Inflammation is the body’s attempt at self-protection; the aim being to remove harmful stimuli, including damaged cells, irritants, or pathogens – and begins the healing process.
Causes: Burns, chemical irritants, frostbite, toxins, infection by pathogens. Physical injury, blunt or penetrating, immune reactions due to hypersensitivity, ionizing radiation, foreign bodies, including splinters, dirt, and debris, stress, trauma, alcohol.
Cardinal signs: Dolor (pain), Calor (heat). Rubor (redness), Tumor (swelling), Functio – laesa (loss of function).
Acute inflammation is a rapid response to an injurious agent that serves to deliver mediators of host defence – leukocytes and plasma proteins – to the site of injury.
Acute inflammation has three major components
1. Alterations in vascular caliber that lead to an increase in blood flow,
2. Structural changes in the microvasculature that permit plasma proteins and leukocytes to leave the circulation,
3. Migration of the leukocytes from the microcirculation, their accumulation in the focus of injury, and their activation to eliminate the offending agent.
Certain terms must be defined before specific features of inflammation are described. The escape of fluid, proteins, and blood cells from the vascular system into the interstitial tissue or body cavities is known as exudation. An exudate is an inflammatory extra-vascular fluid that has a high protein concentration, cellular debris, and a specific gravity above 1. 020. It implies significant alteration in the normal permeability of small blood vessels in the area of injury.
In contrast, a transudate is a fluid with low protein content (most of which is albumin) and a specific gravity of less than 1. 012. It is essentially an ultra-filtrate of blood plasma that results from osmotic or hydrostatic imbalance across the vessel wall without an increase in vascular permeability. Edema denotes an excess of fluid in the interstitial or serous cavities; it can either an exudate or a transudate. Pus, a purulent exudate, is an inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of dead cells and in many cases, microbes.
Stimuli for acute inflammation
Acute inflammatory reactions are triggered by a variety of stimuli:
1. Infections (bacterial, viral, parasitic) and microbial toxins
2. Trauma (blunt and penetrating)
3. Physical and chemical agents (thermal injury, e.g., Burns or frostbite irradiation; some environmental chemicals)
4. Tissue necrosis (from any cause)
5. Foreign bodies (splinters, dirt, sutures)
6. Immune reactions (also called hypersensitivity reactions)
Each of these stimuli may induce reactions with some distinctive feature, our inflammatory reactions share the same basic features. We first describe the characteristic reactions of acute inflammation, and then the chemical mediators responsible for these reactions.
Since the two major mechanisms of host defence against microbes-antibodies and leukocytes are – normally carried in the bloodstream, it is not surprising that vascular phenomena play a major role in acute inflammation, normally, plasma proteins and circulating cells are sequestered inside the vessels and move in the direction of flow. In inflammation, blood vessels undergo a series of changes that are designed to maximize the movement of plasma proteins and circulating cells out of the circulation and into the site of injury or infection.
Changes in Vascular Flow and Caliber: – Changes in vascular flow and caliber begin early after injury and develop at varying rates depending on the severity of the injury. The changes occur in the following order:
1. Vasodilation is one of the earliest manifestations of acute inflammation; sometimes, it follows a transient constriction of arterioles, lasting a few seconds. Vasodilation first involves the arterioles and then results in opening of new capillary beds in the area. Thus comes about increased blood flow, which is the cause of the heat and the redness. Vasodilation is induced by the action of several mediators, notably histamine and nitric oxide, on vascular smooth muscle; these mediators are described later in the chapter,
2. Vasodilation is quickly followed by increased permeability of the micro-vasculature, with the outpouring of protein – rich fluid into the extra-vascular tissues: this; process is described in detail below.
3. The loss of fluid results in concentration of red cells in small vessels and increased viscosity of the blood, reflected by the presence of dilated small Vessels packed with red cells and slower blood flow, a condition termed stasis. With mila stimuli, stasis may not become apparent until 15 to 30 minutes have elapsed, whereas with severe injury, stasis may occur in a few minutes.
