Inflammation I Notes PDF

Inflammation 1 Debra Hazen-Martin, PhD Office: 792-2906 Email: [email protected] Inflammation 1: Acute Inflammation, Vascular Changes and Cellular Events Outline: I. Overview of Inflammation A. Definition B. Components C. Events D. Stimuli E. Significance in disease F. Patterns 1. Acute 2. Chronic...

Inflammation 1 Debra Hazen-Martin, PhD Office: 792-2906 Email: [email protected] Inflammation 1: Acute Inflammation, Vascular Changes and Cellular Events Outline: I. Overview of Inflammation A. Definition B. Components C. Events D. Stimuli E. Significance in disease F. Patterns 1. Acute 2. Chronic II. Acute Inflammation A. Vascular Changes 1. Caliber and flow 2. Permeability B. Cellular Events 1. Margination 2. Rolling 3 Adhesion 4 Transmigration 5. Chemotaxis 6. Activation and Phagocytosis III. Defects in Leukocyte Function Suggested Reading: Robbins Basic Pathology by Kumar, Abbas and Aster, Chapter 3 (57-70) Objectives: 1) Define the steps of acute inflammation and the functional outcomes of the process. 2) Differentiate between acute and chronic inflammation. 3) List the cardinal signs of inflammation and describe the events that cause each. 4) Describe the mechanisms that result in changes in vascular flow, caliber, and permeability during inflammation. 5) Describe the steps in extravasation of leukocytes and the factors that facilitate the movement. 1 Inflammation 1 6) Define leukocyte activation and describe the factors that drive leukocyte chemotaxis. 7) Describe the steps of phagocytosis and the specific mechanisms by which microbes are killed and degraded. 8) Describe 3 genetic defects that lead to leukocyte dysfunction. Describe where the defect occurs within normal leukocyte functional pathways and the outcome for each. I. Overview: Previous lectures described the types of stimuli that cause cell injury and the cellular response to those stimuli. In this lecture we will put the injured cell back into the context of a whole tissue and look again at response to injury. Vascularized connective tissues within tissues respond to injury by a complex chain of events called inflammation. A. Definition: Inflammation is a protective response in vascularized connective tissues that is intended to eliminate the initial cause of injury and remove necrotic debris due to the injury. It is important to understand that while this response is intended to lead to repair and healing, it may initiate or extend harmful events leading to further damage. Examples of this include chronic or persistent inflammation leading to rheumatoid arthritis, acute anaphylactic response, formation of fibrous adhesions due to inflammation of the peritoneum, and many others. Types of cells/tissue? Fig CT comp (4) B. Components of inflammation: The cells and tissues that take part in the inflammatory response are those of connective tissues. They include the cells comprising blood vessels, circulating blood cells, and cells that migrate into the extracellular connective tissue compartment (leukocytes, monocytes/macrophages, and mast cells). In addition, the fibroblast, the resident cell of the connective tissue and the extracellular matrix proteins secreted by fibroblasts are involved in the response. Circulating proteins, many of which are synthesized by the liver, also play a role. 2 Inflammation 1 Fig C. General order of events: The events of inflammation, described simply, include the following - The stimulus for injury also stimulates the release of soluble chemical mediators from plasma or cells in the connective tissues. These mediators stimulate both vascular and cellular stimulate responses which are aimed at removing the initial stimulus for injury. If successful, removal of the stimulus stops further release of chemical mediators and the job is complete. stimulate D. Stimuli for Inflammation: Inflammation may be initiated by many diverse stimuli, but the basic components of all inflammatory responses are similar. E. Why study inflammation? 3 Inflammation 1 A better understanding of the components of inflammation provide the information needed to devise treatment strategies. Heal & repair Further damage AI xter(3) CI xter(3) F. Patterns of Inflammation: Two patterns of inflammation are defined and differentiated by their duration and histologic appearance. Acute inflammation is of short duration (hours to days) and is characterized by infiltration of leukocytes (usually neutrophils) and fluid and proteins from the blood (exudate). Chronic inflammation is of a longer duration (days to months) and is characterized by the infiltration of monocytes (lymphocytes and macrophages), vascular proliferation and scarring. In this slide, note the appearance of the lung with acute inflammation on the left as compared to the lung on the right with a pattern of chronic inflammation. Note the presence of neutrophils within the alveolar spaces in the acute inflammation. In chronic inflammation the most predominant cell type is the lymphocyte. Note the thickening of the alveolar wall on the right. An example of the normal lung tissue is provided in the center. The alveolar spaces are normally empty and clear while the alveolar walls are thin and delicate. 