and other chemical mediators. It occurs rapidly after exposure to the mediator (within 15 to 30 minutes) and is usually short lived. In unusual cases (e.g., in burns), increased vascular permeability may result from direct endothelial injury. In these instances, leakage starts immediately after injury and is sustained for several hours until the damaged vessels become thrombosed or are repaired.

The loss of fluid and increased vessel diameter lead to slower blood flow and higher concentration of red cells in small vessels, raising the viscosity of the blood. Involved small vessels become engorged with red cells, a condition termed stasis , which is seen histologically as vascular congestion and externally as localized redness of the affected tissue.

In addition to the reactions of blood vessels, lymph flow is increased and helps drain edema fluid that accumulates because of increased vascular permeability. The lymphatics may become secondarily inflamed (lymphangitis) , appearing clinically as red streaks extending from an inflammatory focus along the course of lymphatic channels. Involvement of the draining lymph nodes may lead to enlargement (because of increased cellularity) and pain. The associated constellation of pathologic changes is termed reactive or inflammatory lymphadenitis ( Chapter 10 ).

Leukocyte Recruitment to Sites of Inflammation

The journey of leukocytes from the vessel lumen to the tissue is a multistep process that is mediated and controlled by adhesion molecules and cytokines. Leukocytes normally transit rapidly through small vessels. In inflammation, they have to be stopped and then brought to the offending agent or the site of tissue damage, outside the vessels. This process can be divided into phases, consisting first of adhesion of leukocytes to endothelium at the site of inflammation, then transmigration of the leukocytes through the vessel wall, and finally, movement of the cells toward the offending agent ( Fig. 2.3 ).

When blood flows from capillaries into postcapillary venules, under conditions of normal laminar flow, red cells are concentrated in the center of the vessel, displacing leukocytes toward the vessel wall. As the rate of flow slows early in inflammation (stasis), leukocytes, being larger than red cells, slow down more and assume a more peripheral position along the endothelial surface, a process called margination . By moving close to the vessel wall, leukocytes are able to detect and react to changes in the endothelium. When 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 cells finally come to rest at some point where they adhere firmly (resembling pebbles over which a stream runs without disturbing them).

The initial weak binding of leukocytes and their rolling on the endothelium are mediated by a family of proteins called selectins ( Table 2.3 ). Selectins are receptors expressed on leukocytes and endothelium that have an extracellular domain that binds carbohydrates (hence the lectin part of the name). The ligands for selectins are sialic acid–containing oligosaccharides attached to glycoprotein backbones; some are expressed on leukocytes and others on endothelial cells. Endothelial cells express two selectins, E- and P-selectins, as well as the ligand for L-selectin, whereas leukocytes express L-selectin. E- and P-selectins are typically expressed at low levels or not at all on nonactivated endothelium but are upregulated 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 nonactivated endothelial cells, P-selectin is found primarily in intracellular membrane-bound vesicles called Weibel-Palade bodies; however, within minutes of exposure to mediators such as histamine or thrombin, P-selectin traffics to the cell surface. 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 tumor necrosis factor (TNF), which are produced by tissue macrophages, dendritic cells, mast cells, and endothelial cells after encountering microbes or dead tissues. Selectin-mediated interactions have a low affinity with a fast off-rate, and they are easily disrupted by the flowing blood. As a result, the leukocytes bind, detach, and bind again to endothelium. These weak rolling interactions slow down the leukocytes sufficiently for them to recognize additional adhesion molecules on the endothelium.

Firm adhesion of leukocytes to endothelium is mediated by a family of leukocyte surface proteins called integrins (see Table 2.3 ). Integrins are transmembrane two-chain 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, bind to endothelial cell proteoglycans, and are displayed at high concentrations on the endothelial surface. When the rolling leukocytes encounter the displayed chemokines, the cells are activated and their integrins undergo conformational changes and cluster together, thereby 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. The combination of cytokine-induced expression of integrin ligands on the endothelium and increased affinity of integrins on the leukocytes results in firm integrin-mediated binding of the leukocytes to the endothelium at the site of inflammation. The leukocytes stop rolling, and engagement of integrins by their ligands delivers signals to the leukocytes that lead to cytoskeletal changes that arrest the leukocytes and firmly attach them to the endothelium.

A telling indication of the importance of leukocyte adhesion molecules is the existence of mutations affecting integrins and selectin ligands that result in recurrent bacterial infections as a consequence of impaired leukocyte adhesion and defective inflammation. These leukocyte adhesion deficiencies are described in Chapter 5 . Antagonists of integrins are approved for the treatment of some chronic inflammatory diseases, such as multiple sclerosis and inflammatory bowel disease.