4. As stasis develops, leukocytes, principally neutrophils, accumulate along the vascular endothelium. Leukocytes then stick to the endothelium, and soon afterward they migrate through the vascular wall into the interstitial tissue, in processes that are described later.
A hallmark of acute inflammation is increased vascular permeability leading to the escape of a protein – rich fluid (exudate) into the extra-vascular tissue. The loss of protein from the plasma reduces the intravascular osmotic pressure and increases the osmotic pressureof the interstitial fluid. Together with the increased hydrostatic pressure owing of fluid and it`s accumulation in the interstitial tissues. The net increase of extravascular fluid results in edema.
Normal fluid exchange and micro-vascular permeability are critically dependent on an intact endothelium. How then does the endothelium become leaky in inflammation? The following mechanisms have been proposed:
1. Formation of endothelial gaps in venules: – This is the most common mechanism of vascular leakage and is elicited by histamine, bradykinin, leukotrienes, the neuropeptide substance P, and many other classes of chemical mediators. It occurs rapidly after exposure to the mediator and is usually reversible and short – lived (15 to 30 minutes); it is thus known as the immediate transient response.
Classically, this type of leakage affects venules 20 to 60 µm in diameter, leaving capillaries and arterioles unaffected. The precise reason for this restriction to venules is uncertain: it may be because there is a greater density of receptors for the mediators in venular endothelium. Parenthetically, many of the later leukocyte events in inflammation adhesion and emigration also occur predominantly in the venules in most organs.
Binding of mediators, such as histamine to their receptors on endothelial cells activates intracellular signaling pathways that lead to phosphorylation of contractile and cytoskeletal proteins, such as myosin. These proteins contract, leading to contraction of the endothelial cells and separation of intercellular junctions.
Thus, the gaps in the venular endothelium are largely intercellular or close to the intercellular junctions, Cytokines such as interleukin – 1 (IL – 1), tumor necrosis factor (TNF), and interferon – Y (IFN – 7) also increase vascular permeability by inducing a structural reorganization of the cytoskeleton, such that the endothelial cells retract from one another. In contrast to the histamine effect, the cytokine – induced response is somewhat delayed (4 to 6 hours) and long – lived (24 hours or more),.
2. Direct endothelial injury, resulting in endothelial cell necrosis and detachment: – This effect is usually encountered in necrotizing injuries and is due to direct damage to the endothelium by the injurious stimulus. as, for example, in severe burns or lytic bacterial infections. Neutrophils that adhere to the endothelium (discussed below) May also injure the endothelial cells. In most instances, leakage starts immediately after injury and is sustained at a high level for several hours until the damaged vessels are thrombosed or repaired. The reaction is known as the immediate sustained rasnonse. All levels of the microcirculation are affected, including venules, capillaries and arterioles. Endothelial cell detachment is often associated with platelet adhesion and thrombosis.
3. Delayed prolonged leakage: – This is a curious but relatively common tvpe of increased permeability that begins after a delay of 2 to 12 hours, lasts for several hours or even days, and involves venules as well as capillaries. Such leakage is for example, by mild to moderate thermal injury, X – radiation or ultraviolet and certain bacterial toxins. Late – appearing sunburn is a good example of a delayed reaction. The mechanism of such leakage is unclear. It may result from the direct effect of the injurious agent, leading to delayed endothelial cell damage (perhaps by apoptosis), or the effect of cytokines causing endothelial retraction, as described earlier.
4. Leukocyte-mediated endothelial injury: – Leukocytes adhere to endothelium relatively early in inflammation. As discussed later, such leukocytes may be activated in the process, releasing toxic oxygen species and proteolytic enzymes, which then cause endothelial injury or detachment, resulting in increased permeability. In acute inflammation, this form of injury is largely restricted to vascular sites, such as venules and pulmonary and glomerular capillaries, where leukocytes adhere for prolonged periods to the endothelium.
