Inflammation and Healing

CHAPTER 3


Inflammation and Healing



Injury or death of cells caused by infectious microbes, mechanical trauma, heat, cold, radiation, or cancerous cells can initiate a well-organized cascade of fluidic and cellular changes within living vascularized tissue called acute inflammation (Fig. 3-1). These changes result in the accumulation of fluid, electrolytes, and plasma proteins, as well as leukocytes, in extravascular tissue and are recognized clinically by redness, heat, swelling, pain, and loss of function of the affected tissue. Inflammation is often a protective mechanism whose biologic purpose is to dilute, isolate, and eliminate the cause of injury and to repair tissue damage resulting from the injury. Without inflammation, animals would not survive their daily interactions with environmental microbes, foreign materials, and trauma and with degenerate, senescent, and neoplastic cells.



Acute inflammation, a provoked response, is the progressive reaction of vascularized living tissue to injury over time. This process is usually a well-ordered cascade mediated by chemoattractants, vasoactive molecules, proinflammatory and antiinflammatory cytokines and their receptors, and antimicrobial or cytotoxic molecules. Acute inflammation has a short duration, ranging from a few hours to a few days, and its main characteristics are exudation of electrolytes, fluid, and plasma proteins and leukocytic emigration, principally neutrophils from the microvasculature, followed by rapid repair and healing. For convenience, acute inflammation is divided into three sequential phases: fluidic, cellular, and reparative.


Chronic inflammation is considered to be inflammation of prolonged duration, usually weeks to months and even years, in which the response is characterized predominately by lymphocytes and macrophages, tissue necrosis, and accompanied by tissue repair, such as healing, fibrosis, and granulation tissue formation, all of which may occur simultaneously. Chronic inflammation can be a sequela to acute inflammation if there is failure to eliminate the agent or substance that incites the process. With such persistent substances, the inflammatory reaction and exudates gradually transition from seroproteinaceous fluids and neutrophils to macrophages, lymphocytes, and fibroblasts with the potential for formation of granulomas. Alternatively, some inciting substances can invoke chronic inflammation directly and almost immediately. Examples include infections by Mycobacterium spp.; exposure to foreign materials, such as silicates and grass awns; and immune-mediated diseases, such as arthritis.





Evolution of the Current Understanding of Inflammation


The current understanding of inflammation has evolved over the last 3600 years of recorded history (Web Table 3-1). Clinical signs attributable to inflammation were first described in Egypt in 1600 bc. Aulus Cornelius Celsus, 25 bc to 50 ad, was a Roman writer (de Medicina) and the first individual to describe the four cardinal signs of inflammation (redness, heat, swelling, and pain) that are commonly used today to diagnose inflammation in medicine. In the mid-1800s, Rudolf Virchow, the founder of modern pathology, added the fifth cardinal sign of inflammation: loss of function. In addition to his numerous contributions to cellular pathology, Dr. Virchow, by the age of 25, had discovered fibrinogen, described the processes of leukocytosis, and later characterized pus and necrosis. In 1859, his book, Cell Pathology, became the basis for all microscopic study of disease. Phagocytosis by macrophages, an important component of inflammation and immunologic responses, was first described by Illya Mechnikov (Elie Metchnikoff) in 1883, and he was awarded the Nobel Prize in Medicine in 1908. Jules Bordet, a Belgian scientist, demonstrated antibacterial activity of serum, which Paul Ehrlich, of Germany, later defined as complement. The first experiment to demonstrate the role of a chemical mediator (histamine) in inducing vascular changes (flare and wheal reactions) was conducted by Sir Thomas Lewis in 1927. The work of these pioneers, as well as additional experimental studies conducted during the past century, has provided (1) an in-depth and clearer understanding of inflammation and (2) the foundation for development of therapeutic compounds to treat undesirable effects of inflammatory responses. In fact, such treatments are so widely used and commonplace in veterinary medicine today that the contributions and discoveries of these scientists are often taken for granted.



Beneficial and Harmful Aspects of Inflammation


As a general rule, inflammatory responses are beneficial in the following ways:



However, in some instances, an excessive and/or prolonged inflammatory response can be detrimental and even more harmful than that of the inciting agent/substance. In several disorders of humans, such as myocardial infarction, cerebral thrombosis and infarction, and atherosclerosis, excessive and prolonged inflammatory responses can exacerbate the severity of the disease process. In veterinary medicine, exuberant or uncontrolled inflammatory responses occurring in the diseases listed in Box 3-1 can also result in increased severity of disease.



