Inflammation and Healing

• Diluting and/or inactivating biologic and chemical toxins

• Killing or sequestering microbes, foreign material, necrotic tissue (e.g., bone sequestrum), and neoplastic cells

• Degrading foreign materials

• Providing wound healing factors to ulcerated surfaces and traumatized tissue

• Restricting movement of appendages and joints to allow time for healing and repair

• Increasing temperature in the body or locally to induce vasodilation and inhibiting replication of some microbial agents

• Physical barriers and microenvironments provided by epithelia of the skin (low pH, lactic and fatty acids) and mucosae such as in the respiratory mucociliary escalator, reproductive tracts (secretions), and alimentary system (gastric and duodenal secretions, peristalsis, saliva)

• Molecular products released by mucosae including lactoferrin, antimicrobial peptides (α- and β-defensins, cathelicidins), and collectins. These products have immune activity but also contribute to proinflammatory and antiinflammatory reactions, leukocyte activation, and wound healing.

• Effector molecules in the blood, such as plasma proteases (complement, kinin, and clotting systems), and inflammatory mediators released from nerve fibers (sensory fibers, C-reactive fibers), such as substance P.

• Effector cells in mucosae and vascularized connective tissue

• Effector molecules in the blood, on endothelial cells

• A combination of these components

• Visible and ultraviolet light spectra (sunburn and photosensitization)

• Radiation, blunt force trauma (abrasion, bruising, incision, and laceration)

• Thermal injury (hot and cold)

• Chemotherapeutics

• Environmental chemicals

• Microbial molecules (lipids and proteins)

• Venom (insect, snake, and reptile)

• Responses of the adaptive immune system (type I to IV hypersensitivities) to microbial and environmental antigens

• Vasoactive amines such as histamine and serotonin

• Plasma proteases such as complement, kinin, and clotting system proteins

• Lipid mediators such as arachidonic acid metabolites and platelet-activating factor (PAF)

• Cytokines

• Chemokines

• Chemerin

• Nitric oxide (NO)

• Increased blood flow (active hyperemia) to the site of injury

• Increased permeability of capillaries and postcapillary venules to plasma proteins and leukocytes through release of inflammatory mediators

• Emigration of leukocytes (via the leukocyte adhesion cascade) into the perivascular area

Endothelial Cell Dynamics During the Acute Inflammatory Response

Endothelial cells are the interface between plasma in the lumen and the perivascular connective tissue. They are polarized cells that have specific luminal versus abluminal surfaces, which serve the physiologic needs of the vascular bed of each organ. Transport across the endothelial cell layer occurs by (1) transcytosis (transcellular passage) via small vesicles and caveolae or (2) paracellular passage. Transcytosis, the process of transporting substances across the endothelium by uptake into and release from coated vesicles, facilitates the transport of albumin, low-density lipoproteins (LDLs), metalloproteinases, and insulin. Paracellular passage allows transport of water and ions between cells (cell junctions). Paracellular passage is especially active in postcapillary venules. Roughly 30% of endothelial cell junctions in postcapillary venules can open to a width of 6 mm, roughly the width of a red blood cell. Leakage of fluid from the vasculature can occur within seconds after the acute inflammatory response is induced.

The mechanisms of leakage (Fig. 3-5) depend on the biologic and physical characteristics of the inciting agent or substance and include the following:

Formation of Endothelial Cell Gaps

Endothelial gaps, resulting in vascular leakage, can occur by (1) contraction (actin/myosin) of adjacent endothelial cells and through (2) the reorganization of the cytoskeletal microtubule and microfilament proteins within the endothelial cells. With both of these changes, gaps result from the opening of junctional complexes between endothelial cells. Gaps by cell contraction in postcapillary venules where there is a high density of receptors for histamine, serotonin, bradykinin, and angiotensin II. Gaps formed by cytoskeletal reorganization occur most commonly in postcapillary venules and to a lesser extent, in capillaries in response to cytokines, such as interleukin-1 (IL-1) and TNF and hypoxia. Gap formation is transient and lasts 15 to 30 minutes after the stimulus occurs.

Vascular leakage resulting from direct injury to endothelial cells can cause detachment of the cell from the underlying basement membrane. Such damage establishes conditions favorable for the activation and attachment of platelets, clotting, and complement cascades. This type of extravasation usually occurs immediately after necrotizing injury induced by, for example, thermal injury, chemotherapeutic drugs, radiation, bacterial cytotoxins, and inhaled gases such as hydrogen sulfide. It affects arterioles, capillaries, and postcapillary venules. Vascular leakage resulting from leukocyte-induced damage occurs secondary to neutrophils and other leukocytes interacting with endothelial cells during the leukocyte adhesion cascade. Activated leukocytes release reactive oxygen species, such as singlet oxygen and oxygen free radicals, and proteolytic enzymes, such as matrix metalloproteinases and elastase from lysosomes during degranulation of the cells, which then result in endothelial cell necrosis and detachment and thus an increase in vascular permeability. This type of extravasation usually affects capillaries and postcapillary venules.

Leukocyte Adhesion Cascade

The movement of leukocytes from the lumina of capillaries and postcapillary venules into the interstitial connective tissue occurs through a process called the leukocyte adhesion cascade (Fig. 3-6). Chemokines, cytokines, and other inflammatory mediators influence this process by modulating the surface expression and/or avidity of adhesion molecules on both endothelial cells and leukocytes. It has a well-characterized sequence of events, including margination, rolling, activation and stable adherence (adhesion), and transmigration of leukocytes toward a chemotactic stimulus.

This process is actually initiated during the fluidic phase of the acute inflammatory response and is driven by chemokines, cytokines, and chemoattractant substances such as complement. Temporally, margination, rolling, activation and firm adhesion, and transmigration all occur concurrently, involving different leukocytes in the same capillaries and postcapillary venules. This process is largely mediated by the interaction of ligands expressed on the surface of neutrophils, lymphocytes, and macrophages and their receptors expressed on luminal surfaces of activated endothelial cells (see Table 3-1). Adhesion molecules are divided into (1) selectins (E-, L-, and P-selectin), (2) integrins (VLA family of β₁ integrins; β₂ integrins [Mac-1, LFA-1, p150,95, αdβ₂]), (3) cytoadhesin family (vitronectin, β₃ integrins, and β₇ integrins used predominately by lymphocytes), (4) the immunoglobulin superfamily (ICAM-1 to ICAM-3, VCAM-1, PECAM-1) and mucosal addressin adhesion molecule-1 (MAdCAM-1), and (5) other molecules such as CD44 (Web Table 3-2).

Leukocyte Adhesion Deficiencies

The three types (I, II, and III) of leukocyte adhesion deficiencies (LADs) are the result of one or more defects in the sequence of steps leading to migration of leukocytes into the site of inflammation. These deficiencies are rare in humans, cattle, and dogs (Web Table 3-3), although debilitation often occurs in humans and animals with severe forms of LADs. These clinical conditions underscore the importance of leukocytes in host defense and the vital role that these adhesion molecules serve in the transmigration process. Cattle, dogs (Irish setters), and humans with LAD type I lack functional expression of the β₂ integrins. Affected individuals often have high numbers of neutrophils, exceeding 125,000/µL in the blood; however, in cattle, these neutrophils have impaired passage across vascular walls (Web Fig. 3-1) and numerous other functional abnormalities related to inadequate membrane adherence activity. As a result, lesion sites lack significant neutrophils. Cattle with bovine LAD (BLAD) develop severe oral ulcers, gingivitis, tooth loss, enteric ulcers, cutaneous ulcers, abscesses that lack pus formation, and pneumonia (see Web Fig. 3-1). Most affected cattle die within few days or weeks after birth as the result of diarrhea and/or pneumonia. The hallmark lesion histologically is a sparse infiltration of neutrophils into ulcerated mucosal surfaces or pulmonary alveoli, despite high numbers of neutrophils within lumen of submucosal and pulmonary septal blood vessels (Web Figs. 3-2 and 3-3). There are no effective treatments for calves and Irish setters with LAD, although cattle with BLAD can survive into adulthood with intensive medical care.

The deficiency in β₂-integrin expression in Holstein calves is due to a single-point mutation (adenine → guanine) at position 128 resulting in a single amino acid change (aspartic acid → glycine) in the β-subunit (CD18) of the β₂ integrins. This defect occurs in a highly conserved extracellular region (amino acids 96 through 389) of the protein. A second, silent mutation has also been detected in cattle with BLAD and does not alter the amino acid sequence. In Irish setters with canine LAD (CLAD), there is a single missense mutation resulting in a G to C transversion at nucleotide 107 of the cDNA sequence, resulting in a serine that replaces a highly conserved cysteine. In 1992, a second form of LAD was discovered in humans and called LAD type II (see Table 3-3). In LAD type II, patients lack fucosylated sialyl Lewis X used for selectin-mediated adherence caused by a mutation in a fucose transporter gene. Their CD18 binding remains intact; however, these patients suffer from the same types of lesions and infections, as do patients with CD18 deficiency. Therefore these observations demonstrate the importance of CD18-independent adherence. More recently, a third type of LAD (LAD type III) has been identified. LAD type III is caused by a defect in kindlin-3, which is a cytoplasmic protein essential for integrin activity in the cytosol. LAD type III patients have defects in leukocyte function and platelet aggregation. Both LAD types II and III have not been identified in animals.