5. Increased transcytosis across the endothelial cytoplasm: – Transcytosis occurs across channels consisting of clusters of interconnected, uncoated vesicles and vacuoles called the vesiculo-vacuolar organelle, many of which are located close to in intercellular junctions. Certain factors, for example, vascular endothelial growth factor (VEGF), appear to cause vascular leakage by increasing the number and perhaps the size of these channels. It has been claimed that this is also a mechanism of increased permeability induced by histamine and most chemical mediators.
6. Leakage from new blood vessels: – During repair, endothelial cells proliferate and form new blood vessels, a process called angiogenesis. New vessel sprouts remain leaky until the endothelial cells mature and form intercellular junctions. In addition, Certain factors that cause angiogenesis (e. g. VEGF) also increase vascular permeability, and endothelial cells in foci of angiogenesis have increased density of receptors for vasoactive mediators, including histamine, substance P, and VEGE. All these factors account for the edema that is characteristic of the early stages of healing that follow inflammation.
Note: In summary, in acute inflammation, fluid loss from vessels with increased permeability occurs in distinct phases:
1. An immediate transient response lasting for 30 minutes or less, mediated mainly by the actions of histamine and leukotrienes on endothelium;
2. A delayed response starting at about 2 hours and lasting for about 8 hours, mediated by kinins, complement products, and other factors; and
3. A prolonged response that is most noticeable after direct endothelial injury, for example, after burns.
CELLULAR EVENTS: LEUKOCYTE
A critical function of inflammation is to deliver leukocytes to the site of injury and to activate the leukocytes to perform their normal functions in host defence. Leukocytes ingest offending agents, kill bacteria and other microbes, and get rid of necrotic tissue and foreign substances. A price that is paid for the defensive potency or leukocytes is that they may induce tissue damage and prolong inflammation, since the products that destroy microbes and necrotic tissues can also injure normal, host tissues.
The sequence of events in the journey of leukocytes from the vessel lumen to the interstitial tissue, called extravasation, can be divided into the following steps: –
1. In the lumen: margination, rolling, and adhesion to endothelium. Vascular endothelium normally does not bind circulating cells or impede their passage. In inflammation, the endothelium has to be activated to permit it to bind leukocytes, as a prelude to their exit from the blood vessels.
2. Transmigration across the endothelium (also called diapedesis).
3. Migration in interstitial tissues toward a chemotactic stimulus.
In normally lowing blood in venules, erythrocytes are confined to a central axial column, displacing the leukocytes towards the wall of the vessel. Because blood flow slows early in inflammation (stasis), hemodynamic conditions changes (wall shear stress decreases), and more white cells assume a peripheral position along the endothelial surface. This process of leukocyte accumulation is called margination. Subsequently, individual and then rows of leukocytes tumble slowly along the endothelium and adhere transiently (a process called rolling), finally coming to rest at some point where they adhere firmly (resembling pebbles over which a stream runs without disturbing them).
In time, the endothelium can be virtually lined by white cells, an appearance called pavementing. After firm adhesion, leukocytes insert pseudopods into the junctions between the endothelial cells, squeeze through inter-endothelial junctions, and assume a position between the endothelial cell and the basement membrane. Attacking, they traverse the basement membrane and escape into the extra-vascular space. Neutrophils, monocytes, lymphocytes, eosinophils, and Basophils all use the same pathway to migrate from the blood into tissues. We now examine the molecular mechanisms of each of the steps.
Leukocyte adhesion and transmigration
Leukocyte adhesion and transmigration are regulated largely by the binding of complementary adhesion molecules on the leukocyte and endothelial surfaces and chemical mediators – chemoattractants and certain cytokines – affecting these processes by modulating the surface expression or avidity of such adhesion molecules. The adhesion receptors involved belong to four molecular families the selectins, the immunoglobulin superfamily, the integrins, and mucin – like glycoproteins. The most important of these are listed:
1. P – selectin,
2. E – selectin,
3. ICAM – 1
4. VCAM – 1
5. GlyCam – 1
6. CD31 (PECAM)
(ICAM-1, VCAM-1, and CD31 belong to the immunoglobulin family of proteins; PSGL-1, P-Selectin gheoprotein ligandi 1.)