BOX 3-1


Selected Disorders that Are Induced or Exacerbated by Inflammatory Responses





Modified from Nathan C: Nature 420:846-851, 2002.



Acute Inflammation


The acute inflammatory response (Fig. 3-2) can be initiated by a variety of exogenous and endogenous substances that injure vascularized tissue. The response to injury begins as active hyperemia, characterized by an increased flow of blood to injured tissue secondary to dilation of arterioles and capillaries (vasodilation), and it is this response that is responsible for redness and heat. It is facilitated by chemical mediators such as prostaglandins, endothelin, and nitric oxide (Box 3-2). With vasodilation, vascular flow is slowed (vascular congestion), allowing time for fluid leakage that occurs as a result of changes in junctional complexes of endothelial cells induced by vasoactive amines, complement components C3a and C5a, bradykinin, leukotrienes, prostaglandins, and platelet-activating factor (PAF), resulting in leakage of plasma and plasma proteins into the extracellular space (swelling and pain [stretching of pain receptors]) mainly from interendothelial cell gaps in the postcapillary venules.




The volume and protein concentration of leaked fluid is a function of the size of gaps between endothelial cells and the molecular weight, size, and charge of electrolytes and plasma proteins, such as albumin and fibrinogen. With more severe injury resulting in destruction of individual endothelial cells, hemorrhage, as well as plasma and plasma proteins, can leak directly through a breach in the wall of the capillary or venule. Once activated, endothelial and perivascular cells, such as mast cells, dendritic cells, fibroblasts, and pericytes, can produce cytokines and chemokines that regulate the expression of receptors for inflammatory mediators and adhesion molecules within the lesions.


The plasma proteins and fluid that initially accumulate in the extracellular space in response to injury is classified as a transudate (Fig. 3-3). A transudate is a fluid with minimal protein (specific gravity <1.012 [<3 g of protein/dL]) and cellular elements (<1500 leukocytes/mL) and is essentially an electrolyte solution similar to that of plasma. Most commonly, the formation of a transudate occurs with hypertension, hypoproteinemia, and/or early in the acute inflammatory response. With these conditions, there is increased permeability because of small physiologic gaps between endothelial cells. Hypertension in veins and capillaries can be secondary to arterial hypertension or venous/lymphatic obstruction. Hypoproteinemia is often due to loss of albumin, the major intravascular colloidal protein, and an inability of the liver to rapidly synthesize replacement albumin. The loss of albumin allows intravascular fluid to move toward the extravascular colloids (extravascular proteins). Loss of albumin and other intracellular proteins can occur secondary to renal disease (urinary loss), severe burns, and severe hepatic disease (decreased albumin production). During the early stages of the acute inflammatory response, intercellular gaps form between endothelial cells caused by endothelial cell contraction. The gaps are very small and only allow water and electrolytes to pass through them. With persistent and widening endothelial gap formation or with endothelial cell injury, neutrophils and additional protein can enter injured areas resulting in the formation of an exudate (see Fig. 3-3). An exudate is an opaque and often viscous fluid (specific gravity >1.020) that contains more than 3 g of protein/dL and more than 1500 leukocytes/mL. As discussed in a later section, the morphologic classification of inflammatory responses into categories, such as serous, fibrinous, and/or suppurative, is based on the character of the fluid that leaks from the vessel and of the leukocytes that migrate from the vascular lumen into the extracellular space.



Fibrinogen is an important plasma protein in exudates that polymerizes in extravascular tissues to form fibrin (Fig. 3-4). Plasma dilutes the effects of the inciting stimulus, whereas polymerized fibrin confines the stimulus to an isolated area, thus preventing its movement into adjacent tissue. This confinement provides leukocytes with a well-defined target for migration during the cellular phase of the acute inflammatory response. Neutrophils are the first leukocytes to enter the exudate, and their accumulation in the exudate after they liquefy is termed pus. Neutrophils have a variety of cytoplasmic granules, such as lysosomes, that contain antimicrobial peptides and proteins, as well as matrix metalloproteinases, elastases, and myeloperoxidases. They kill pathogens and degrade foreign material by two mechanisms: (1) phagocytosis and fusion with primary and secondary lysosomes and (2) secretion of the contents of granules into the exudate. Because of the enzymes released, these cells can contribute to tissue injury. Fibrin and its products have additional activities, including chemotactic properties and blood clot formation. Fibrin also forms a framework/scaffold for fibroblast and endothelial cell migration during the initial stages of wound healing.