Therapeutic Strategies to Modulate Leukocyte Infiltration

Inhibition of leukocyte infiltration may be useful treating diseases such as stroke, myocardial infarction, asthma, and autoimmune diseases in humans and laminitis, reperfusion injury of intestine after colic, gastric dilation/volvulus, mastitis, enteritis, allergic lung disease, pneumonia, and autoimmune diseases in domestic animals. In animal models, antibodies and small molecule antagonists have diminished the severity of rheumatoid arthritis, type 1 diabetes, asthma, and other conditions. In humans, antibody (efalizumab) to αLβ₂ integrin (LFA-1) is effective against psoriasis, whereas antibody (natalizumab) to α₄β₁ integrin reduces the severity of multiple sclerosis and Crohn’s disease. However, complete inhibition of integrin activity can have side effects. Additional strategies to modulate integrin function therapeutically include inhibition of integrin-associated proteins, such as paxillin, to regulate α₄β₁ integrin, talin effects on β₂ integrins, and inhibition of membrane protein CD98 to regulate β₁ and β₃ integrins.

Vascular Endothelial Cells

Central to the integrity of the vasculature and any type of acute inflammation is the endothelial cell. Once considered a cell that, in the most simplistic view, forms separation between the blood and the surrounding tissue, endothelial cells are now known to have an extremely sophisticated role in regulating (1) hemostasis/coagulation, (2) vascular pressure, (3) angiogenesis during wound healing, (4) carcinogenesis, (5) leukocyte homing, and (6) inflammation. Under physiologic conditions, transcytosis (transcellular passage) of albumin, LDLs, metalloproteinases, and insulin occur via small vesicles and caveolae in the cytoplasm of the endothelial cell. Paracellular passage (between cell junctions) of water and ions occurs with endothelial cell contraction secondary to physiologic stimuli and/or inflammatory mediators. Secondary to inflammatory mediators, endothelial cells are activated and contract, allowing fluid to lead into the extravascular tissue. Vascular tone is held in check in part by endothelin, a vasoconstrictive molecule and angiotensin II, both of which are produced by endothelial cells, along with vasodilatory substances such as NO and prostacyclin (PGI₂). Activated endothelial cells release these chemical mediators and express adhesion molecules and receptors, including E-selectin, P-selectin, L-selectin ligand, PECAM-1, JAM A, JAM B, and JAM C, and the immunoglobulin superfamily such as ICAM-1. These adhesion molecules serve as ligands for leukocyte adherence. With the onset of inflammation, endothelial cells tend to increase procoagulative properties through release of tissue factor and other procoagulative substances.

Mast Cells and Basophils

The origin and relationship between mast cells and basophils has been a traditional point of debate and confusion. Current research clearly indicates that mast cells and basophils represent distinct cell types even though they share several morphologic and functional characteristics. Mast cells and basophils, along with other granulocytes and monocytes, originate and differentiate in bone marrow from a common CD34⁺ precursor cell. Differentiation of CD34⁺ precursor cells into mast cells or basophils depends on stem cell factor, a glycoprotein that acts with other cytokines and is produced in the bone marrow by fibroblasts and vascular endothelial cells. There is no evidence to suggest that basophils differentiate into tissue mast cells.

Mammalian mast cells are normally distributed throughout connective tissue adjacent to small blood and lymphatic vessels of skin and mucous membranes. In this location, they respond rapidly to foreign proteins, microbes, and other substances and contribute significantly to the initiation of acute inflammation. This location also allows mast cells to interact with resident dendritic cells and release inflammatory mediators that activate endothelial cells. Experimental studies suggest that cutaneous mast cells in tissue have a lifespan of 4 to 12 weeks, depending on their location. Mast cells represent an extremely heterogeneous population of cells. In the 1960s, Enerback identified two separate types of mast cells: mucosal and connective tissue. The mucosal mast cells are typically located in the respiratory and intestinal mucosa and can increase in numbers during some types of T helper 2 (T_H2) lymphocyte–dependent immune responses. In contrast, the connective tissue mast cells show little or no T lymphocyte dependence. Mast cells express high affinity receptors for immunoglobulin E (IgE; Fc ε-RI) on their surface, and the release of mast cell granules is stimulated by the cross-linking of IgE receptors by antigens such as pollens, allergens, and parasites. Substance P released from sensory (C-reactive) nerve fibers and macrophages also causes degranulation of mast cells. Degranulation results in the release of preformed TNF-α histamine, neutral proteases, proteoglycans (chondroitin sulfates and heparin), serotonin (in rodent species but not humans), tryptase, chymase, and stem cell factor into tissue. Histamine and substance P activity appears interrelated because histamine released by mast cells can downregulate the release of substance P by nerve fibers, thereby reducing excessive amounts of the two proinflammatory molecules. The mast cell–substance P fiber interrelationship is an often-cited example of the neuroinflammatory-neuroimmune pathway.

Mast cells also synthesize leukotriene (LT) C₄ (LTC₄), PAF, prostaglandin (PG) D₂ (PGD₂), numerous cytokines, serotonin (in some species), heparin, and C-C chemokines (macrophage inflammatory protein [MIP]-1-α and macrophage chemotactic protein [MCP]-1). The release of these mediators contributes significantly to the initiation of the acute inflammatory response. In addition, at physiologic concentrations these products likely counteract the effects of dense populations of mast cells in tissue and thus assist in regulating vascular permeability. Mast cells also release proteolytic enzymes, such as tryptase and chymase, which are involved with remodeling of the ECM. Tryptase is mitogenic to epithelial cells and likely contributes to proliferation of epithelial cells during wound repair.

Basophils are similar to neutrophils and eosinophils in that they mature in the bone marrow, circulate in the peripheral blood, are recruited into the tissue, and have a lifespan of several days in tissue. Basophils express high affinity IgE receptors, similar to mast cells, and release granules and inflammatory mediators. Basophils appear to lack heparin, have a more limited cytokine repertoire than mast cells, and release mainly IL-4 and IL-13. Basophils express CD40L and CCR3 (eotaxin receptor). The presence of these suggests that they have a role as a cell that can enter sites of inflammation, release regulatory cytokines where they upregulate VCAM-1 expression by endothelial cells, and switch B lymphocytes to produce IgE, further contributing to the IgE type of response. Basophils can be prominent in IgE-mediated leukocyte infiltration into the mucosa of the nose, sinuses, respiratory tract, and skin, and all of these sites are particularly predisposed to allergic conditions.

The role of mast cells and basophils in IgE-mediated hypersensitivity reactions has been known for decades. These cells are critical effector cells in disorders of IgE-dependent immediate type I hypersensitivities (see Chapter 5). The release of their granules and mediators at inflammatory concentrations in the lung, for example, results in mucus secretion, accumulation of seroproteinaceous fluid in airways, bronchoconstriction, and vasodilation. The excessive release of tryptase and chymase by mast cells may enhance degradation of the ECM, which contributes to fibrosis and tissue remodeling. Chemokines and cytokines from mast cells and basophils contribute to innate immune defenses through chemotaxis and release of antimicrobial peptides. The mediators also enhance adhesion molecule expression on endothelial cells of nearby blood vessels and leukocytes that enter the area.

Neutrophils

Neutrophils are often the first type of leukocyte recruited into the inflammatory exudate. Their purpose is to (1) kill microbes, such as bacteria, fungi, protozoa, and viruses; (2) kill tumor cells; or (3) eliminate foreign materials. The biologic activities of neutrophils are primarily designed to kill microbes through lysosomal degradation, but if killing does not occur, neutrophils can limit the growth of microbes, allowing time for adaptive immunologic responses to develop.

Neutrophils perform two important functions to accomplish their effects: (1) phagocytosis of microbes or foreign material and then fusion of the phagosome with primary lysosomes to form a phagolysosome in which the microbes or foreign material are killed or degraded, respectively (Web Fig. 3-4), and (2) secretion and/or release of the contents of their granules into the inflammatory exudate to enhance the acute inflammatory response. They also infiltrate areas of acute tissue necrosis, such as those that occur in infarcts and necrotic areas of tumors.

Neutrophils are produced in the bone marrow, circulate in the bloodstream, and if not recruited into tissue by an acute inflammatory response, can enter tissue where they eventually are destroyed by macrophages via apoptosis and phagocytosis or are lost from the body by migration across mucosae such as the alimentary and respiratory tracts. The average transit time in the blood is 10 hours, and the half-life in the blood varies between species but ranges from 5 to 10 hours; neutrophils within tissue survive from 1 to 4 days. Cytokines, such as IL-1 and TNF, and growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and IL-3 can increase release of neutrophils from the bone marrow and induce granulopoiesis in 2 to 4 days. GM-CSF and G-CSF are also present in tissue during acute inflammatory response and prevent apoptosis of tissue neutrophils. Within areas of intense inflammation, neutrophils and other leukocytes must function under hypoxic conditions and do so through the stabilization of hypoxia-inducible factor-1α (HIF-1α). HIF-1α induces transcription of genes that promote phagocytosis, inhibition of apoptosis, release of antimicrobial peptides, granule proteases, VEGF, cytokine release, and inducible NO synthetase (iNOS). Growth factor withdrawal, which occurs during resolution of acute inflammation, induces apoptosis, and this outcome can be accelerated by TNF. During apoptosis, neutrophils lose the capacity to degranulate and become activated, which prevents release of their lysosomal enzymes and thus excessive tissue damage and allows for their phagocytosis by macrophages.