4 Inflammation 1 Types(2) vas(2) celluar(2) cardinal signs vas c(3) cm(2) vas change(3) VCM fxn II. Acute Inflammation: This type of inflammation is an early response. Leukocytes rush to an area to remove microbes and clean up necrotic debris. This process may be separated into 2 components. A vascular component involves alterations in the caliber and permeability of vessels. The cellular component involves the recruitment and activation of leukocytes to the site of injury. Vascular changes are responsible for development of 3 of the cardinal signs of inflammation: calor, rubor, and tumor. Dolor and functio laesa follow and are due to additional chemical mediators. A. Vascular Changes: Sir Thomas Lewis described simple events following a scratch to the skin, noting that a fine red line was followed by a halo of redness (flare) and then swelling (wheal). The events, known as the “triple response” were the basis for his theory that a vasoactive chemical mediator caused vasodilation and increased vascular permeability at the injury. 1. Changes in caliber and flow Following injury there is an immediate, but transient, period of vasoconstriction. This is followed by dilation of the arteriolar side of the capillary bed. The vasodilation serves to increase blood flow to an affected area, accounting for the signs of redness (erythema) and warmth. Increased flow and vasodilation in turn lead to increased permeability allowing blood proteins and fluid to leave the vessel. 5 Inflammation 1 The loss of fluid to the interstitial compartment tends to concentrate the blood cells in the vessel. Flow becomes slower (stasis). As flow slows, the leukocytes settle to the vessel wall (margination) and then migrate out of the vessel. 2. Changes in vascular permeability - What are the mechanisms responsible for increased vascular permeability that produce the distinctive loss of fluid and proteins in acute inflammation? I choose to group them into hydrostatic mechanisms and mechanisms that affect endothelial integrity. Mec(3) Normal blood flow - In this diagram normal blood flow from arteriole to capillary to venule is illustrated. Hydrostatic pressure at the arteriolar end of the capillary bed results in flow of a watery filtrate from blood plasma into the interstitial space at a rate of 14 ml/min. This fluid is largely recovered at the venular end of the capillary bed where the blood, having lost fluid, has a colloid osmotic pressure that results in movement of the interstitial fluid back into the plasma at a rate of 12 ml/min. The net loss of fluid to the interstitial space is about 2ml/min. This fluid is taken up into the lymphatic system and ultimately returned to the venous 6 Inflammation 1 circulation. Any time the net loss exceeds 2ml/min fluid will accumulate in the interstitial space and this is called edema. What happens when arteriolar hydrostatic pressure increases? During acute inflammation the arteriole dilates and more blood fills the vessel resulting in a local increase in hydrostatic pressure. This generates a greater-than-normal movement of fluid into the interstitial space from the blood plasma. Initially this fluid is an watery ultrafiltrate of the plasma which is very low in protein content - a transudate. With continued increase in Edema(2) hydrostatic pressure (and subsequent endothelial changes) proteins begin to leave the blood plasma along with fluid and the resulting interstitial fluid is called an exudate. Formation of an exudate decreases the colloid osmotic pressure of the plasma in the venule. Without adequate osmotic pressure, the interstitial fluid is not recovered and continues to collect in the interstitium accounting for the swelling and other gross changes associated with acute inflammation. Mechanisms affecting endothelial integrity Proper fluid flow between the vascular compartment and the interstitial area is very dependent on maintenance of an intact endothelial lining with normal intercellular junctions and transport properties. During acute inflammation a number of mechanisms may alter the integrity of the vascular endothelium resulting in “leakiness.” Different areas (arteriole, capillary, venule) are affected by different agents and the proposed changes have been categorized as follows: 7 Inflammation 1 Endothelial gap formation by contraction - Vasoactive mediators (histamine and others) stimulate an immediate but transient opening of endothelial intercellular junctions by receptor-mediated phosphorylation of junction-associated cytoskeletal proteins. This response in limited to post-capillary venules. Endothelial gap formation by retraction - In a delayed, but sustained response, cytokines (TNF, IL-1) stimulate reorganization of the endothelial cell cytoskeleton and cells round up