PHAGOCYTOSIS It comprises of three steps. 1. Recognition and attachment of Micro- organism to the surface of phagocyte. Engulfment. Killing and degradation of ingested par- ticle, 2. 3. ATTACHMENT AND RECOGNITION Attachment of micro-organisms is facilitated by a process in which they are coated with opsonins in serum (e.g. IgG, C3b). Cell sur- faces of the neutrophils and macrophages have receptors for these opsonins (IgG and C3b). Receptor mediated attachment of op- sonized bacteria is called Recognition. ENGULFMENT ‘The phagocyte flow partially around the bac- teria to create a deep pocket. This invagina- tion is closed by formation of microfilaments. The particle is now enclosed vesicle called Phagosome. Granules of Phagocyte fuse with phagosome and discharge their contents into it i.e, de- granulation. In cytoplasm of Neutrophil, following two types of granules are present: Azurophil granules (Lysosomes) They contain acid hydrolases, neutral proteases, proteins, mycloperoxidase and ly- sozyme., Specific granules They contain lysozyme and lactoferrin, Macrophages also have azurophil granules but macrophages do not have myeloperoxi- dase since they lose it after changing into macrophages from monocytes (which have this enzyme). (KILLING AND DEGRADATION Fate of engulfed microbe is as: Most are destroyed by phagocyte. 49 + They can destroy the leuk ON le a fast bacilli of TB. and leprosy” “® ®t + They can survive within phagooye, transport of such infected leukgc, through lymphatics can spread infection, Microbicidal mechanisms are following: Oxygen dependent mechanism There is rapid activation of a 4 zyme called "NADPH Oxidase”. It oxidian, NADPH to NADP* and H* and in the roo. reduces oxygen to superoxide 09°, In phagosome, Op” is converted into Hy0> (Oy: + Ht = Hy02 + Op). 2" and Hy0y both are weak microbicidal, In the presence of myeloperoxide, HO) reac with CI° to form hypochlorite. This is power. ful microbicidal. Oxygen independent —_mechanism (Lysosomal agents) a) Phagocytic (protein). b) Enzymes such as lysozyme, elastase and lactoferrin. Lysosomal enzymes have 2 actions: a) Direct killing of bacteria. b) Digestion and degradation of killed m= cro-organism. ' Phagocytosis means ingestion of small part cles such as bacteria, protozoal parasites of other microorganims, tissue cells (usually dead), dust, pigment and other foreign male rial by phagocytes. The phagocytes f° microphages and macrophages. The pros is as follows: If the particles are large such as clumps rd bacteria, tissue cells or a large single bale the cytoplasm of the phagocyte flows One oF more processes or pscudoposia : he surround the particle and draw it withll © body of the cell, A vacuole or of th? formed which appears in the cytoplas™ Dhagocytes, a— cles are smaller suck as ferritin or If the Par molecules, these : ce taken large Peis and in the process smal pinoy the union of these small vesicles a ferge vacuole is formed which is called phagosome sre vacuole oF phagosome now come in con- thet with lysosome. The lysosomes are rich in ide variety of catalytic enzymes as well as in antibacterial substances such as hydrogen peroxide, basic peptides and a basic protein in), These are released and act on the foreign body. If the material is digestible, ‘and neutrophils soon digest the fnaterial, Dead bacteria are digested rapidly nd their degradation products such as nu- ‘leic acids, fats and proteins appear in the cy- toplasm of phagocyte within. two hours and these materials are utilized as a source of en- ergy. If neutrophils or macrophages are un- able to kill the microorganism, the microor- ganism e.g. tubercle bacillus survive happily within the phagocyte. The persistence of mi- croorganism within phagocytes poses a prob- Jem in the eradication of such infection as tu- berculosis. Thus enclosed, the microorganism is protected against the action of antibacterial agents as well as other defense mechanism. When these phagocytic cells migrate through lymphatic pathways, infections such as tuber- culosis may spread. A type of phagocytosis is surface phagocyto- sis. In free fluid a phagocyte is unable to en- gulf encapsulated microorganisms unless they are sensitized by serum; it merely pushes the organism ahead of it. The organism is en- trapped between some barrier and then Phagocytosis readily occurs. For this reason fibrin strands in an effective support to the Phagocytic process. This phenomenon is es- Pecially important in the