Neutrophils entering tissue become activated, which enhances migration, phagocytosis, and microbial killing, through stimulation by inflammatory mediators and by adherence to ligands by surface adhesion molecules. Inflammatory mediators bind receptors on neutrophils, such as receptors for PAF, C5a, IL-8 and substance P (the neurokinin-1 receptor), leukotrienes, kallikrein, GM-CSF, and cytokines such as TNF. Many of these mediators induce chemotaxis; when leukocyte adhesion molecules, such as the selectins and integrins, bind to their respective ligands, they induce mitogen-activated protein kinase (MAPK) and G proteins, resulting in migration of neutrophils, usually toward a chemotactic gradient and activation.

Neutrophils can internalize large particles up to 0.5 µm in diameter by phagocytosis, including microbes, foreign bodies, senescent cells, and debris. Although neutrophils can internalize nonopsonized particles, opsonization greatly facilitates phagocytosis. The principal opsonin receptors present on neutrophil membranes are complement (CR1 and CR3) and Fc receptors (Fc γ-receptor I, IIA, IIIB), which bind complement fragments (C3b and C3bi) and the Fc portion of immunoglobulins such as IgG1 and IgG3. Such binding initiates activity of the guanosine triphosphatase (GTPase) Rac1 and the β2 integrin Mac-1 (CD11b/CD18), which also binds complement fragment C3bi and initiates GTPase-ρ. Such binding-inducing proteins and lipid kinases (e.g., protein kinase C and phosphatidylinositol 3-kinase) mediate actin assembly for formation of filopodia or lamellipodia, which surround and then internalize particles via phagocytosis by activated neutrophils. The activation process also leads to the release of calcium stored in the endoplasmic reticulum, which induces a respiratory (oxidative) burst (Table 3-2). Oxidative burst is the process by which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase composed of five phox protein subunits in the membrane of phagosomes is formed. It catalyzes the formation of superoxide free radical that is used to kill microbes or degrade internalized material. Superoxide can react to form hydrogen peroxide, and additional free radicals such as hydroxyl radical, and hypochlorous acid. Neutrophils also express iNOS, which generates NO, and myeloperoxidase, which also produces hypochlorous acid. Superoxide anion and NO can form peroxynitrite, which is highly reactive.

Once a particle is internalized, phagosomes can “mature” by fusing with lysosomes and endosomes or remove parts of internalized particles. The fusion process is likely mediated by calmodulin, a calcium-binding protein, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNARE; a fusion protein) that bind ligands on another vesicle to bring the membranes together for fusion. The maturation process results in lowering of the pH within the phagosome and the activation of microbicidal enzymes, including NADPH oxidase and myeloperoxidase complexes. Smaller particles are internalized by receptor-mediator endocytosis.

The ability of neutrophils to kill microbes or to degrade foreign material depends largely on the contents of the neutrophil granules, which store degradative enzymes, peroxidative enzymes, adhesion molecules, and antimicrobial peptides and/or proteins (Web Box 3-1). Myeloperoxidase is an enzyme used to convert hydrogen peroxide to hypochlorous acid. Hypochlorous acid, hydrogen peroxide, and a halide cofactor (chloride) form the myeloperoxidase system, which is an effective microbicidal mechanism used by neutrophils to kill internalized microbes and degrade internalized substances. Defensins, cathelicidins, and antimicrobial proteins contribute to the degradation of microbes by forming pores in microbial membranes. They also affect chemotaxis and activation of the adaptive immune response. Lactoferrin inhibits the growth of phagocytosed bacteria by sequestering free iron, and elastase hydrolyzes bacterial cell wall proteins and tissue elastin. The enzymatic contents of granules, such as gelatinase (matrix metalloproteinase-9 [MMP-9]) and myeloperoxidase, and nonenzymatic substances, such as antimicrobial peptides and lactoferrin, are also commonly released by the cell into the extracellular space and contribute to killing of extracellular microbial pathogens and with degradation of the ECM. The effects of extracellular neutrophil proteases, if not inactivated, can cause serious injury to tissue; therefore protease inhibitors are present in plasma and are present in inflammatory lesions after vascular leakage.

Neutrophil granule formation begins during myeloid cell differentiation in the bone marrow (see Web Box 3-1). Granules are observed initially in myeloblasts and promyelocytes when immature transport vesicles bud from Golgi and fuse to form primary granules. Primary granules are also called azurophilic granules because of their affinity for the dye azure A. These granules contain myeloperoxidase, elastase, defensins, and small amounts of lysozyme. Myelocytes and metamyelocytes form secondary (specific) granules that contain defensins, lactoferrin, lysozyme, and lesser amounts of myeloperoxidase, CD11b/CD18, and elastase. Band cells, the penultimate stage of neutrophil development, form tertiary (gelatinase) granules that contain lysozyme, gelatinase (MMP-9), cysteine-rich secretory protein-3 (CRISP-3), and adhesion molecules CD11b/CD18 (Mac-1) but have smaller amounts of myeloperoxidase, lactoferrin, proteinase 3, elastase, and defensins. Band and mature neutrophils also have secretory vesicles that contain plasma proteins, alkaline phosphatase, and numerous CD antigens, including CD11b/CD18 adhesion molecules. The secretory vesicles are mobilized quickly after neutrophil activation resulting in rapid expression of adhesion molecules, which mediate leukocyte infiltration.

Neutrophil granules have evolved phylogenetically and are specially adapted for each species. In most mammals, enzymes released into an exudate from neutrophil granules cause liquefaction of the exudate and the process results in the formation of pus. Reptiles and birds either lack or have reduced concentrations of these enzymes, particularly myeloperoxidase, and cannot liquefy the exudate. Thus a caseous material forms to be degraded by the next available line of inflammatory cells, macrophages. Granules in chicken heterophils (the avian, rabbit, and guinea pig neutrophil equivalent is termed heterophils) have little myeloperoxidase, but concentrations are also reduced in neutrophils of cattle and pigs. Cattle and sheep neutrophils have limited lysozyme levels. α-Defensins are present in rabbits, guinea pigs, hamsters, rats, and cattle neutrophils but have not been identified in those of dogs, cats, mice, pigs, and horses. The effect of these granule differences in various animal species on host defense and neutrophil function is not fully understood.

On cell death, neutrophils can release neutrophil extracellular traps (NETs) composed of a DNA backbone embedded with antimicrobial peptides and proteins and peptides that include histones, primary granule contents, lactoferrin, gelatinase, cathelicidins, and α-defensins. NETs entrap bacteria and can be microbicidal. Actin is also released from dead neutrophils, and in the lung, it can increase the viscosity of respiratory mucus. This outcome may occlude airways in dehydrated animals.

Eosinophils

Eosinophils are recruited from the bloodstream into vascularized connective tissue of most organs in response to eosinophil chemoattractants present in allergic and parasitic diseases. Eosinophils frequently enter lesions during the transition from acute to chronic inflammation. Eosinophils have prominent granules that release basic proteins and when activated produce cytokines, chemokines, proteases, and oxidative radicals. This array of mediators is often released in response to helminthic infections, and eosinophilic infiltration has been more recently implicated in resistance to the development of some cancers. On the other hand, eosinophil products contribute to tissue damage in several organs, including the lungs (asthma), heart, skin, and gastrointestinal tract.

Eosinophils were first recognized as blood cells (leukocytes) having numerous cytoplasmic granules with affinity for acidic dyes such as eosin. Therefore the name eosinophil (“eosin-loving”) was proposed by Ehrlich in the late 1800s for these unique cells. By 1939, eosinophils were postulated to have a role in the immune response to helminths, and by the 1970s, eosinophils were well known to increase in the blood (eosinophilia) in parasitic and allergic diseases. Eosinophils are slightly larger than neutrophils. The nucleus is lobulated (bilobed) and composed primarily of heterochromatin (condensed). Eosinophil granules are known for their large size, especially in horses, and are rich in arginine with reddish brown tinctorial properties.

Eosinophils have several types of granules listed in Web Table 3-4, including large specific granules, small granules, primary granules, and secondary granules. Large specific granules contain four distinct basic proteins: (1) major basic protein, (2) eosinophil cationic protein, (3) eosinophil-derived neurotoxin, and (4) eosinophil peroxidase. These proteins exert biologic effects on microbes and on the tissue in which the microbes replicate by damaging lipid membranes. In addition, histaminase and a variety of hydrolytic lysosomal enzymes, such as collagenase and gelatinase, are also present in the large specific granules. Small granules contain enzymes such as arylsulphatases, acid phosphatases, MMPs, and gelatinases. Eosinophils also elaborate cytokines such as IL-1 to IL-6, IL-8, IL-10, IL-12, IL-16, GM-CSF, TGF-α and TGF-β, and chemokines. The content of eosinophil granules are released in response to inflammatory stimuli in a manner similar to those used to activate neutrophils. However, products of eosinophil granules can result in extensive tissue degradation, including the degradation of collagen, which is commonly seen in eosinophilic granulomas of cats, horses, and dogs. Nearly all mast cell tumors in dogs and some mast cell tumors in cats contain eosinophils.