After arresting on the endothelial surface, leukocytes migrate through the vessel wall, primarily by squeezing between interendothelial junctions. This extravasation of leukocytes is called transmigration or diapedesis . 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 crossing the endothelium, leukocytes pierce the basement membrane, probably by secreting collagenases, and enter the extravascular tissue. The directionality of leukocyte movement within tissues is controlled by locally produced chemokines, which create a diffusion gradient that the cells migrate along.

After exiting the circulation, leukocytes move in the tissues toward the site of injury by a process called chemotaxis , defined as locomotion along a chemical gradient. Among the many chemoattractants known, the most potent are bacterial products, particularly peptides with N -formylmethionine termini; cytokines, especially those of the chemokine family; components of the complement system, particularly C5a; and leukotrienes. These chemoattractants, which are described in more detail later, are produced in response to infections and tissue damage and during immunologic reactions. All of them bind to G protein–coupled receptors on the surface of leukocytes. Signals initiated from these receptors activate second messengers that induce the polymerization of actin at the leading edge of the cell and the localization of myosin filaments at the back. The leukocyte moves by extending 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 front wheels. The net result is that leukocytes migrate toward the inflammatory stimulus in the direction of the locally produced chemoattractants.

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. 2.4 ). There are several reasons for the early preponderance of neutrophils: they are more numerous in the blood than other leukocytes, they respond more rapidly to chemokines, and they may attach more firmly to adhesion molecules that are rapidly induced on endothelial cells, such as P- and E-selectins. After entering tissues, neutrophils are short lived; they undergo apoptosis and disappear within a few days. Monocytes develop into macrophages in tissues that not only survive longer but may also proliferate, 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 allergic reactions and infections with certain parasites, eosinophils may be the main cell type.

The molecular understanding of leukocyte recruitment and migration has provided a large number of therapeutic targets for controlling harmful inflammation. As we discuss later, agents that block TNF, one of the major cytokines in leukocyte recruitment, are extremely useful therapeutics for chronic inflammatory diseases such as rheumatoid arthritis. Antibodies that block integrins were mentioned earlier.

Phagocytosis and Clearance of the Offending Agent

Neutrophils and monocytes that have been recruited to a site of infection or cell death are activated by products of microbes and necrotic cells and by locally produced cytokines. Activation induces several responses ( eFig. 2.1 ), of which phagocytosis and intracellular killing are most important for destruction of microbes and clearance of dead tissues.

Phagocytosis

Phagocytosis is the ingestion of particulate material by cells. The body’s most important phagocytes are neutrophils and macrophages ( Table 2.4 ). Neutrophils are rapid responders but relatively short-lived. In inflammatory reactions, macrophages are derived from blood monocytes and can live for days or months. (As we will discuss later, some long-lived tissue-resident macrophages are derived from embryonic precursors that seed the tissues in early life and remain for years.) Macrophage responses tend to be slower but more long lasting.

Table 2.4

Properties of Neutrophils and Macrophages

	Neutrophils	Macrophages
Origin	HSCs in bone marrow	HSCs in bone marrow (in inflammatory reactions) Stem cells in yolk sac or fetal liver (early in development, for some tissue-resident macrophages)
Life span in tissues	1–2 days	Inflammatory macrophages: days or weeks Tissue-resident macrophages: years
Responses to activating stimuli	Rapid, short-lived, mostly degranulation and enzymatic activity	More prolonged, slower, often dependent on new gene transcription
Reactive oxygen species	Rapidly induced by assembly of phagocyte oxidase (respiratory burst)	Less prominent
Nitric oxide	Low levels or none	Induced following transcriptional activation of iNOS
Degranulation	Major response; induced by cytoskeletal rearrangement	Not prominent
Cytokine production	Low levels or none	Major functional activity, requires transcriptional activation of cytokine genes
NET formation	Rapidly induced, by extrusion of nuclear contents	Little or none
Secretion of lysosomal enzymes	Prominent	Less

HSC , Hematopoietic stem cell; iNOS , inducible nitric oxide synthase; NET , neutrophil extracellular traps.

This table lists the major differences between neutrophils and macrophages. Note that the two cell types share many features, such as phagocytosis, ability to migrate through blood vessels into tissues, and chemotaxis.