Neutrophils and other leukocytes leave capillaries and venules and migrate into tissue exudates in response to chemoattractant molecules released from host cells, microbes, foreign substances, and some neoplastic cells. As would be expected, the greatest concentration of chemoattractant exists nearest the microbes or foreign substance, and the concentration decreases in a gradient-like manner with increasing distances from the source. This forms a “chemotactic gradient” that essentially creates a pathway for leukocytes to follow to reach the site of tissue injury. Chemoattractants activate receptors and molecules on neutrophils that result in neutrophil (1) movement and attachment to the luminal surface of capillaries and venules, (2) migration through the intercellular junctions formed by gaps between endothelial cells, and (3) migration within the exudate up the concentration gradient to the source of injury. This transmigration process, called the leukocyte adhesion cascade, has a well-characterized sequence of events occurring on the luminal surface of endothelial cells. These events, discussed in detail in a later section, lead to the transmigration of leukocytes into the exudate.


The reparative phase of the acute inflammatory response begins early and is only completed after the process or substance causing injury is removed. In the reparative phase, necrotic cells and tissue are replaced by differentiation and regeneration of parenchymal and mesenchymal stem cells, coupled by filling the defect with connective tissue and covering denuded surfaces with a basement membrane and reepithelialization. When the acute inflammatory response has been completed in the proper sequence and the stimulus of injury removed, the inflammatory process is terminated. Failure to remove the stimulus can result in a persistent, unresolved lesion that becomes chronic and can form into granulation tissue or fibrosis.



Substances Inducing the Acute Inflammatory Response


There are two classes of substances, endogenous and exogenous, capable of injuring cells and tissue and inducing the acute inflammatory response. Endogenous substances include those that primarily cause autoreactive inflammatory responses, such as those induced by newly developed antigens and intracellular molecules released from degenerate, dysplastic, or neoplastic cells, and hypersensitivity reactions. Exogenous substances include microbes, such as viruses, bacteria, protozoa, and metazoan parasites; foreign bodies, such as plant fibers and suture material; mechanical actions, such as traumatic injury; physical actions, such as thermal or freezing injury, ionizing radiation, and microwaves; chemical substances, such as caustic agents, poisons, and venoms; and nutritive actions, such as ischemia and vitamin deficiencies. These substances or actions trigger cells to release mediators that lead to an acute inflammatory reaction and include preformed (in cytoplasmic granules) and synthesized (released from cell immediately after synthesis) chemical mediators from effector cells such as mast cells (histamine and tumor necrosis factor-α [TNF-α), leukocytes (cytokines, degradative enzymes), macrophages (cytokines), endothelial and epithelial cells (chemokines, interferons). These cells also produce inflammatory mediators such as prostaglandins, leukotrienes, and PAF from products released from plasma membranes.


The acute inflammatory response to either endogenous or exogenous substances occurs simultaneously with activation of the innate immune system (see Chapter 5). Innate immunity is a nonspecific defense against potentially harmful environmental substances and consists of the following:



The physical and biologic processes that activate the acute inflammatory and innate immune responses can exert their actions directly on the following:



The location, severity, and clinical signs of the acute inflammatory response depend on the route of exposure, such as dermal, alimentary, respiratory, urinary, or hematogenous, and the physical or biologic characteristics of the stimulus. More specifically, causes of the acute inflammatory response include but are not limited to the following:



These must either penetrate (light spectra) or break/penetrate (microbes and foreign bodies) epithelial barriers of the skin and alimentary, urinary, and respiratory systems to irritate the tissue and incite an acute inflammatory response. Microbes have a few highly conserved ligands called pathogen-associated molecular patterns (PAMPs), and when in contact with mucosae, they immediately encounter cells that express membrane pattern-recognition receptors (PRRs), which include Toll-like receptors (TLRs), expressed on the cell surface or in its cytoplasm. These patterns include the lipopolysaccharide (LPS; Gram-negative bacterial cell wall), lipoteichoic acids (Gram-positive bacterial cell wall), mannose, peptidoglycan, bacterial DNA, N-formylmethionine (in bacterial proteins), double-stranded RNA (dsRNA; viruses), and glucans (fungal cell walls). When these molecular patterns are recognized by receptors, such as TLRs, on macrophages, leukocytes, and mucosal epithelia, they trigger the release of chemokines and cytokines and cellular activation, all of which initiate and/or participate in the acute inflammatory response and the innate immune response.