Major chemoattractants for eosinophils include histamine and eosinophilic chemotactic factor A (from mast cells), C5a, cytokines (IL-4, IL-5, and IL-13), and chemokines (CCL-5, known as regulated on activation, normal T lymphocytes expressed and secreted [RANTES], and CCL-11 [known as eotaxin]) released from epithelial cells, eosinophils, mast cells, and helminths. 5-Oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE) is a strong activator of human eosinophils with a chemotactic potency comparable with those of eotaxin and RANTES, both of which enhance 5-oxo-ETE–induced chemotaxis. 5-Oxo-ETE and these chemokines contribute to the accumulation of eosinophils in the respiratory system in diseases such as asthma.

Natural Killer Cells and Natural Killer T lymphocytes

Natural killer (NK) cells are sentinels of the immune system named for lysis of tumor cells and virus-infected cells without previous encounter. These cells enter regions of acute inflammation hours and even days after initiation of the lesion. NK cells kill target cells through release of perforin from cytoplasmic granules. NK cells express CD161, a C-type lectin, but do not express CD3, the T lymphocyte antigen. Roughly 95% of NK cells expression CD56 and produce interferon-γ (IFN-γ); these are type I NK cells. Type II NK cells lack CD56 expression and produce IL-4, IL-5, and IL-13, thus supporting a T_H2 response.

IL-21 regulates differentiation and apoptotic death induced by NK cells. Inactive NK cells can be stimulated by Flt-3 ligand, a hematopoietic cytokine that stimulates proliferation of dendritic cells and antitumor immune responses, and also by IL-4, IL-12, IL-15, and IL-21. Once activated, IL-21 induces NK cell differentiation and upregulation of CD16, the low affinity IgG receptor necessary for antibody-dependent cellular cytotoxicity (ADCC), and also NK release of IFN-γ required for macrophage and dendritic cell activation. Finally, IL-21 initiates a delayed apoptotic program for death of the differentiated NK cell and prevents recruitment of uninvolved NK cells. NK-T lymphocytes are T lymphocytes (express CD3 antigen) that have T and NK cell properties. NK-T lymphocytes recognize CD1d molecule, which is an antigen-presenting molecule that binds self and foreign lipids and glycolipids, and on activation of the NK-T lymphocyte, induce release of IFN-γ, IL-4, and GM-CSF. Because of this close discretion of self and nonself, NK-T lymphocytes can have important roles in the development of autoimmune disease.

Monocytes and Macrophages

Macrophages arise from bone marrow–derived monocytes, which circulate hematogenously with some monocytes localizing in tissues physiologically. They enter acute inflammatory lesions roughly 12 to 48 hours after the initiation of a lesion, depending on the inciting agent/substance. Differentiation of monocyte stem cells into blood monocytes proceeds rapidly in the bone marrow (i.e., 1.5 to 3 days) and is regulated by growth and differentiating factors, cytokines, and adhesion molecules such as IL-3, CSFs, and TNF. Under physiologic conditions, monocytes in the blood localize throughout the body and differentiate into tissue macrophages. Recently, nonclassical monocytes have been identified; they migrate slowly along the luminal side of endothelium and monitor healthy tissue. When these monocytes sense damage or infection, they migrate rapidly into the tissue.

There are two types of tissue macrophages: macrophages that reside within specific organs/tissue (free macrophages and fixed macrophages) and macrophages derived from monocytes in response to inflammatory stimuli. Macrophages residing in organs/connective tissue first enter these sites as blood monocytes under physiologic (rather than inflammatory) conditions. These macrophages form the monocyte-macrophage system and include macrophages in connective tissue (histiocytes [free macrophages]), liver (Kupffer cells [fixed macrophages]), lung (alveolar macrophages [free macrophages], and intravascular macrophages [fixed macrophages]), lymph nodes (free and fixed macrophages), spleen (free and fixed macrophages), bone marrow (fixed macrophages), serous fluids (pleural and peritoneal macrophages [free macrophages]), brain (microglial cells), and skin (histiocytes [fixed macrophages]). The number of macrophages in tissue is maintained by (1) influx of monocytes from the blood, (2) proliferation of recruited monocytes locally in tissue, and (3) biologic turnover of macrophages via apoptotic cell death (lifespan in tissue of less than 3 weeks).

During inflammatory responses, monocytes express receptors (IgG Fc-domains, C3b) for chemical mediators of inflammation that exert migratory, chemotactic, pinocytic, and phagocytic activities in response to inflammatory stimuli. Once within inflammatory lesions, monocyte receptors are bound by cytokines, antigens, and other stimuli, which rapidly activate the maturation of monocytes into macrophages. This process can occur virtually anywhere in the body and often sets the stage for the development of chronic inflammation. In chronic inflammatory lesions, macrophages are the cell of last resort and accumulate in sites of persistent antigen, persistent microbes, foreign material, or repeated injury. Functionally, macrophages are a component of the innate immune system in terms of their role in phagocytosis and cytokine release during the acute inflammatory response. However, macrophages are one of the main triggers of the adaptive immune response because of their ability to process and present antigen and regulate T lymphocyte activity.

• Have short half-lives and decay rapidly

• Are destroyed enzymatically

• Are scavenged by protective mechanisms such as antioxidants

• Are blocked by endogenous inhibitors such as complement inhibitors and decoy receptors

• Aggregation and after contact with collagen in an exposed basement membrane from areas of endothelial necrosis and detached cells

• Thrombin from activation of the coagulation cascade

• Adenosine diphosphate (ADP) released from injured endothelial cells

• Immune complex activation of the complement cascade (C3a, C5a)

• Increased vascular permeability

• Vasodilation (venules)

• Increased sensitivity to pain

• Smooth muscle contraction

• Increased arachidonic acid metabolism (stimulation of phospholipase A₂)

• Hypotension

• Bronchoconstriction

Complement Cascade

The complement cascade is a unique sequence of molecular events occurring within the vascular system in which inactive complement plasma proteins synthesized by the liver are activated after tissue injury (Fig. 3-7; Web Fig. 3-6), inflammation, clotting, or immune responses. This cascade results in the generation of numerous biologically active molecules, which have effects that are proinflammatory, chemotactic, opsonizing, antigen solubilizing, antibody inducing, permeability enhancing, and microbicidal (cell lysis) and usually beneficial to the animal (Table 3-3). A large number of plasma proteins make up the complement system, and nearly 10% of serum proteins are complement factors. Divided by the “classical,” “alternative,” and lectin pathways, activation or “fixation” of complement proteins eventually results in formation of a membrane attack complex (MAC) that perforates the cell membranes of foreign invaders and naïve host cells alike. In the generation of MAC, a variety of complement components are elaborated that have important inflammatory and immune effects.

Complement proteins C1 through C9 are inactive components of plasma that are activated by substances, including microbial molecules such as endotoxins, aggregated immunoglobulin, complex polysaccharides, and venoms. The critical step in unleashing the biologic functions of the complement cascade in the classical pathway is activation of C3 through either the classical or alternative complement activation pathways. The classical pathway of the complement cascade can be activated by antibody complexes. Activation occurs when IgG and/or IgM are cross-linked with C1. C1 has three components: C1q, r, and s. C1q binds the Fc regions of IgG and/or IgM and brings C1r, which is proteolytic, into proximity to C1s, which is cleaved, through interactions with C4 and C2. This leads to the formation of classical pathway C3 convertase (C4b2a) and the eventual formation of classical pathway C5 convertase (C4b2a3b). Classical pathway C3 convertase converts C3 to C3a, and classical pathway C5 convertase converts C5 to C5a.

The alternative pathway is initiated by products from microorganisms, including LPS from Gram-negative bacteria and polysaccharides from fungal cell walls (see Web Fig. 3-6). In addition, other activated plasma proteins, including kallikrein, plasmin, and activated factor XII, can cleave C3, resulting in its activation to C3b. C3b combines with factor B and coupled with factor D activity, forms the alternative pathway C3 convertase (C3bBb). Alternative pathway C3 convertase converts C3 to C3b. C3b combines with alternative pathway C3 convertase to form alternative pathway C5 convertase (C3bBb3b), which converts C5 to C5a. Because the alternative pathway can be activated by clotting and kinin factors, once the clotting or the kinin systems are activated, complement activity follows and vice versa. Therefore the clotting, kinin, and complement systems are closely interactive, and often activation of one system leads to activation of others (Fig. 3-8). When bound to a microbial product, mannose-binding lectins (MBLs) and ficolins can also activate complement through MBL-associated serine proteases (MASPs). MASP-2 cleaves C4 and C2 to activate steps of the classical pathway.

C3a increases vascular permeability by inducing histamine release from mast cells. C5a, once formed, is released into the inflammatory exudate and behaves as an anaphylatoxin (a molecule that causes the release of histamine and other chemical mediators from mast cells or basophils), a chemoattractant for leukocytes, and an inducer of adhesion molecule expression by endothelial cells. C3b and C3bi are important opsonins and enhance neutrophil phagocytosis through CR1 and CR3 receptors. The plasma enzyme carboxypeptidase can degrade both C3a and C5b.