Neutrophils and macrophages can ingest microbes following their recognition by phagocyte receptors, such as the mannose receptor (which recognizes terminal mannose residues found in microbial glycoproteins) and so-called “scavenger receptors.” The efficiency of this process is greatly increased if the microbes are coated (opsonized) with molecules called opsonins for which the phagocytes also have specific receptors. Opsonins include antibodies, the C3b cleavage product of complement, and certain plasma lectins. Following binding to phagocyte receptors the particle is ingested into a membrane-bound vesicle called the phagosome , which then fuses with lysosomes, resulting in discharge of lysosomal contents into the phagolysosome ( Fig. 2.5 ). During this process, neutrophils may also release granule contents into the extracellular space.

Intracellular Destruction of Microbes and Debris

Killing of microbes and destruction of ingested materials are accomplished by reactive oxygen species (ROS, also called reactive oxygen intermediates ), reactive nitrogen species (mainly derived from nitric oxide [NO]), and lysosomal enzymes. All these substances are normally sequestered in lysosomes, to which phagocytosed materials are brought. Thus, potentially harmful substances are segregated from the cell’s cytoplasm to avoid damage to the phagocyte while it is performing its normal function.

Reactive Oxygen Species

These free radicals are produced mainly in the phagolysosomes of neutrophils. Upon activation of neutrophils, a multicomponent enzyme called phagocyte oxidase (or NADPH oxidase) is rapidly assembled in the membrane of the phagolysosome ( Fig. 2.5B ). This enzyme oxidizes NADPH (reduced nicotinamide-adenine dinucleotide phosphate) and, in the process, reduces oxygen to superoxide anion $\left({\text{O}}_2^{\overline{\cdot }}\right)$ , which is then converted to H ₂ O ₂ . H ₂ O ₂ is not able to efficiently kill microbes by itself. However, the azurophilic granules of neutrophils contain the enzyme myeloperoxidase (MPO), which, in the presence of a halide such as Cl ⁻ , converts H ₂ O ₂ to hypochlorite (ClO ⁻ ), a potent antimicrobial agent that destroys microbes by halogenation (in which the halide is bound covalently to cellular constituents) or by oxidation of proteins and lipids (lipid peroxidation). The H ₂ O ₂ -MPO-halide system is the most efficient bactericidal system of neutrophils. H ₂ O ₂ is also converted to hydroxyl radical (•OH), another powerful destructive agent. As discussed in Chapter 1 , these oxygen-derived free radicals bind to and modify cellular lipids, proteins, and nucleic acids and thus destroy cells such as microbes. The production of ROS coupled with oxygen consumption is called the respiratory burst . Genetic defects in the generation of ROS are the cause of an immunodeficiency disease called chronic granulomatous disease , described in Chapter 5 .

Nitric Oxide

NO, a soluble gas produced from arginine by the action of nitric oxide synthase (NOS), also participates in microbial killing, especially in macrophages. Inducible NOS (iNOS) is upregulated in macrophages by transcriptional activation of the gene in response to microbial products and cytokines such as IFN-γ ( Fig. 2.5C ). NO reacts with superoxide $\left({\text{O}}_2^{\overline{\cdot }}\right)$ generated by phagocyte oxidase to produce the highly reactive peroxynitrite (ONOO ⁻ ). These nitrogen-derived molecules, similar to ROS, attack and damage the lipids, proteins, and nucleic acids of microbes.

Leukocyte Granule Contents

Neutrophils have two main types of granules containing enzymes that degrade microbes and dead tissues and may contribute to tissue damage. The smaller specific (or secondary) granules contain lysozyme, collagenase, gelatinase, lactoferrin, plasminogen activator, histaminase, and alkaline phosphatase. The larger azurophil (or primary) granules contain myeloperoxidase, bactericidal factors (such as defensins), acid hydrolases, and a variety of neutral proteases (elastase, cathepsin G, nonspecific collagenases, proteinase 3). The contents of both types of granules are released when neutrophils are activated. Phagocytic vesicles containing engulfed material may fuse with these granules (and with lysosomes, as described earlier), allowing the ingested materials to be destroyed in the phagolysomes by the actions of the enzymes. Similarly, macrophages contain lysosomes filled with acid hydrolases, collagenase, elastase, and phospholipase, all of which can destroy ingested materials and cell debris.