Selectins, so called because they are characterized by an extracellular N-terminal domain related to sugar – binding mammalian lectins, consist of E – selectin (CD62E, previously known as ELAM – 1), which is confined to endothelium: P selectin (CD62P, previously called GMP140 or PADGEM), which is present in endothelium and platelets; and L – selectin (CD62L, previously known by many names, in cluding LAM – 1), which is expressed on most leukocyte types. Selectins bind, through their lectin domain, to sialylated forms of oligosaccharides (e.g. sialylated Lewis X), which themselves are covalently bound to various mucin – like glycoproteins (GlyCAM – 1, PSGL – I, ESL – 1, and CD34).
The immunoglobulin family molecules include two endothelial adhesion molecules: ICAM – I (intercellular adhesion molecule 1) and VCAM – 1 (vascular cell adhesion molecule 1). Both these molecules serve as ligands for integrins found on leukocytes.
Integrins are transmembrane heterodimeric glycoproteins, made up of α and β chains,those are expressed on many cell types and bind to ligands on endothelial cells. Other leukocytes, and the extracellular matrix. The β2 integrins LFA – 1 and Mac – 1 (CD11a/CD18 and CD11b/CD18) bind to ICAM – 1, and the β1 integrins (such as VLA – 4) bind VCAM – 1.
Mucin – like glycoproteins, such as heparin sulfate, serve as ligands for the leukocyte adhesion molecule called CD44. These glycoproteins are found in the extracellular matrix and on cell surfaces.
The recruitment of leukocytes to sites of injury and infection is a multistep process involving attachment of circulating leukocytes to endothelial cells and their migration through the endothelium. The first events are the induction of adhesion molecules on endothelial cells, by a number of mechanisms. Mediators such as histamine, thrombin, and platelet activating factor (PAF) stimulate the redistribution of P selectin from its normal intracellular stores in granules (Weibel-Palade bodies) to the cell surface.
Resident tissue macrophages, mast cells, and endothelial cells respond to injurious agents by secreting the cytokines TNF, IL-1, and chemokines (chemoattractant cytokines). TNF and IL-1 act on the endothelial cells of post-capillary venules adjacent to the infection and induce the expression of several adhesion molecules. Within 1 to 2 hours, the endothelial cells begin to express E-selectin. Leukocytes express at the tips of their microvilli carbohydrate ligands for the selectins, which bind to the endothelial selectins. These are low-affinity interactions with a fast off-rate, and they are easily disrupted by the flowing blood. As a result, the bound leukocytes detach and bind again, and thus begin to roll along the endothelial surface.
TNF and IL-1 also induce endothelial expression of ligands for integrins, mainly VCAM-1 (the ligand for the VLA-4 integrin) and ICAM-1 (the ligand for the LFA-1 and Mac-1 integrins). Leukocytes normally express these integrins in a low affinity state. Meanwhile, chemokines that were produced at the site of injury enter the blood vessel, bind to endothelial cell heparan sulfate glycosaminoglycans, and are displayed at high concentrations on the endothelial surface. These chemokines act on the rolling leukocytes and activate the leukocytes.
One of the consequences of activation is the conversion of VLA-4 and LFA-1 integrins on the leukocytes to a high affinity state. The combination of induced expression of integrin ligands on the endothelium and activation of integrins on the leukocytes results in firm integrin-mediated binding of the leukocytes to the endothelium at the site of infection. The leukocytes rolling their cytoskeleton is reorganized, and they spread out on the endothelial surface.