The MAC results from the cleavage of C5 by C5 convertase, leading to the formation of C5a and C5b. C5b serves as the anchor for the assembly of a single molecule composed of C6, C7, and C8. This MAC (C5b with C6, C7, C8) facilitates polymerization of C9 (up to 18 molecules of C9) into a tube that is inserted into the lipid bilayer of the plasma membrane of, for example, a bacterium. A channel is formed through the cell membrane allowing the passage of ions, small molecules, and water into the bacterium by osmosis. Bacterial lysis ensues. This process can also injure naïve host cells such as occurs in hemolytic anemia.

Arachidonic Acid Metabolites

When inflammation or inflammatory mediators injure cells, the cell membrane lipids are rapidly rearranged to create a variety of biologically active lipid mediators derived from arachidonic acid. Arachidonic acid metabolites are lipid-derived autocoid (acting as a local hormone) mediators of inflammation that serve as intracellular and extracellular signals influencing the coagulation cascade and mediating nearly every step of the acute inflammatory response (Fig. 3-9; Web Fig. 3-7). Effects are short-lived because these lipid metabolites decay rapidly or are destroyed by enzymes. Arachidonic acid metabolites include prostaglandins, leukotrienes, and lipoxins that are produced by cyclooxygenase (COX) and lipoxygenase enzyme pathways.

Arachidonic acid (eicosapentaenoic acid) is an essential 20-carbon, polyunsaturated fatty acid derived from linoleic acid, which is present in plasma membranes of dietary red meats. Arachidonic acid is an integral component of esterified membrane phospholipids that, when cleaved from the plasma membrane by phospholipase, serves as the major precursor for eicosanoids. Eicosanoids are synthesized by two main classes of enzymes: (1) COXs and (2) lipoxygenases and also (3) cytochrome P450 enzymes. Their respective products (eicosanoids) are (1) prostaglandins and thromboxanes and (2) leukotrienes and lipoxins. These molecules are synthesized in endothelial cells, leukocytes, and platelets and principally exert their biologic effects on vascular and airway smooth muscle cells, endothelial cells, and platelets during the acute inflammatory response.

Arachidonic acid is released from membrane phospholipids of many cell types, but particularly endothelial cells and leukocytes, through the action of cytoplasmic phospholipase A₂ (cPLA₂) and to a lesser degree, soluble (extracellular) phospholipase A₂ (sPLA₂). This occurs in response to physical and chemical stimuli, including C5a. cPLA₂ is translocated from the endoplasmic reticulum to plasma membrane when intracellular calcium concentrations increase. The activity of sPLA₂ also requires the participation of calcium; however, its contribution to the formation of intracellular arachidonic acid varies among the different cell types when compared with that of cPLA₂. Membrane phospholipids contain a glycerol backbone attached to which is often a saturated fatty acid at the sn-1 position, an unsaturated fatty acid at the sn-2 position, and a base at the sn-3 position. Arachidonic acid is often in the sn-2 position and released by cPLA₂ or sPLA₂ to become free arachidonic acid.

Free arachidonic acid is metabolized in one of three pathways: (1) the COX pathway for the formation of prostaglandins and thromboxanes, (2) the lipoxygenase pathway for the formation of leukotrienes and lipoxins, and (3) the cytochrome p450 pathway for the formation of epoxyeicosatrienoic acids (hydroperoxyeicosatetraenoic acid [HPETE] and hydroxyeicosatetraenoic acid [HETE]). There are three COX isoenzymes—COX-1, COX-2, and COX-3—that are actually components of prostaglandin H synthase and work in concert with a peroxidase heme group. The COX-1 isoenzyme is constitutively expressed, is present in almost all tissues, and is considered a housekeeping enzyme with physiologic roles in hemostasis and gastric mucosa protection. COX-2 isoenzyme expression is induced by exogenous and endogenous stimuli and occurs locally in sites of inflammation. It is present in leukocytes, endothelial cells of blood vessels, and synovial fibroblasts. COX-3 isoenzyme is a splice variant of COX-1 (and also termed COX-1b or COX-1v). It is present in greatest abundance in the cerebral cortex of dogs and humans and is also detected in human aortas and rodent cerebral endothelia, heart, kidney, and neuronal tissues.

Prostaglandin Formation and Inhibition

Arachidonic acid metabolites from COX isoenzymes induce an intermediate prostaglandin, PGH₂, that is converted into at least five metabolites (PGD₂, PGF₂, PGE₂, PGI₂, and thromboxane A₂ [TXA₂]) by prostanoid synthase enzymes unique for each of these five metabolites (see Web Fig. 3-7). The relative concentration of each of these five types of prostaglandins synthesized after stimulation depends on the cell type stimulated. For example, PGI₂—a thromboresistant prostaglandin termed prostacyclin—is produced by endothelial cells via PGI₂ (prostacyclin) synthase, whereas TXA₂—a thrombogenic prostaglandin termed thromboxane—is produced by platelets via TXA₂ (thromboxane) synthase. PGD₂ is the major prostanoid produced by mast cells; PGE₂ is the major prostanoid produced by epithelial cells, fibroblasts, and smooth muscle cells. Specific prostaglandins inhibit (PGI₂) or induce (TXA₂) coagulation/thrombosis, whereas others affect vascular permeability (PGD₂ and PGE₂). Prostaglandins bind GPCRs that are specific for each prostaglandin. Activated prostaglandin receptors trigger cyclic adenosine monophosphate (cAMP) or increased cytoplasmic calcium. PGE₂ is long known for immunomodulatory activity; mechanistically, recent work suggests that PGE₂ receptor EP4 promotes differentiation of both T helper 1 (T_H1) and T helper 17 (T_H17) CD4⁺ T lymphocytes.

Prostaglandins contribute to the following:

Aspirin, indomethacin, ibuprofen, and naproxen are COX-1 inhibitors. Aspirin, naproxen, and ibuprofen also inhibit COX-2, as do the highly selective COX-2 inhibitor drugs celecoxib, rofecoxib, valdecoxib, lumiracoxib, and etoricoxib. Acetaminophen was thought to inhibit COX-3; however, acetaminophen’s activity in humans and rodents is not fully defined. Because COX-3 is present in greatest abundance in the cerebral cortex and acetaminophen crosses the blood-brain barrier (unlike other nonsteroidal antiinflammatory drugs [NSAIDs]), these observations were initially thought to explain why acetaminophen was sometimes more effective for treating headaches and pain relief and less effective in inhibiting inflammation in the body. Although this may be the case in dogs, the mechanistic basis of acetaminophen’s activity in humans is not fully understood at this time. Acetaminophen may inhibit COX-2 to a minor degree and also slightly inhibit COX-3 (COX-1b), and yet have other activity/activities. Much of the antiinflammatory effect of corticosteroids is due to their inhibition of phospholipase A₂, the enzyme that releases arachidonic acid from membrane phospholipids. Corticosteroids signal the cell to synthesize a polypeptide known as lipocortin (lipomodulin), which then acts to inhibit phospholipase A₂. Therefore the antiinflammatory effect of corticosteroids is delayed. A variety of other natural and synthetic compounds can inhibit phospholipase A₂.

Leukotriene Formation and Inhibition

In leukocytes, arachidonic acid can be metabolized through the 5-lipoxygenase pathway to form leukotrienes and lipoxins (see Web Fig. 3-7), which have profound effects on inflammation. 5-Lipoxygenase works in concert with 5-lipoxygenase-activating protein as an arachidonic acid binding protein, which optimizes the proximity of 5-lipoxygenase to interact with arachidonic acid. The complex formed by 5-lipoxygenase and 5-lipoxygenase-activating protein (HPETE) is converted to an intermediate complex, LTA₄, which is subsequently converted to either LTB₄ or LTC₄, via LTA₄ hydrolase or LTC₄ synthetase, respectively. LTC₄ synthetase is a glutathione transferase, and once LTC₄ is secreted from a cell, it is metabolized to LTD₄ and LTE₄ by sequential removal of the glutathione, glutamic acid, and glycine moieties.

The major effects of the leukotrienes include (1) increased vascular permeability, (2) chemotaxis for leukocytes, and (3) vasoconstriction, all of which exacerbate the acute inflammatory response. Neutrophils produce primarily LTB₄, which is one of the most potent chemotactic factors for neutrophils and macrophages. LTB₄ also stimulates leukocytes to release their lysosomal contents further contributing to inflammation. Mast cells and eosinophils produce primarily leukotriene C₄, and the group of leukotrienes—LTC₄, LTD₄, and LTE₄, formerly known as “slow-reacting substance”—causes intense vascular leakage from the venules. Macrophages produce both LTB₄ and LTC₄. In addition to the leukotrienes, 5-HPETE and its end-product 5-HETE are highly chemotactic for neutrophils and macrophages. Leukotrienes also bind GPCR. LTC₄, LTD₄, and LTE₄ bind the leukotriene receptors CysLT₁ and CysLT₂, which mediate bronchoconstriction and vasoconstriction, and LTB₄ binds two receptors, BLT₁ and BLT₂, which mediate neutrophil chemotaxis.