lungs where the large alveolar spaces permit bacteria to escape the leukocytes until they become enmeshed in the fibrin coagulum. Bacteria very in their susceptibility to phago- cytosis. General the more virulent the organ- ism, the more likely will it resist phagocyto- sis, Indeed the virulence is often related to the presence of outer polysaccharide capsules which in some way hamper oreven block phagocytosis. Phagocytosis is an energy dependent process and the energy is derived from glycolytic pathway. Many factors affect the phagocytosis which are given below: CONDITIONS AFFECTING PHAGOCYTOSIS There are many factors which affect phagocy- tosis. ‘xogenous Factors i. Certain serum proteins promote phagocy- tosis through their adsorption on the sur- face of particles such as bacteria. These proteins are beta-globulins, alpha- globulins, protamines, globins, lysozymes ‘and some other basic proteins because surfaces of bacteria have free acid radi- cals. ii, Some bacteria can be phagocytosed in the absence of serum, others must be brought in contact with proteins which are viru- lent especially if encapsulated, can only be ingested in the presence of specifically immune serum. The antibodies them- selves do not have bactericidal action but they serve as poisons. iii, Also mechanical conditions available, fi- brin threads or any other anchoring material, if available makes even virulent micro-organisms ingestible without the help of specific immune serum. iv. pH Range. : vy. Temperature Range: Phagocytosis is more at high temperatures. vi. Opsonins: These coat bacteria and make them more prone. to phagocytosis. These are found in serum. vii. Calcium promotes the process.viii.Presence of oxygen promotes the act. Endogenous Factors i. More the number of particles engulfed by the phagocyte, greater the activity of en- gulfing further particle. This is called ad- aptation response. ii. Presence of enzymes e.g. lysozymes, HO etc. help in destruction and diges- tion of the particles. They also help in the 51 iii. iv. ——s release of phagocytin which Promotes phagocytosis. Nucleic acid break down products. Various stimuli from outside in the form of pyrogens which lead to injury of leu- kocytes leading to endogenous pyrogens which in turn leads to stimulation of theromoregulatory centre in the brain. This cause fever leading to phagocytosis promotion and bacterial death on account of increased temperature.Les Tolbte | Feceptor Recognition = °f microbes, Mediators —_ Cellular Cytosk ‘eletal changes, response Signal transduction Production of Production of reactive Phagocytosis (eee ‘=YGSR speces (ROS); meen a acid metabolites, °SMAlereymes ” —ohagosone cytokines) __ Increased Chemotaxis integrin avidi i Functional ity Microbicidal activity of ukocytes Adhesion to Migration Amplification of the Killing of microbes endothelium into tissues inflammatory reaction Fig. 3.6 Leukocyte activation. Various types of leukocyte cell responses that mediate leukocyte functions. Only some receptors shown). IFN-y, Interferon- LPS, lipopolysaccharide Surface receptors recognize different agonists. Once stimulated, the receptors inte are depicted (see text for details) LPS first binds to a circulating LPS-bndng protein (ot(A. 1. RECOGNITION AND ATTACHMENT ‘Microbes bind to phagocyte receptors Phagocytic“ receptor Phagosome with ingested microbe }>.0.—> [OCF ‘Membrane { Fert OFF ‘Membrane B PHAGOCYTIC VACUOLE _ - Degradation of microbes: by lysosomal enzymes: Im phagolysosome 3. KILLING AND DEGRADATION Fig.3.7 Phagocytosis and intracellular destruction of microbes. (A) Phagocytosis ofa particle (eg. bacterium) involves binding to receptors on che leukocyte ‘membrane, engufment, and fusion of the phagocytic vacuoles with lysosomes. This is followed by destruction of ingested particles within the phagolysosomes by lysosomal enzymes and by reactive oxygen and nitrogen species. (B) In activated phagocytes, cytoplasmic components of the phagocyte oxidase enzyme assemble in the membrane of the phagorome to form the active enzyme, which catalyzes the conversion of oxygen into superoxide (O; ) and HO, Myelo- peroxidase, present in the granules of neutrophil, converts H;O; to hypochlorite. (C) Microbicidl reactive oxygen species (ROS) and neric oxide (NO) kill ingested microbes. During phagocytosis, granule contents may be released into extracellular tissues (not showa).iNOS, Inducible NO synthase: MPO, myelo~ peroxidase: ROS, reactive oxygen