drawing away from intercellular contacts. This response occurs primarily in venules. Stimulus -necrosis -apoptosis Direct endothelial injury - This type of injury involves endothelial cell death. Severe burns and infection result in an immediate and sustained response that culminates in cell death by necrosis. Thermal injury, bacterial toxins, and UV radiation (sunburn) results in a delayed but prolonged response that includes apoptotic endothelial cell death. Arterioles, capillaries and venules are all subject to this type of injury and response. Leukocyte-dependent endothelial injury - This response is secondary to adhesion and activation of leukocytes that occurs following arteriolar vasodilation. Upon activation, leukocytes will release both reactive oxygen species and proteolytic enzymes. Both result in endothelial cell injury and detachment. This is a late response that is of long duration affecting mostly venules and, in the lung, capillaries. 8 Inflammation 1 4 Increased endothelial transcytosis This mechanism for fluid transport involves the fusion of pinocytotic vesicles to form channels through the endothelial cell. It is stimulated by mediators like vascular endothelial growth factor (VEGF) and targets mainly venules. Leakage due to angiogenesis - One component of repair following inflammation involves the formation of new blood vessels by sprouting or outgrowth of existing vessels (angiogenesis). VEGF is one mediator that stimulates endothelial mitosis in this process. As new vessels form they have immature or leaky junctions and probably increased intracellular channels for transcytosis. This allows considerable fluid leakage into the interstitium until the vessels mature. B. Cellular Events: The most important event in acute inflammation involves the movement of leukocytes from the blood within vessels to the extra-vascular space (extravasation). Once in the extra-vascular space, the leukocyte goes about the task of degrading and eliminating insulting substances (microbes, foreign bodies, debris) by phagocytosis. In the process, lysosomal proteins and toxic radicals are released which may prolong inflammation and/or add to tissue damage. 9 Inflammation 1 We will first look at the 4 steps of extravasation of leukocytes (margination, rolling, adhesion, and transmigration) and then activation of the leukocyte in the extra-vascular space. Note: Cell adhesion molecules facilitate extravasation. Chemical mediators modulate expression of these adhesion molecules and play an important role in activation of the leukocyte. 1. Margination - Normal blood flow is laminar. Smaller formed elements (RBC) tend to flow in the central axis of the column while larger elements (leukocytes) are moved to the periphery. In acute inflammation, vasodilation and subsequent stasis permit extended interaction between the leukocyte and endothelial surface. Table 2. Rolling - Leukocytes normally “bounce” along the endothelial surface by forming loose, transient adhesions with those cells. The transient interactions are facilitated by a class of adhesion molecules called selectins. Selections are protein receptors located on leukocytes (L-selectin) and endothelial cells (E or P-selectin). The receptors bind specific sugars (sialylated oligosaccharides) in the glycoproteins of cell membranes of the opposite cell. Leukocytes -> L-Selectin Endothelial -> E/P-Selectin 10 Inflammation 1 Normally endothelial selectins are expressed at low levels or may be absent. During inflammation, chemical mediators will upregulate expression and/or availability of these selectins for binding. P selectin is normally found in an intracellular compartment called the Weibel-Palade body. In the presence of histamine or thrombin this compartment translocates to the surface. E-selectin synthesis may be upregulated by endothelial cells in the presence of cytokines like TNF or IL-1. Table 3. Adhesion - Firm adhesion between activated endothelial cells and leukocytes is mediated by endothelial adhesion molecules (ICAM and VCAM) and the integrins found in the membrane of leukocytes. Integrins are heterodimers of 2α and 2βsubunits with an intracytoplasmic domain that binds cytoskeleton and an extracellular domain that binds endothelial adhesion molecules. Cytokines (TNF, IL-1) increase integrin’s affinity for ICAM and VCAM by causing conformational changes in the integrin molecules. The endothelial adhesion molecules belong to the immunoglobulin superfamily. 4. Transmigration - Leukocytes primarily move through venule walls (exception is the pulmonary capillary) at the site of intercellular junctions. This process, also known as diapedesis, is mediated by PECAM-1 on both the endothelial and leukocyte membranes. 