Lipoxins, secreted primarily by platelets, are also produced from arachidonic acid via the lipoxygenase pathway. Platelets cannot form lipoxins directly, but through cell-to-cell interactions with leukocytes and the transfer of arachidonic acid metabolites to platelets, cooperation between different cells types can result in the formation of eicosanoids via transcellular biosynthesis. Neutrophils can transfer their LTA₄ to platelets. Platelets contain 12-lipooxygenase, which converts the neutrophil derived and transferred LTA₄ to lipoxin A₄ and lipoxin B₄. Lipoxins have proinflammatory and antiinflammatory effects and appear to counteract the effects of leukotrienes. They stimulate macrophage adhesion to endothelial cells in blood vessels, but inhibit LTB₄ activation of neutrophils, thus decreasing neutrophil adhesion and chemotaxis, and also inhibit angiogenesis. Lipoxin A₄ causes vasodilation and dampens the actions of LTC₄–induced vasoconstriction. Resolvin is a product of eicosapentaenoic acid which also has antiinflammatory effects, including inhibition of BLT₁ (LTB₄ receptor) and dendritic cell migration.

The 5-lipoxygenase pathway can be inhibited by a variety of new chemical agents (5-lipoxygenase inhibitors and leukotriene receptor antagonists) (see Fig. 3-9). In addition, eosinophils release arylsulfatase from granules, which inactivates leukotrienes.

Omega-3 Fatty Acids (Fish Oils) and Inhibition of Eicosanoid Activity

Many studies have demonstrated that omega-3 fatty polyunsaturated fatty acids, which are in high concentrations in certain fish, particularly salmon, can reduce inflammatory responses mediated by prostaglandin and leukotriene activity (see Web Fig. 3-7). This effect occurs because omega 3 fatty acids, when acted on by phospholipase A and subsequent enzymes, form the “3” series of prostaglandins (TXA₃) and the “5” series of leukotrienes (LTB₅) instead of the “2” series of prostaglandins (TXA₂) and the “4” series of leukotrienes (LTB₄), which occur with omega-6 fatty acids. The “3” and “5” series of prostaglandins and leukotrienes derived from omega-3 fatty acids are less biologically active than their omega-6 series counterparts. Roughly two-thirds of the polyunsaturated fatty acids in the body have an omega-6 structure; the remaining have an omega-3 structure. The two-thirds to one-third ratio can be altered by consumption of diets high in the proper fish oils and thereby result in more omega-3 in the tissue and less active prostaglandin and leukotriene products.

Platelet-Activating Factor

PAF is another potent molecule of phospholipid origin derived from cell membranes of platelets, basophils, mast cells, neutrophils, macrophages, and endothelial cells. PAF has potent pathophysiologic effects and contributes to inflammation, endotoxic shock, and allergic reactions (asthma) through vasoconstriction, bronchoconstriction, platelet aggregation, and leukocyte adhesion, chemotaxis, and degranulation. However, at low concentrations (experimentally induced), PAF can cause vasodilation and increased vascular permeability. PAF also contributes to the inflammatory response by increasing the oxidative burst in neutrophils following phagocytosis of bacteria and enhances the synthesis of eicosanoids by leukocytes. During IgE-mediated hypersensitivity reaction in the lung, PAF induces mast cell release of serotonin and histamine as well as platelet aggregation. Thus platelet aggregation can increase vascular permeability in acquired immunologic responses and result in inflammation.

Two enzymes, lysoPAF acetyltransferase (lysoPAF-AT) and PAF-synthesizing phosphocholinetransferase (PAF-PCT), control the final synthesis of PAF from lipid membranes and thus promote PAF production. PAF mediates its effects on target cells through a single GPCR. These receptors have been identified on endothelium, neutrophils, eosinophils, macrophages, smooth muscle, and glial cells in the brain. PAF activity is reduced/regulated by PAF acetylhydrolase, which is expressed by cells expressing PAF receptor. PAF acetylhydrolase degrades PAF through hydrolysis of its acetate moiety in the sn-2 position of glycerol, which inhibits the proinflammatory activity of PAF. PAF acetylhydrolase is thus potentially an enzyme that could be used for the development of drugs to reduce inflammatory responses.

Cytokine Family

Oxygen-Derived Free Radicals and Nitric Oxide

Free radicals, such as superoxide anion, hydroxy radical, and NO derivatives, can (1) injure vascular endothelial cells, leading to increased vascular permeability; (2) inactivate antiproteases, such as α₁-antitrypsin, resulting in damage to ECM proteins; (3) enhance cytokine and chemokine expression secondary to signaling changes and cell damage; (4) activate endothelial cells and increase adhesion molecule expression; and (5) increase the formation of chemotactic factors (LTB₄). They can also inactivate neurotransmitters (adrenaline and noradrenaline), leading to hypotension.

Oxygen-derived free radicals are released from neutrophils and macrophages after exposure to chemokines and immune complexes and after phagocytosis by the leukocyte (see Table 3-2). They damage cells through peroxidation of cell membrane lipids, cross-linking of proteins and oxidizing thiol groups on methionine and cysteine, cleaving glycoconjugates, directly damaging DNA phosphate backbone and bases, and inducing the formation of DNA adducts. Because of damage to proteins, the activity of key enzymes and transcription factors needed by the cell can be impaired. Fortunately, the body has antioxidants that are (1) enzymatic, such as superoxide dismutase (SOD) isoforms 1, 2, and 3, catalase, thioredoxin, peroxiredoxins, and glutathione reductase; (2) nonenzymatic endogenous substances, such as ceruloplasmin, transferrin, metallothionein, uric acid, and melatonin; and (3) nonenzymatic dietary substances, such as vitamins A, C, and E and lycopenes, flavonoids, resveratrol genistein, anthocyanins, naringenin, and reserpines. All of these minimize the damage to tissue caused by free radicals.

NO is a chemical mediator of inflammation that causes vasodilation by relaxing vascular smooth muscle cells. In response to injury and inflammatory stimuli, NO derivatives are synthesized by endothelial cells, macrophages, and specific populations of neurons in the brain from l-arginine, molecular oxygen, NADPH, other cofactors, and the enzyme NO synthetase (NOS). There are three forms of NOS that mediate NO formation: neuronal (nNOS), iNOS, and endothelial (eNOS). In addition to its vasodilatory activities, NO inhibits platelet aggregation and adhesion, inhibits mast cell-induced inflammation, oxidizes lipids and other molecules, and regulates leukocyte chemotaxis.

Receptors for Exogenous and Endogenous Inflammatory Stimuli and Toll-Like Receptors

Inflammatory responses occur in response to endogenous and exogenous substances. The body’s inflammatory reaction to exogenous pathogens has been understood and studied for many years. Increasingly, it is clear that the body also produces inflammatory reactions to host endogenous molecules released under sterile conditions. Thus both endogenous and exogenous stimuli produce “danger” signals, termed danger-associated molecular patterns (DAMPs).

Exogenous microbial products, often with redundant molecular structure, are PAMPs and include substances such as LPS, peptidoglycan, and lipoteichoic acid. PAMPs can bind several types of PRRs (Fig. 3-11; Table 3-4; Web Fig. 3-9). These receptors include secreted receptors that circulate in the blood (LPS binding protein), surface receptors (macrophage mannose receptors; and TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), and endosomal receptors (TLR and endosomal viral receptors). These receptors have different mechanisms by which they activate the cell and some work together. LPS, for example, is bound by LPS-binding protein (LBP), which in turn binds CD14 and TLR4. The formation of a PRR-PAMP complex initiates transmembrane signaling that often involves a MyD88 protein event leading to NF kappa B activation and MAPK (p38) signaling. The NF kappa B family of transcription factors initiates gene transcription and translation, resulting in the expression of proteins involved in many cellular processes such as cell proliferation, differentiation, apoptosis, and cell responses to injury, stress, and external pathogens. NF kappa B and p38 then can induce phagocytosis by leukocytes, dendritic cell activation, release of inflammatory cytokines and chemokines, and the activation of the innate (defensin and antimicrobial peptide release) and adaptive (T_h1, T_h2, T_h17, T_FH activity) immune systems. Alternatively, TLR4 signaling does not engage MyD88 (MyD88-independent signaling) and results in the formation of IFN-β and IFN-inducible gene products.

In addition to LBP, other secreted PRRs that can trigger inflammatory reactions through complement activation or receptor binding include collagenous lectins such as mannan-binding lectins A and C, ficolins, surfactant proteins A (SP-A) and D (SP-D), and conglutinin (cattle), which bind glycans of chitin, bacterial capsules, viruses, C-reactive protein, and serum amyloid–binding protein. SP-A, for example, can bind, aggregate, and inhibit respiratory syncytial virus. SP-A can also activate macrophages.

Leukocytes, epithelia, mucosal-lining cells, and other cells can also express PRR on the plasma membrane, including TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11. TLRs are a family of PRRs in mammals that can distinguish between chemically diverse classes of genetically conserved microbial products (see Table 3-4). For example, bacterial lipoprotein binds TLR1 and TLR2/6, flagellin binds TLR5, and LPS binds TLR4. These receptors have a central role in the release of inflammatory cytokines from the innate immune system in response to microbial structures, such as exogenous microbial substances and endogenous products (Web Fig. 3-10). Also, TLRs likely play a role in the adaptive immune response, which develops during the acute inflammatory response.