The next step in the process is migration of the leukocytes through endothelium, called transmigration or diapedesis. Chemokines act on the adherent leukocytes and stimulate the cells to migrate through interendothelial space towards the chemical concentration gradient, that is, toward the site of injury or infection. Certain homophilic adhesion molecules (i.e., adhesion molecules that bind to each other) present in the intercellular junction of endothelium are involved in the migration of leukocytes.
One of these molecules is a member of the immunoglobulin superfamily called PECAM – 1 (platelet endothelial cell adhesion molecule) or CD31. Leukocyte diapedesis, similar to increased vascular permeability, occurs predominantly in the venule (except in the lungs, where it also occurs in capillaries). After traversing the endothelium, leukocytes are transiently retarded in their journey by the continuous basement membrane of the venules, but eventually the cells pierce the basement membrane, probably by secreting collagenases. The net result of this process is that leukocytes rapidly accumulate where they are needed.
Once leukocytes enter the extravascular connective tissue, they are able to adhere to the extracellular matrix by virtue of B1 integrins and CD44 binding to matrix proteins. Thus, the leukocytes are retained at the site where they are needed. The most telling proof of the importance of adhesion molecules is the existense of genetic deficiencies in the leukocyte adhesion proteins, which result in impaired leukocyte adhesion and recurrent bacterial infections.
In leukocyte adhesion deficiency type 1 (LAD1), patients have a defect in the biosynthesis of the β2 chain shared by the LFA-1 and Mac-1 integrins. Leukocyte adhesion deficiency type 2 (LAD2) is caused by the absence of sialyl-Lewis X, the fucose-containing ligand for E-selectin, owing to a defect in a fucosyl transferase, the enzyme that attaches fucose moieties to protein backbones. In addition, antibodies to adhesion molecules abrogate leukocyte extravasation in experimental models of acute inflammation, and gene knockout mice deficient in these molecules show defects in leukocyte adhesion and extravasation.
The type of emigrating leukocyte varies with the age of the inflammatory response and with the type of stimulus. In most forms of acute inflammation, neutrophils predominate in the inflammatory infiltrate during the first 6 to 24 hours, and then are replaced by monocytes in 24 to 48 hours.
Several reasons account for this sequence-neutrophils are more numerous in the blood, they respond more rapidly to chemokines, and they may attach more firmly to the adhesion molecules that are rapidly induced on endothelial cells, such as P- and E-selectins. In addition, after entering tissues, neutrophils are short-lived; they undergo apoptosis and disappear after 24 to 48 hours, whereas monocytes survive longer. There are exceptions to this pattern of cellular exudation, however. In certain infections for example, those produced by Pseudomonas organisms-neutrophils predominate over 2 to 4 days; in viral infections, lymphocytes may be the first cells to arrive: in some hypersensitivity reactions, eosinophilic granulocytes may be the main cell type.
After extravasation, leukocytes emigrate in tissues toward the site of injury, by a process called chemotaxis, defined most simply as locomotion oriented along a chemical gradient. All granulocytes, monocytes and, to a lesser extent, lymphocytes respond to chemotactic stimuli with varying rates of speed. Both exogenous and endogenous substances can act as chemoattractants.
The most common exogenous agents are bacterial products. Some of these are peptides that possess an N-formyl–methionine terminal amino acid. Others are lipid in nature. Endogenous chemoattractants which pre detailed later, include several chemical mediators:
1. Components of the complement system, particularly Csa;
2. Products of the lipoxygenase pathway, mainly leukotriene B4 (LTB4); and
3. Cytokines, particularly those of the chemokine family (e.g., IL-8).
How does the leukocyte sense the chemotactic agents, and how do these substances induce directed cell movement?
All the chemotactic agents mentioned above bind to specific seven transmembrane G-protein-coupled receptors (GPCRs) on the surface of leukocytes. Signals initiated from these receptors result in recruitment of G-proteins and activation of several effector molecules, including phospholipase C (PLC) and phosphoinositol-3 kinase (PI3K), as well as protein tyrosine kinases. PLAy and PI3K act on membrane inositol phospholipids to generate lipid second messengers that increase cytosolic calcium and activate small GTPases as well as numerous kinases.