In contrast to the innate immune system, adaptive immunity results in an antigen-specific immune response characterized by the production of protective antibodies and effector leukocytes that attempt to eliminate the inciting cause of injury and the generation of memory cells that make subsequent adaptive immune responses against a specific microbial antigen more efficient and effective (see Chapter 5). Because acute inflammation is a vasocentric response, it would be reasonable to assume that just about any exogenous or endogenous cause could induce inflammation. This is true, but because the inflammatory response has numerous redundant checks and balances that regulate the occurrence and severity of expression of the response, harmful effects of inflammation are minimized.


The effects of inflammation are mediated through chemical mediators of inflammation, which include the following:



Mast cells are rich in histamine and many of the chemical mediators listed previously and are widely distributed in connective tissue adjacent to blood vessels. Changes in the permeability of these vessels in the fluidic phase of the acute inflammatory response often occur as the result of mast cell activation. Histamine, preformed in mast cell granules, is released through a process called degranulation. Bradykinin, another vasoactive amine, is produced where there is vascular and/or endothelial cell injury. Both histamine and bradykinin cause changes in the caliber of arterioles, capillaries, and postcapillary venules and permeability in capillaries and postcapillary venules. These occur early in the fluidic phase of the acute inflammatory response and are quickly followed by the cellular phase.



Fluidic (Exudative) Phase of the Acute Inflammatory Response


The principal function of the fluidic phase of the acute inflammatory response is to dilute and localize the inciting agent/substance. In this phase, there is an immediate vasocentric reaction (arterioles, capillaries, and postcapillary venules) to the inciting agent/substance. The sequence of vascular events in the acute inflammatory response includes the following:



Initially, arterioles dilate and capillary beds in the affected area expand in volume to accommodate an increased blood flow (heat and redness) in response to the stimulus. Second, as a result of permeability changes induced by inflammatory mediators, blood flow through the capillary beds is slowed as a result of increased viscosity and hemoconcentration after leakage of water from capillary beds into extracellular space. Because of the reduced blood flow and pressure, microscopically, capillaries are often packed with erythrocytes and the microenvironment facilitates leukocytic margination along the luminal surface of endothelial cells. This stage precedes leukocyte emigration through intercellular junctions of endothelial cells into the extravascular space. Inflammatory mediators induce endothelial cell contraction, resulting in the formation of interendothelial cell gaps, which further allow fluid leakage and leukocyte migration. In general, the endothelium of the normal vascular capillaries limits exchange of molecules to those less than 69,000 MW, the size of albumin. The exchange of small molecules and water between the vessel lumen and the interstitial space is extremely rapid. For example, the water of plasma is exchanged with the water of the interstitial space 80 times before the plasma can move the entire length of the capillary. Physiologically, increased amounts of fluid can pass across the vascular wall when there is (1) excessive hydrostatic pressure caused by hypertension and/or sodium retention, (2) decreased plasma proteins (colloid), or (3) lymphatic and/or venous obstruction. If fluid leakage is excessive, edema (transudate) develops (see Fig. 3-3). If the leakage is not excessive and postcapillary venules and lymphatic vessels are functioning normally, all of the fluid released from arterioles and small capillaries is returned to the circulation via paracellular gaps of postcapillary venular and lymphatic vessels. During acute inflammatory responses, there is a net outflow of fluid from arterioles, capillaries, and venules into extracellular tissue, which overwhelms the capacity for resorption by postcapillary venules and lymphatic vessels).



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.



Cellular Phase of the Acute Inflammatory Response


The principal function of the cellular phase of the acute inflammatory response is to deliver leukocytes into the exudate at the site of injury so they can internalize agents/substances through phagocytosis and as required, for killing and/or degradation. Neutrophils, eosinophils, basophils, monocytes, mast cells, lymphocytes, natural killer T (NK-T) cells, and dendritic cells play an integral role in protecting mucosa, skin, and other surfaces of the body, as well as the pleura, pericardium, and peritoneum, from infection by microbes through phagocytosis or release of proteolytic degradative enzymes, chemical mediators, and reactive oxygen species. Neutrophils also have an important role in responding to foreign materials and toxins and in responding to neoplastic cells.