C-type lectin receptors are also present on the surface of monocytes, macrophages, neutrophils, and dendritic cells and include dectin 1 that binds beta 1,3 glucan, as well as dectin 2, MBL, and dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrins (DC-SIGNs) that bind high mannose and fructose; both glucans and mannose/fructose residues are produced and released by mycobacterial and fungal organisms. Once bound, both TLR and C-type lectins regulate a wide range of innate and adaptive immune responses through NF kappa B, transcription factor activators protein-1 (TFAP-1), and interferon regulatory factors (IRFs).

Intracellular receptors, such as the NLRs, have a leucine-rich repeat domain that binds PAMPs and xenocompounds to induce and/or regulate inflammation. NOD1 binds to peptidoglycan-derived tripeptide structures, whereas NOD2 binds peptidoglycan-derived muramyl dipeptides. Bound NOD1 and NOD2 activate the receptor-interacting protein (RIP) family of serine/threonine protein kinase (RIPK2), which then induces NF kappa B activity and eventual secretion of antimicrobial peptides and inflammatory cytokines such as IL-1β and IL-18 and IL-33, invoking inflammation. Muramyl dipeptides and a variety of xenocompounds and endogenous stimuli can activate another family of NLRs that contain a pyrin domain NLRP3 (also termed NALP3), which triggers caspase 1 activation and activation of IL-1β, IL-18, and IL-33.

PRRs for certain viral infections include retinoic acid-inducible gene I (RIG-I) (e.g., paramyxoviridae, influenza viruses) and melanoma differentiation-associated gene 5 (MDA-5) (picornaviridae). These molecules sense viral RNA, alter mitochondrial activity through IPS-1, and invoke IRF3 for IFN-α and -β release and NF kappa B for inflammatory cytokine production along with antimicrobial peptides.

Endosomes express PRRs that also have important roles in sensing viral infection and triggering a cellular response that often results in inflammatory and immune activity. TLR3, TLR7, TLR8, and TLR9 are present along the inner endosomal membrane and bind double-stranded RNA (dsRNA) (TLR3) or single-stranded RNA/DNA (ssRNA/DNA) (TLR7, TLR8, TLR9) and activate IRF7 and NF kappa B.

Endogenous molecules (not produced by microbes) are released in response to noninfectious injury (trauma, toxins, neoplasia, necrosis, or irritation). Because endogenous molecules can trigger an inflammatory reaction, they have been termed alarmins. Alarmins include sulfate, hyaluronan, HSP 60 (mitochondria), HSP 70 (cytoplasm), Gp 96 (endoplasmic reticulum), fibronectin, fibrinogen, and SP-A, all of which can bind PRRs or other receptors and initiate cell signaling, inflammation, and activation of the innate immune system (see Table 3-4). These molecules are often overlooked when considering acute inflammation in the context of microbial infections because microbial products, such as teichoic acid and LPS, are very potent activators of acute inflammation. However, endogenous molecules likely have a significant role in inflammation generated against neoplastic cells, toxins, and mechanical injury. For example, a wide variety of xenocompounds, including apoptotic cells, toxins (maitotoxin, valinomycin, nigericin, aerolysin), stress molecules (ultraviolet [UV] radiation, monosodium urate, calcium pyrophosphate, beta amyloid fibrils), xenogeneic compounds (silicates, asbestos, aluminum hydroxide), as well as microbial products (lipoteichoic acid, LPS), can affect lysosomal function which in turn induces NLRP3 (NALP3) activation (Fig. 3-12). Furthermore, adenosine, a by-product of cell injury CD39 hydroxylation of ATP, can bind adenosine receptors present on the surface membrane of leukocytes and modulate inflammatory and immune responses. Endogenous molecules mitigate the intensity of the inflammatory response by regulating signaling intensity.

Antimicrobial Peptides and Collectins

Antimicrobial peptides, such as the α- and β-defensins, cathelicidins, and others—such as anionic peptides, histatin, and dermacidins—are small peptides with microbicidal activity against Gram-negative and -positive bacteria, fungi, mycobacteria, and some enveloped viruses such as human immunodeficiency virus (HIV) (Web Table 3-9). They are encoded by genes of the cells involved in the inflammatory response, especially neutrophils, and by epithelial cells forming the skin and the mucosal barriers of the respiratory and alimentary systems. The microbicidal activity likely occurs through the formation of pores within bacterial membranes and viral envelopes. In addition to the microbicidal activity, antimicrobial peptides are increasingly appreciated for their role in other nonmicrobial activities related to inflammation and wound repair. These activities include chemotaxis of leukocytes and dendritic cells, cell proliferation, wound repair, cytokine release, and the protease-antiprotease balance. There is also much evidence that antimicrobial peptides link the innate and adaptive immune responses.

There are a wide variety of antimicrobial peptides; however, the defensins have received the most attention for activities other than microbial killing. There are three types of defensins: the α-, β- and θ-defensins. α-Defensins are produced by neutrophils and Paneth cells. β-Defensins are produced by neutrophils and epithelial cells. θ-Defensins are produced in neutrophils of primates. The defensins are cationic proteins with three pairs of intramolecular disulfide bonds. α-Defensins and β-defensins can stimulate mast cell degranulation, induce IL-8 synthesis, induce T-lymphocyte chemotaxis, and activate T lymphocytes, macrophages, and dendritic cells, thus interconnecting innate and adaptive immunity. The importance of defensins in immunity is underscored by the fact that HIV-1 infected individuals that are healthy and in “remission” have high concentrations of α-defensins, which are thought to enhance T-lymphocyte activity and may also have direct anti–HIV-1 activity.

Acute Phase Proteins

Acute phase proteins are plasma proteins synthesized in the liver whose concentrations increase (or decrease) by 25% or more during inflammation. These proteins serve as inhibitors or mediators of the inflammatory processes and include C-reactive protein, α₁-acid glycoprotein, haptoglobin, mannose-binding protein, fibrinogen, α₁-antitrypsin, and complement components C3 and C4. The concentration of these acute phase proteins usually increases during inflammation, whereas the concentration of prealbumin and albumin (also acute phase proteins) decreases in inflammation. Acute inflammatory conditions that are severe enough to raise blood plasma concentrations of cytokines, such as IL-1 and TNF, increase the blood concentrations of acute phase proteins and elevation of fibrinogen in the blood of cattle is used clinically as an indicator of systemic inflammation. C-reactive protein has recently received attention as a marker of inflammatory conditions, especially atherosclerosis in humans. In addition to its role diagnostically, C-reactive protein binds to bacteria and fungi and also activates complement. Once acute phase proteins and systemic levels of inflammatory cytokines are elevated, they affect the heart rate, blood pressure, and the hypothalamic regulation of temperature by directly or indirectly stimulating neurons within specific hypothalamic nuclei. The previously described changes also affect respiratory rates and gaseous exchange.

Summary of the Chemical Mediators of Acute Inflammation

Various exogenous and endogenous stimuli can activate soluble, surface, or cytoplasmic and endosomal receptors or simply cause mechanical or other damage to invoke an acute inflammatory response. This response can occur very rapidly as the result of the release of preformed or rapidly activated inflammatory mediators such as histamine, kinins, complement factors like C3a and C5a, and tachykinins (substance P). These molecules generally affect vascular caliber and permeability and activate leukocytes and endothelial cells. Concurrently, lipid-based products, such as prostaglandins, leukotrienes, and PAF, contribute to chemotaxis, vascular tone, and leukocyte activity, all of which work in concert with chemokines and cytokines to activate endothelial cells and leukocytes and enhance neutrophil infiltration. NO released by macrophages and endothelia induce vasodilation and also can contribute to tissue damage caused by other reactive oxygen species. Inflammatory mediators bind receptors that subsequently induce cytoplasmic signaling and cellular activation, resulting in the production of additional cytokines, chemokines, adhesion molecules, antimicrobial peptides, and other inflammatory mediators that may either exacerbate or inhibit the inflammatory process. Activated neutrophils expressing leukocyte adhesion molecules, such as the integrins, enter inflamed tissue and can release hydrolytic enzymes and other granule contents that further damage tissue. Systemically, increases in acute phase proteins, cytokines, chemokines, complement, and inflammatory proteins can affect body temperature, cardiovascular function, locomotion, sleep, appetite, and other activities (Table 3-5).

Outcomes of the Acute Inflammatory Response

The four main outcomes of acute inflammation are as follows:

The severity of tissue damage, the ability of cells to regenerate, and the physical or biologic characteristics of the cause of the injury determine these outcomes. In the acute inflammatory response (Fig. 3-13), the desired outcome is resolution, that is, the complete return of normal structure and function. Resolution occurs if the following occur:

Mechanistically, the critical first stage of resolution involves the killing and/or removal of the inciting cause, removal of chemical mediators by neutralization or decay, return to normal vascular flow and capillary permeability, cessation of leukocyte emigration, apoptotic cell death of remaining neutrophils in the exudate, the removal of the exudate by monocyte-macrophage phagocytosis, and drainage to regional lymph nodes (Fig. 3-14). Inflammatory responses of neutrophils, monocytes, and macrophages are further inhibited by chemokine inactivation and release of products such as lipoxin A4, resolvins, annexin A, lactoferrin, and lysophosphatidylcholine.