The GTPase induce polymerization of actin, resulting in increased amounts of polymerized actin at the leading edge of the cell. The leukocyte moves by extending filopodia that pull the back of the cell in the direction of extension. Actin reorganization may also occur at the trailing edge of the cell. Locomotion involves rapid assembly of actin monomers into linear polymers at the filopodium’s leading edge, followed by cross-linking of filaments, and disassembly of such filaments away from the leading edge. A number of actin-regulating proteins, such as filamin, gelsolin, profilin, and calmodulin, interact with actin and myosin in the filopodium to produce contraction.
Microbes, products of necrotic cells, antigen-antibody complexes, and cytokines, including chemotactic factors, induce a number of responses in leukocytes that are part of the defensive functions of the leukocytes (neutrophils and monocytes/macrophages) and are referred to under the rubric of leukocyte activation. Activation results from several signaling pathways that are triggered in leukocytes, resulting in increases in cytosolic Ca2+ and activation of enzymes such as protein kinase C and phospholipase A2.
The functional responses that are induced on leukocyte activation include the following:
a) Production of arachidonic acid metabolites from phospholipids, as a result of activation of phospholipase A2 by increased intracellular calcium and other signals.
b) Degranulation and secretion of lysosomal enzymes and activation of theoxidative burst (discussed below under phagocytosis).
c) Secretion of cytokines, which amplify and regulate inflammatory reactions. Activated macrophages are the chief source of the cytokines that are involved in inflammation, but mast cells and other leukocytes may contribute.
d) Modulation of leukocyte adhesion molecules. As stated earlier, different cytokines cause increased endothelial expression of adhesion molecules and increased avidity of leukocyte integrins, allowing firm adhesion of activated neutrophils to endothelium.
Leukocytes express a number of surface receptors that are involved in their activation
1. Toll-like receptors (TLRS), which are homologous to a Drosophila protein called Toll, function to activate leukocytes in response to different types and componets of microbes.
2. Different seven-transmembrane G-protein-coupled receptors recognize microbes and some mediators that are produced in response to infections and tissue injury.
3. Phagocytes express receptors for cytokines that are produced during immune-responses.
4. Receptors for opsonins promote phagocytosis of microbes coated with various proteins and deliver signals that activate the phagocytes.
Phagocytosis and the release of enzymes by neutrophils and macrophages are responsible for eliminating the injurious agents and thus constitute two of the major benefits derived from the accumulation of leukocytes at the inflammatory focus. Phagocytosis involves three distinct but interrelated steps:
a) Recognition and attachment of the particle to be ingested by the leukocyte;
b) Its engulfment, with subsequent formation of a phagocytic vacuole; and
c) Killing or degradation of the ingested material.
Defects in Leukocyte Function
Leukocytes play a central role in host defense, therefore, defects in leukocyte function, genetic and acquired, lead to increased vulnerability to infections. These defects may be:
1. Defects in leukocyte adhesion
2. Defects in phagolysosome function
3. Defects in microbicidal activity
4. Bone marrow suppression,
Termination of the inflammatory response It is predictable that such a powerful system of host defense, with its inherent capacity to cause tissue damage, needs tight controls to minimize the damage. In part, inflammation declines simply because the mediators of inflammation have short half lives, are degraded after their release, and are produced in quick bursts, only as long as the stimulus persists. In addition as inflammation develops, the process also triggers a variety of stop signals that serve to actively terminate the reaction.
These active mechanisms include a switch in the production of pro-inflammatory leukotrienes to anti-inflammatory lipoxins from arachidonic acid; the liberation of an anti-inflammatory cytokine, transforming growth factor-ß (TGF-B), from macrophages and other cells; and neural impulses (cholinergic discharge) that inhibit the production of TNF in macrophages.