11 Inflammation 1 5. Chemotaxis and leukocyte activation- Leukocytes migrate toward the site of injury once they reach the extravascular space. Migration occurs along a gradient of chemotactic substances emitted from the site of injury. Exogenous chemotactic substances may include soluble bacterial products. Endogenous chemotactic substances include components of the complement system, by-products of arachidonic acid metabolism, and cytokines. What is the mechanism that propels and activates the leukocyte to/at the site of injury? Activation of G protein: Many chemotactic molecules bind to leukocyte 7-transmembrane receptors that are coupled to G protein. G protein is activated by the release of GDP for GTP. The active alpha subunit of G protein separates and interacts with other targets. Activation of phopholipase C: One target of active G protein is phospholipase C which hydrolyzes phophotidylinositol biphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). Both will activate leukocytes in different ways. IP3 triggers a release of intracellular calcium stores. The increase in intracellular calcium, in turn, triggers cytoskeletal reorganization required for pseudopod formation and movement. In addition the increased calcium will modulate leukocyte adhesion factors. 12 Inflammation 1 DAG activates protein kinase C and subsequent phosphorylation events result in degranulation/secretion of lysosomal enzymes and an oxidative burst. DAG also activates phospholipase A2 and in the presence of elevated calcium this enzyme metabolizes membrane phospholipids producing arachidonic acid metabolites. 6. Phagocytosis and Degranulation - The purpose of leukocyte recruitment to the site of injury is phagocytosis of debris and insulting sutstances. Phagocytosis is divided into the following 3 steps: a. Recognition and attachment of the particle for ingestion - Opsonins are proteins found in the serum that bind specific molecules on the surface of a microbe. Opsonins include IgG (Fc fragment), C3b (a fragment of complement), and collectins (carbohydrate-binding lectins). In fact binding of IgG may stimulate the complement cascade resulting in subsequent binding of C3b. We will discuss the complement cascade in the next hour. The leukocyte has specific receptors that bind these substances. b. Engulfment and formation of the phagolysome vacuole - Binding of the opsonized particle stimulates engulfment with formation of pseudopods that surround the particle and form a vacuole (phagosome). The phagosome membrane then fuses with the membrane of a lysosome and the contents of the two are mixed forming a phagolysosome. These events are mediated in response to activation of the G protein - phospholipase C - IP3 (pseudopod formation) or G protein-protein kinase C- DAG (degranulation) chain of events discussed above. 13 Inflammation 1 c. Microbe killing and degradation - An oxidative burst is stimulated by leukocyte activation and phagocytosis resulting in generation of reactive oxygen species. Activation of phagocytic oxidase and oxidation of NADPH converts oxygen to superoxide ion. Superoxide is then converted to hydrogen peroxide by spontaneous dismutation. The lysosomes of neutrophils then contribute myeloperoxidase (MPO), which in the presence of a halide Cl-, converts the hydrogen peroxide to a very lethal free radical HOCl- (hypochlorous radical). The anti-microbial activity of the free radicals is followed by microbial degradation by the lysosomal acid hydrolases. Other lysosomal enzymes that play a role even in the absence of an oxidative burst include bactericidal permeabilityincreasing protein (stimulates phospholipase degradation of membrane phospholipid), major basic protein (a cytotoxic substance in the granules of eosinophils), and defensins (peptides that form holes in bacterial membranes). III. Defects in Leukocyte Function: During upcoming blocks, you will study various diseases that may contribute to neutrophil dysfunction. Although rare, genetic and acquired defects in leukocyte function may contribute to significant clinical problems. Several genetic defects are considered below to illustrate the importance of the neutrophil and the early events in acute inflammation as part of the normal response to microbial infection. 14 Inflammation 1 A. Defects in adhesion: 1. LAD -1 is a defect where the β subunit of integrin is defective. This leads to deficiencies in adhesion and migration of the leukocytes from the vessel. In turn there is no oxidative burst or phagocytosis. Table 2. LAD -2 is a defect in fucose metabolism that leads to absence of sialyl-Lewis X. This inhibits leukocyte binding to endothelial cells selectin. So initial rolling and subsequent adhesion and migration is inhibited. B. Defect in microbicidal activity: CGD (chronic granulomatous disease) involves a deficiency in NADPH oxidase so superoxide is not formed. When the microbe is engulfed oxygen-dependent radicals are not active. In this disease, and adequate acute inflammatory response is not effective. The disease process leads to a chronic pattern of tissue inflammation with macrophages congregating around the area of microbes to form granulomas. You will learn more about granuloma formation in an upcoming lecture. 15