1. Basic Immunology University of Tabuk Faculty of Applied Medical Science Department of Medical Laboratory Technology Mr.AYMAN.S.YOUSIF MSc.Medical Microbiology & Immunology Academic Year: 1433-1434 (2012-2013)

2. Mr.AYMAN.S.YOUSIF 17-18/02/2013 Lecture 3 : part 1 Innate Immunity PHAGOCYTOSIS

3. Objectives At the end of this lecture, you should be able to: 1.Recognize the significance of the phagocytosis and inflammation in combating infection and disease. 2.Define the phagocytosis. 3.know the types and characteristics of phagocytic cells. 4.Understanding the mechanism of action of phagocytosis. 5.know the function of inflammation. 6.Explain the different stages of inflammation and inflammatory response. 1

4. Phagocytosis  Is process by which particular substances, such as bacteria, are engulf by a cell and destroyed.  Phagocytosis is one form of endocytosis (internalise solid matter).The other form - pinocytosis – is the internalization of fluids and solutes.  Phagocytosis requires : 1. Energy generated through glucose metabolism. 2. Synthesis of a new cell membrane. 3. An active cytoplasmic contractile protein system. 2

5. Types of phagocytic cells  Most cells are capable of phagocytosis.  The professional phagocytes of the immune system, including.  Neutrophils.  Monocytes/Macrophages. 3

6. Types of phagocytic cells 4

7. Neutrophiles/Polymorphonuclear cells (PMNs)  Are granulocytes that circulate in the blood and migrate quickly in response to local invasion by microorganism.  Cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66.  They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells. 5

8. Neutrophiles/Polymorphonuclear cells (PMNs) 1.The primary or azurophilic granules which are abundant in young newly formed PMNs, contain :- A. Cationic proteins and Defensins that can kill bacteria. B. Proteolytic Enzymes like  Elastase, and Cathepsin G to break down proteins.  Lysozyme to break down bacterial cell.  Myeloperoxidase, which is involved in the generation of bactericidal compounds. 6

9. Neutrophiles/Polymorphonuclear cells (PMNs) 2. The second type of granule Found in more mature PMNs is the secondary or specific granule. These contain. A. Lysozyme. B. NADPH oxidase components, which are involved in the generation of toxic oxygen products. C. Lactoferrin and B12-bindin protein. 7

10. Monocytes/Macrophages  Monocytes also circulate in blood, but in much lower numbers than neutrophils. They migrate to the tissue, where they differentiate into Macrophage, which reside in all body tissues. For example  Kupffer cells are macrophage in the liver.  Histiocytes are macrophage in connective tissue. 8

11. Monocytes/Macrophages  Macrophages are characteristic kidney- shaped nucleus. They can be identified morphologically or by the presence of the CD14 cell surface marker.  Unlike PMNs they do not contain granules but they have numerous lysosomes which have contents similar to the PNM granules. 9

12. Movement of phagocytic cells 1. Ameboid movement. Phagocytic cells migrate in and out of blood vessels and through out the tissue. The process of cellular emigration from capillaries is called diapedesis. 2. Chemotaxis. Phagocytes move toward other cells or organisms by cytoplasmic streaming in response to chemical agent called chemotaxin. 10

13. DIAPEDESIS  The movement or passage of blood cells, especially white blood cells, through intact capillary walls into surrounding body tissue. Also called migration. 11

14. CHEMOTAXIS. During chemotaxis cells move in response to chemical signals. The action of neutrophils is just one example of how the body uses chemotaxis to respond to an infection 12

15. Factors that chemotactic phagocytic cells 13

16. Mechanism of Phagocytosis Macrophage 14

17. Mechanism of Phagocytosis  Microbes adhere to the phagocyte.  Pseudopods engulf the particle (antigen) and creation of phagosome.  Phagosomes fuse with a lysosome to form a phagolysosome.  Invaders in the phagolysosome are digested by proteolytic enzymes.  Indigestible and residual material is removed by exocytosis. Endocytosis is the movement of a substance into a cell by a vesicle. while the movement of a substance by a vesicle tot eh outside of a cell is called exocytosis 15

18. Initiation of Phagocytosis  Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:  Fc receptors. Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes.  Complement receptors – Phagocytic cells have a receptor for the 3 rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst. 16

19. Phagocytic cells receptors Fc receptors Complement receptors 17

20. Initiation of Phagocytosis  Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.  Toll-like receptors – Phagocytes have a variety of Toll- like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll- like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-á and IL-6) by the phagocytes. ( InterLeukin 1-6 & Tumor Necrosis Factor)  18

21. Phagocytic cells receptors Toll-like receptors 19

22. Intracellular Killing by phagocytosis 1. Oxygen-dependent intracellular  When a phagocyte ingests bacteria (or any material), its oxygen consumption increases.  The increase in oxygen consumption, called a respiratory burst, produces reactive oxygen-containing molecules that are anti-microbial.  The oxygen compounds are toxic to both the invader and the cell itself, so they are kept in compartments inside the cell.  This method of killing invading microbes by using the reactive oxygen-containing molecules is referred to as oxygen-dependent intracellular killing. 20

23. Intracellular Killing by phagocytosis 2. Oxygen-independent intracellular  Phagocytes can also kill microbes by oxygen-independent methods, but these are not as effective as the oxygen-dependent ones. There are four main types.  The first uses electrically charged proteins which damage the bacterium's membrane.  The second type uses lysozymes; these enzymes break down the bacterial cell wall.  The third type uses lactoferrins, which are present in neutrophil granules and remove essential iron from bacteria.  The fourth type uses proteases and hydrolytic enzymes; these enzymes are used to digest the proteins of destroyed bacteria. 21

24. Mr.AYMAN.S.YOUSIF 17-18/02/2013 Part 2 Innate Immunity Inflammation

25.  The function of inflammation is to localize tissue damage, localize responses, Prevents spread of agents and then to restore tissue function (tissue repair).  The action of localized leukocytes is augmented via the attraction of neutrophils and monocytes normally found in circulation.  Microbial materials such as LPS, flagellin (making up bacterial flagella), activated complement, and even bacterial DNA serve as indicators of infection which in turn activates the production of pro-inflammatory cytokines (immune- system activating chemicals).  In addition to the cell-to-cell interactions underlying inflammation, the inflammatory response involves localized increases in blood flow, leakage of blood vessels, and attraction of leukocytes from the blood. 21

26. The Three Stages of Inflammation 1. Vasodilatation and increased vessel permeability Due to histamine (and other cytokine) release  edema 2. Phagocyte migration and phagocytosis.  Margination and diapedesis (emigration)  Chemotaxis(due to various cytokines and components of complement system)  Pus formation.  Factors challenging effectiveness of phagocytosis 3. Tissue repair and regeneration depends on type of tissue 22

27. Hallmarks of Inflammation  Redness – erythema & hyperemia  Heat – due to dilated BVs  Swelling – exudates/edema  Dilutes  O 2 and nutrients for repair  Clotting proteins enter  Pain – pressure on localized nerve endings 23

28. Inflammatory Response Histamine & prostaglandins released Capillaries dilate Clotting begins Chemotactic factors attract phagocytic cells Phagocytes consume pathogens & cell debris 24

29. Inflammation 25 Margination Diapedesis

30. Inflammation 26

31. Figure 21.3 27

32. Thank You