Regeneration is the second stage of resolution and depends on the availability of progenitor epithelial cells and the presence of a supportive stroma and intact basement membrane for orderly cellular migration. As an example, acute renal tubular necrosis can be caused by aminoglycoside antibiotics and results in detachment and necrosis of tubular epithelial cells from the tubular basement membrane. If the supporting stroma and basement membrane remain intact, progenitor epithelial cells can divide and migrate to replace lost cells and thus return the tubule to normal function. If the basement membrane is not intact to guide the proliferating cells, functional tubules will not form. Instead, regenerative tubular epithelial cells will atrophy or form into small aggregates with syncytial giant cells. Simultaneously, the microvascular supply to the region must be restored, which occurs mechanistically by the proliferation of endothelial cells in response to molecules such as VEGF.

Serous Inflammation

Serous inflammation is the term used to describe a pattern of acute inflammation in which the tissue response consists of the leakage or accumulation of fluid with a low concentration of plasma protein and no to low numbers of leukocytes. This watery material is released from small gaps between endothelial cells and from hypersecretion of inflamed serous glands. This response is essentially a transudate (specific gravity <1.012) and is seen with (1) thermal injury to skin, such as burns and photosensitization, in which the lesion can appear as a fluid-filled blisters, or (2) acute allergic responses characterized by watery eyes and a runny nose with a clear, colorless transudate.

Grossly, lesions, for example, with serous inflammation consist of tissue that (1) contains excessive clear to slightly yellow, watery fluid that leaks from the tissue on cut section or (2) forms raised fluid-filled vesicles protruding above the surface of the mucous membrane of the nasal cavity (serous rhinitis) or of the skin (Fig. 3-16). Microscopically, connective tissue fibers are separated, often widely, and capillaries and postcapillary venules are dilated with erythrocytes (active hyperemia). Endothelial cells lining these vessels can vary from flattened to hypertrophied.

Catarrhal Inflammation

Catarrhal inflammation, or mucoid inflammation, is the term used to describe a pattern of acute inflammation in which the tissue response consists of the secretion or accumulation of a thick gelatinous fluid containing abundant mucus and mucins from a mucous membrane. This response occurs most commonly in tissue with abundant goblet cells and mucus glands, such as in certain types of chronic allergic and autoimmune gastrointestinal diseases and with chronic inflammation of the airways of the respiratory system (chronic asthma). Grossly, the surface or cut surface of affected tissue may be covered with or contain a clear to slightly opaque, thick fluid (Fig. 3-17). Microscopically the lesion can include hyperplastic epithelial cells of mucus glands and goblet cells, as well as connective tissue fibers separated by mucins.

Fibrinous Inflammation

Fibrinous inflammation is the term used to describe a pattern of acute inflammation in which the tissue response consists of the accumulation of fluid with a high concentration of plasma protein (specific gravity >1.02) and no to low numbers of leukocytes. This response is an exudate. Fibrinous inflammation occurs with more severe endothelial cell injury that allows leakage of large molecular weight proteins such as fibrinogen. Fibrinogen leaks from capillaries and postcapillary venules during the fluidic phase of the acute inflammatory response and polymerizes outside of the vessels to fibrin, a homogeneous and vividly pink (eosinophilic) protein when stained with hematoxylin and eosin (H&E). This lesion is most commonly caused by infectious microbes and is seen in the serous membranes of the body cavities, such as those lined by pleura (fibrinous pleuritis), pericardium (fibrinous pericarditis), peritoneum (fibrinous peritonitis), and in the synovial membranes of joints (fibrinous synovitis) and the meninges (fibrinous leptomeningitis). Common examples include the lesions in pulmonary alveoli in fibrinous pneumonia (Mannheimia haemolytica), atypical interstitial pneumonia (3-methyl indol), and respiratory viral infections (bovine herpesvirus-1 in cattle). When fibrin forms a distinct layer covering an ulcer, it is referred to as a fibrinous pseudomembrane, whereas when it lines the pneumonocyte surface of lung alveoli in a curvilinear fashion and is mixed with necrotic cell debris, such as occurs with bovine respiratory syncytial virus (BRSV) infection, it is called a hyaline membrane.

Grossly, the surfaces of affected tissue are red (active hyperemia) and covered with a thick, stringy, elastic, white-gray to yellow exudate that can be removed from the surface of the tissue (in contrast to a fibrous response) (Fig. 3-18; see Fig. 3-4). A classic example of fibrinous inflammation occurs in fibrinous pneumonia caused by acute Mannheimia haemolytica infection, in which both alveoli and stromal connective tissue (interlobular septa and pleura) contain notable fibrinous exudate that rapidly becomes infiltrated by neutrophils, resulting in a fibrinosuppurative exudate. Another example of fibrinous inflammation occurs in bovine herpesvirus-1 infection. This virus injures epithelial cells of the respiratory tract, resulting in an acute fibrinous inflammatory response. Noninfectious causes, such as heat and smoke inhalation, can cause fibrinous exudate in the trachea. Microscopically, capillaries and postcapillary venules are dilated with erythrocytes (active hyperemia), and endothelial cells are reactive (hypertrophied). The stromal connective tissue or the mesothelial surfaces of the affected organ contains or is covered by red to vividly red layers of coagulated and/or polymerized fibrin, albumin, and other plasma proteins. The fibrinous exudate is often rapidly infiltrated by neutrophils, resulting in fibrinosuppurative inflammation.

Suppurative Inflammation

Suppurative inflammation is the term used to describe a pattern of acute inflammation in which the tissue response consists of the accumulation of fluid with a high concentration of plasma protein (specific gravity >1.02) and high numbers of leukocytes, predominately neutrophils. This material is an exudate commonly known as pus. Pus can be a creamy liquid, but if dehydrated, it can be more caseous and firm in consistency and occasionally laminated in diseases such as ovine caseous lymphadenitis. A collection of pus circumscribed by a fibrous capsule that is visible grossly is called an abscess, whereas, if visible only microscopically, it is called a microabscess.

Dense neutrophil accumulation (pus) can also be distributed in tissue layers such as fascial planes and subcutaneous connective tissue and is referred to as cellulitis or phlegmonous inflammation. Instead of producing a focal abscess, the neutrophils evoke a watery suppurative exudate distributed along fascial planes and tissue spaces, as in some cases of blackleg or extensive Gram-positive (staphylococcal) infection. Suppurative inflammation, microabscesses, abscesses, and exudates are most commonly caused by bacteria, including Staphylococcus spp., Streptococcus spp., and Escherichia coli, and can occur in many organs. These genera can also cause suppurative bacterial meningitis in the central nervous system (CNS). Abscesses in the brains of horses caused by Streptococcus equi and microabscesses in the brains of cows caused by Listeria monocytogenes are good examples of suppurative inflammation. Bacteria-induced suppurative inflammation also occurs commonly in (1) the pelvis and tubules (pyelonephritis) of the kidney, (2) the bronchi of the lungs (bronchopneumonia), (3) the nasal and sinus cavities (rhinitis and sinusitis), (4) the glandular epithelium of the prostate (prostatitis), (5) the lumina of the gall bladder (cholecystis) and urinary bladder (urocystitis), and (6) the acini and ducts of the mammary gland (mastitis). Unresolved suppurative inflammation can progress to chronic inflammation.

Grossly the surfaces and/or connective tissues of affected organs are hyperemic and covered by or contain, respectively, a thick white-gray to yellow pus (Fig. 3-19). In some cases, pus will be mixed with fibrin, forming a fibrinosuppurative exudate. Microscopically, affected tissues have large numbers of neutrophils; many degenerate and often are mixed with necrotic cellular debris, bacteria, plasma proteins, and fibrin.

• Persistence/resistance: Persistent infections, such as those caused by Mycobacterium spp.; Nocardia spp.; deep-seated mycoses, such as Blastomyces dermatitidis (Web Fig. 3-11) and Histoplasma capsulatum; and parasites, such as Toxocara canis larvae, can avoid and/or resist phagocytosis by neutrophils and macrophages—or once internalized by these cells, can prevent fusion of primary and secondary lysosomes or killing by lysosomes. Such microbes also usually do not produce biologic molecules that cause severe tissue injury, but their presence continually incites chronic inflammatory and immune responses. Some microbial agents can induce macrophages to undergo apoptosis and subsequent internalization by adjacent macrophages. Tissue destruction, granulomatous inflammation, and fibrosis are common sequelae of persistent/resistant infectious agents.

• Isolation: Some microbes, such as Streptococcus and Staphylococcus spp., are not naturally resistant to phagocytosis and/or destruction but are able to isolate themselves from effective innate and adaptive immune responses and from antimicrobial drugs by “hiding” themselves in pus.

• Unresponsiveness: Certain foreign materials are virtually indestructible and therefore are unresponsive to phagocytosis and/or enzymic breakdown. These include plant material, grass awns, silica dust, asbestos fibers, some suture materials, and surgical prostheses.

• Autoimmunity and leukocyte defects: Alterations in the regulation of adaptive immune responses to self-antigens result in autoimmune diseases, such as polyarteritis nodosa, with a chronic inflammatory response. Defects in leukocyte function can also result in chronic inflammation. For example, loss of NADPH oxidative function in human patients with chronic granulomatous disease, impair leukocyte free radical formation and oxidative killing, thus allowing persistence of microbial agents or internalized material.

• Unidentified mechanisms: In some diseases, such as canine granulomatous meningoencephalitis, the cause of chronic inflammation remains unknown.