species, intracellular killing. Several other responses aid in the defensive functions of inflammation and may contribute to its injurious consequences, Phagocytosis Phagocytosis involves three sequential steps: (1) recogni- tion and attachment of the particle to be ingested by the leukocyte; (2) engulfment, with subsequent formation of a phagocytic vacuole; and (3) killing or degradation of the ingested material (Fig. 3.7). These steps are triggered by activation of phagocytes by microbes, necrotic debris, and various mediators. Recognition by Phagocytic Receptors. Mannose receptors, scavenger receptors, and receptorsfor variousopsoninsbind and ingest microbes. The macrophage mannose receptor is a lectin that binds terminal mannose and fucose residues of glycoproteins and glycolipids. These sugars are typically part of molecules found on microbial cell walls, whereas mammalian glycoproteins and glycolipids contain termi- nal sialic acid or N-acetylgalactosamine. Therefore, the mannose receptor recognizes microbes and not host cells. Scavenger receptors bind and ingest low-density lipopro- tein (LDL) particles as well as a variety of microbes. The effi- ancy of phagocytosis is greatly enhanced when microbes are opsonized (coated) by specific proteins (opsonins) for which the phagocytes express high-affinity receptors. ‘The major opsonins are immunoglobulin (Ig)G antibod- ies, the C3b breakdown product of complement activa- tion, and certain plasma lectins, notably mannose-binding lectin, all of which are recognized by specific receptors on leukocytes, Engulfinent. After a particle is bound to phagocyte recep- tors, extensions of the cytoplasm (pseudopods) flow around it, and the plasma membrane pinches off to form a cytosolic vesicle (phagosome) that encloses the particle. ‘The phagosome then fuses with lysosomes, resulting in the discharge of lysosomal contents into the phagolysosome (Fig. 3.7). During this process the phagocyte also may release some granule contents into the extracellular space, thereby damaging innocent bystander normal cells,The killi i ng of microbes and the destruction of ingested materials : (Ros, alao ead eked by reactive oxygen species nitrogen species, oa oxygen intermediates), reactive and lysosomal en: ainly derived from nitric oxide (NO), inte cee (Fig. 3.7). This is the final step The Kling sah of infectious agents and necrotic cells. of dead-cel Ae eaestn of microbes and elimination occur most effici is within neutrophils and macrophages pact ciently after their activation. All these killing which pha are normally sequestered in lysosomes, to tially ete teed materials are brought. Thus, poten- es nful substances are segregated from the cell’s (Hoplasm and nucleus to avoid damage to the phagocyte ile it is performing its normal function. Reactive Oxygen Species. ROSare produced by the rapid assembly and activation of a multicomponent enzyme, Phagocyte oxidase (also called NADPH oxidase), which oxidizes NADPH (reduced nicotinamide-adenine dinu- cleotide phosphate) and, in the process, reduces oxygen to the superoxide anion (O3) (Fig. 3.7B). In neutrophils, this oxidative reaction is tightly linked to phagocytosis, and is called the respiratory burst. Phagocyte oxidase is an enzyme complex consisting of at least seven proteins. In resting neutrophils, different components of the enzyme are located in the plasma membrane and the cytoplasm. In response to activating stimuli, the cytosolic protein com- ponents translocate to the phagosomal membrane, where they assemble and form the functional enzyme complex. Thus, the ROS are produced within the phagolysosome, where they can act on ingested particles without damag- ing the host cell. O} so produced is then converted into hydrogen peroxide (H:0;), mostly by spontaneous dis- mutation, a process of simultaneous oxidation and reduc- tion, H,Or is not able to kill microbes efficiently by itself. However, the azurophilic granules of neutrophils contain the enzyme myeloperoxidase (MPO), which, in the presence of a halide such as Cl-, converts HO; to hypochlorite (OCIs , the active ingredient in household bleach). The latter is a potent anti-microbial agent that destroys microbes by halo- sgenation (in which the halide is bound covalently to cellul: constituents) or by oxidation of proteins and lipids (lipi peroxidation). The H,O.