In addition to granule contents, activated neutrophils liberate chromatin components, including histones, which form fibrillar networks called neutrophil extracellular traps (NETs) ( eFig. 2.2 ). These networks bind and concentrate antimicrobial peptides and granule enzymes, forming extracellular sites for the destruction of microbes. In the process of NET formation, the nuclei of the neutrophils are lost, leading to death of the cells. NETs have also been detected in the blood during sepsis, as a consequence of widespread neutrophil activation.

Leukocyte-Mediated Tissue Injury

Leukocytes are important causes of injury to normal cells and tissues. This happens during normal defense reactions against microbes, especially if the microbes are resistant to eradication, such as mycobacteria. It is also the basis of tissue damage when the response is inappropriately directed against self antigens (as in autoimmune diseases) or against normally harmless environmental antigens (as in allergic diseases).

The mechanism of leukocyte-mediated tissue injury is release of the contents of granules and lysosomes. To some extent, this happens normally, when activated leukocytes try to eliminate microbes and other offenders. The process is exaggerated if phagocytes encounter materials that cannot be easily ingested, such as antibodies deposited on indigestible flat surfaces, or if phagocytosed substances, such as urate and silica crystals, damage the membrane of the phagolysosome. The harmful proteases released by leukocytes are normally controlled by a system of antiproteases in the blood and tissue fluids. Foremost among these is α ₁ -antitrypsin, which is the major inhibitor of neutrophil elastase. A deficiency of these inhibitors may lead to sustained protease activity, as is the case in patients with α ₁ -antitrypsin deficiency ( Chapter 11 ).

While we have emphasized the role of neutrophils and macrophages in acute inflammation, other cell types also serve important roles. Some T cells, called Th17 cells , secrete cytokines such as IL-17 that recruit neutrophils and stimulate production of antimicrobial peptides that directly kill microbes. In the absence of effective Th17 responses, individuals are susceptible to fungal and bacterial infections. The skin abscesses that develop lack the classic features of acute inflammation, such as warmth and redness. Eosinophils are especially important in reactions to helminthic parasites and in some allergic disorders, and mast cells and basophils are critical cells of allergic reactions.

Once the acute inflammatory response has eliminated the offending stimulus, the reaction subsides because there is no further leukocyte recruitment, mediators are short lived and decline if they are no longer produced, and neutrophils have short life spans.

Vasoactive Amines: Histamine and Serotonin

The major vasoactive amine is histamine , which is stored as a preformed molecule in the granules of mast cells, blood basophils, and platelets. It is rapidly released when these cells are activated, so it is among the first mediators to be produced during inflammation. The richest source of histamine is the mast cell, which is normally present in the connective tissue adjacent to blood vessels. Mast cell degranulation and histamine release occur in response to a variety of stimuli, including binding of IgE antibodies to mast cells, which underlies immediate hypersensitivity (allergic) reactions ( Chapter 5 ); products of complement called anaphylatoxins (C3a and C5a), described later; and physical injury induced by trauma, cold, or heat, by unknown mechanisms. 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. Its effects on blood vessels are mediated mainly via binding to histamine receptors called H ₁ receptors on microvascular endothelial cells. Common antihistamine drugs that treat inflammatory reactions, such as allergies, bind to and block the H ₁ receptor. Histamine also causes contraction of some smooth muscles, but leukotrienes, described later, are much more potent and relevant for causing spasms of bronchial smooth muscle, such as in asthma.

Serotonin (5-hydroxytryptamine) is a preformed vasoactive mediator present in platelets and certain neuroendocrine cells, for example, in the gastrointestinal tract. It is a vasoconstrictor, but its importance in inflammation is unclear.

Arachidonic Acid Metabolites

Prostaglandins and leukotrienes are lipid mediators produced from arachidonic acid (AA) present in membrane phospholipids that stimulate vascular and cellular reactions in acute inflammation. AA is a 20-carbon polyunsaturated fatty acid that is released from membrane phospholipids through the action of cellular phospholipases, mainly phospholipase A ₂ , that are activated by inflammatory stimuli, including cytokines, complement products, and physical injury. AA-derived mediators, also called eicosanoids (from the Greek, eicosa , meaning 20, as they are derived from 20-carbon fatty acids), are synthesized by two major classes of enzymes, cyclooxygenases (which generate prostaglandins) and lipoxygenases (which produce leukotrienes and lipoxins) ( Fig. 2.6 ). Eicosanoids bind to G protein–coupled receptors on many cell types and can mediate virtually every step of inflammation ( Table 2.6 ).

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 5 . Here the cytokines involved in acute inflammation are reviewed ( Table 2.7 ).