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.



image


Fig. 3-6 Leukocyte adhesion cascade.
Steps involved with neutrophil migration across the vascular wall include rolling, activation, stable adhesion, and migration across the endothelial layer and vascular wall. Neutrophils express receptors for E- and P-selectin, which bind to ligands expressed on endothelial cells. L-selectin is also expressed by bovine neutrophils and to a varying degree in other species. The rolling process slows neutrophil movement within the vessel and brings the neutrophil closer to the vascular endothelial cell surface. Activation is induced by inflammatory mediators, including chemokines, such as interleukin-8 (IL-8), and cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which are released by nearby leukocytes and endothelial cells. These activate both neutrophils and endothelial cells which then changes the conformation of integrins allowing for enhanced binding avidity to integrin receptors and increased expression of additional adhesion molecules. Binding of β2 integrins (Mac-1; CD11a/CD18) expressed by leukocytes to intercellular adhesion molecule-1 (ICAM-1) expressed by endothelial cells leads to stable adherence of the cell to the endothelial cell surface. Once leukocytes are attached to the vascular endothelium, they adhere to platelet-endothelial cell adhesion (PECAM-1) and other junctional adhesion molecules present at the endothelial cell junction and transmigrate through the junction into the perivascular tissue, where they express β1 integrins that adhere to extracellular matrix proteins such as laminin, fibronectin, vitronectin, and collagen. The process is mediated by chemokines (CXCL8; IL-8), complement fragments, vasoactive amines, cytokines, and membrane-derived mediators such as platelet-activating factor (PAF) and leukotrienes. (From Kumar V, Abbas A, Fausto N, et al: Robbins & Cotran pathologic basis of disease, ed 8, Philadelphia, 2009, Saunders.)



• Margination. As vessels vasodilate and have reduced hydrostatic pressure and blood flow, leukocytes exit the central region of the vascular lumen and move to the periphery of the vascular lumen near the endothelial cell surface (marginization).


• Rolling. The initial contact between leukocytes and endothelial cells occurs by transient, weak binding interactions between the selectin family of adhesion molecules and their receptors. During rolling, leukocytes temporarily bind to endothelium and then release, which brings the leukocyte close to endothelial cell surface and reduced velocity of the traveling leukocyte. This process is mediated by selectins, including L-selectin expressed by neutrophils and P-selectin, a carbohydrate binding molecule stored in Weibel-Palade bodies of endothelial cells and α-granules of platelets, as well as E-selectin expressed by endothelial cells. L-selectin is expressed on all leukocytes, but at low concentrations in normal human neutrophils and binds sialyl Lewis X receptor (and other receptors) on endothelial cells. P-selectin molecules expressed on endothelial cell surfaces bind to P selectin glycoprotein ligand 1 (sialyl Lewis X–modified proteins) present on neutrophils, eosinophils, monocytes, and lymphocytes. E-selectin also mediates leukocyte-endothelial cell adherence and is expressed on endothelial cell surfaces for binding glycoprotein receptors expressed on leukocytes. Selectin-mediated attachments are formed at the leading edge of the rolling leukocyte and broken at the trailing edge. Even slight disturbances, such as surgical manipulation, heat, temporary ischemia, and mast cell products, induce rolling of neutrophils along the surface of endothelial cells. By slowing leukocyte transit time through capillaries and postcapillary venules, combined with the continual close proximity of slow rolling leukocytes to the endothelium, and the continued release of chemokines and proinflammatory cytokines, the proper microenvironment for progression to the “stable adhesion” stage occurs.


• Stable adhesion. For stable adhesion to occur, neutrophils and endothelial cells become activated by a variety of cytokines (such as IL-1, interleukin-6 [IL-6], TNF), complement factors (C5a), PAF, platelet-derived growth factor (PDGF), chemokines, and other inflammatory mediators. Once neutrophils are activated, L-selectin molecules are proteolytically cleaved from the neutrophil surface by ADAM17, and the neutrophils express a new set of membrane proteins (integrins) by rapid exocytosis of cytoplasmic vesicles. Firm adhesion of neutrophils to endothelial cells is mediated by binding of β2 integrin molecules, such as Mac-1 (CD11a/CD18), that are expressed on stimulated neutrophils in an active conformation, to intercellular adhesion molecule-1 (ICAM-1) and other ICAM molecules on endothelial cells. P- and E-selectin adherence also contributes to the process of firm adhesion. There are four β2 integrins (lymphocyte function antigen-1 [LFA-1], Mac-1, p150,95, and αdβ2), each of which are heterodimers that differ only in their subunit CD11 a, b, c, and d for LFA-1, Mac-1, p150,95, and αdβ2, respectively. CD18 (the β-subunit) is identical in all four β2 integrins. Three β2 integrins (LFA-1, Mac-1, and p150,95) are involved with leukocyte adherence; however, the β2 integrin αdβ2, which was first identified in dogs and subsequently in humans, is apparently not meaningfully involved with the adherence of neutrophils or other leukocytes to endothelium. Once stable adherence is achieved, neutrophils move to endothelial cell junctions for transendothelial cell migration.