-MPO-halide system is the most Fificient bactericidal system of neutrophils. Nevertheless, inherited deficiency of MPO only causes a modest increase in susceptibility to infection, emphasizing the redundancy of microbicidal mechanisms in leukocytes. H,0, also is converted to hydroxyl radical (OH*), another powerful destructive agent. As discussed in Chapter 2, these oxygen- derived free radicals bind to and modify cellular lipids, proteins, and nucleic acids, and thus destroy cells such as microbes. Oxygen-derived radicals may be released extracellu- larly from leukocytes after exposure to microbes, chemo- kines, and antigen-antibody complexes, or following a phagocytic challenge. These ROS are implicated in tissue damage accompanying inflammation. Serum, tissue fluids, and host cells possess anti-oxidant mechanisms that protect against these potentially harmful wen-derived radicals. These anti-oxidants are discussed > they include (1) the enzyme superoxide activated ina oxifies H,Oy and rful HO; detersifies dismutase, which is found in or can be of cell types; (2) catalase, which det glutathione peroxidase, another powe' The role of oxygen-derived free radicals in any given faction depends on the balance between inflammatory re ation of these metabolites by cell production and inactivi and tissues. Genetic defects in the of an immunodeficiency disease call tous disease, described in Chapter 5. Nitric Oxide, NO, a soluble gas produced from arginine by the action of nitric oxide synthase (NOS), also partic. ‘ates in microbial killing. There are three different types of NOS: endothelial (¢NOS), neuronal (nNOS), and inducible (iNOS). eNOS and nNOS are constitutively expressed at low levels, and the NO they generate acts to maintain vas- cular tone and as a neurotransmitter, respectively. iNOS, the type that is involved in microbial killing, is expressed when macrophages are activated by cytokines (e.g., IFN-y or microbial products, and induces the production of NO. In macrophages, NO reacts with superoxide (03) to gener. ate the highly reactive free radical peroxynitrite (ONOO’) (Fig. 37C). These nitrogen-derived free radicals, similar to ROS, attack and damage the lipids, proteins, and nucleic acids of microbes and host cells. In addition to its role as a microbicidal substance, NO produced by endothelial cells relaxes vascular smooth muscle and promotes vasodilation. It is not clear if this action of NO plays an important role in the vascular reac- tions of acute inflammation. Granule Enzymes and Other Proteins. Neutrophils and monocytes contain granules packed with enzymes and anti-microbial proteins that degrade microbes and dead tissues and may contribute to tissue damage. These gran- ules are actively secretory and thus distinct from classical lysosomes. Neutrophils have two main types of granules. The smaller specific (or secondary) granules contain lyso- zyme, collagenase, gelatinase, lactoferrin, plasminogen activator, histaminase, and alkaline phosphatase. The larger azterophil (or primary) granules contain MPO, bacte- ricidal factors (such as defensins), acid hydrolases, and a variety of neutral proteases (elastase, cathepsin G, nonspe- cific collagenases, proteinase 3). Phagocytic vesicles con- taining engulfed material may fuse with these granules (and with lysosomes, as described earlier), and the ingested materials are destroyed. In addition, both types of granules also undergo exocytosis (degranulation), leading to the extracellular release of granule contents Different granule enzymes serve different functions. Acid proteases degrade bacteria and debris within phagoly- sosomes, which are acidified by membrane-bound proton pumps. Neutral proteases are capable of degrading various extracellular components, such as collagen, basement membrane, fibrin, elastin, and cartilage, resulting in the tissue destruction that accompanies inflammatory pro- cesses, Neutrophil elastase combats infections by degrad- ing virulence factors of bacteria. Macrophages also contain acid hydrolases, collagenase, elastase, phospholipase, and generation of ROS are the cause led chronic granuloma plasminogen activator. Because of the destructive effects of granule enzymes, the initial leukocytic infiltration, if unchecked, can potenti- ate further inflammation by damaging tissues. Thesethe neutrophils Sometimes called NETosi 8, which includes hist A, may be a source of nuclear autoimmune diseases, Viduals react against (Chapter 5) ‘ones and associated antigens in systemic particularly lupus, in which indi- heir own DNA and nucleoproteins ae