• Transendothelial cell migration. In postcapillary venules, neutrophil movement decreases from 10 µm/sec to a complete stop after margination. Firmly adhered leukocytes emigrate (transmigrate) across the endothelial layer by passing between endothelial cells. A number of leukocyte adhesion molecules are involved in this process (Table 3-1). Adhesion molecule activity and expression differ slightly for different tissues and cell types. In noninflamed skin, for example, there is a higher level of E- and P-selectin expression on endothelial cells, which facilitates rolling of leukocytes. In inflamed liver, CD44 of neutrophils binds serum-derived hyaluronan-associated protein (SHAP) that is bound to hyaluronic acid present on the luminal surface of endothelial cells. Nonactivated lymphocytes and monocytes utilize L-selectin to mediate adherence to high endothelial venules (HEVs) in lymph nodes. These cells also utilize α4β1 (very-late antigen-4 [VLA-4]) to mediate stable adherence to the endothelial ligand, vascular cell-adhesion molecule-1 (VCAM-1). Neutrophils and other leukocytes transmigrate between endothelial cells at the intercellular junctions. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a molecule that is present on endothelial cell membranes, and junctional adhesion molecules (JAM) A, B, and C mediate adherence activities and the adherence process. Contributions to this process also include β2 integrin binding of ICAM-1 and E-selectin binding. Pseudopodia of neutrophils and other leukocytes extend between endothelial cells and come into contact with and bind to the basement membrane (composed of laminin and collagens) and subjacent extracellular matrix (ECM) proteins (proteoglycans, fibronectin, and vitronectin). This binding interaction is mediated, at least in part, by the β1 integrins. Neutrophils that pass across the vascular wall accumulate in the perivascular connective tissue stroma within the inflammatory exudate. Once within the perivascular stroma, neutrophils migrate along a pathway established by the chemotactic gradients and inflammatory mediators.



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.



Additional Regulation of Inflammation


There are many regulatory mechanisms that closely control the onset, intensity, duration, and resolution of inflammation. In addition to those discussed, additional factors influence the type of inflammatory response by specific individuals. These include mechanisms that affect the expression level and activity of individual inflammatory genes and gene products. First, single nucleotide polymorphisms (SNPs) within the pyrin domain of the inflammasome receptor allow enhanced IL-1β production in patients with familial Mediterranean fever. SNPs in TLR are associated with altered responses to ligands and thus altered inflammatory responses. Similarly, SNP alterations in expression or regulation cytokines, chemokines, and their receptors can change the type and magnitude of inflammatory reactions. Second, premature truncation codons (PTC) are genes with stop codons within a critical location resulting in truncation of the mRNA and protein product. Thus the protein product, whether a cytokine or other inflammatory molecule, has an altered function or may even lack function. Third, gene copy number (the number of copies of a gene in a chromosome) directly affects the amount of protein produced. Some antimicrobial peptides have low gene copy numbers, therefore there is a very limited amount of peptide produced even in the presence of inflammatory stimuli. Fourth, there are latent forms of some molecules such as transforming growth factor-β (TGF-β). The latent forms are present within the tissue stroma and become functional on activation by matrix metalloproteinases. Fifth, there are isoforms of some genes that inhibit activity. VEGF-A, for example, has an isoform that when bound to the VEGF receptor-2 (VEGFR-2) reduces rather than enhances signaling. Sixth, there are soluble receptors such as soluble ICAM-1, which bind ligands but do not transmit signals because the receptor is detached from a cell.



Effector Cells of the Acute Inflammatory Response



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 (TH2) 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.

Sep 17, 2016 | Posted by in GENERAL | Comments Off on Inflammation and Healing
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