ACUTE INFLAMMATION [Autosaved].pptx pathology

Response of vascularised tissues to infections & damaged tissues, that brings cells and molecules of host defence from circulation to the site where they are needed in order to eliminate the offending agent

Serves to get rid of both initial cause & consequences of injury

1. ACUTE INFLAMMATION NOORI MOINUDEEN 1YR M.D.S

2. CONTENTS INTRODUCTION HISTORICAL HIGHLIGHTS CLASSIFICATION CAUSES & STEPS OF INLAMMATION ACUTE AND CHRONIC INFLAMMATION CELLS OF ACUTE INFLAMMATION COMPONENTS OF ACUTE INFLAMMATION VASCULAR CHANGES CELLULAR CHANGES CHANGES IN EXTRACELLULAR MATRIX KILLING MECHANISM NET LEUCOCYTE MEDIATED TISSUE INJURY TERMINATION OF ACUTE INFLAMMATION FATE OF ACUTE INFLAMMATION MORPHOLOGICAL PATTERNS OF ACUTE INFLAMMATION REFERENCES

3. INFLAMMATION Response of vascularised tissues to infections & damaged tissues, that brings cells and molecules of host defence from circulation to the site where they are needed in order to eliminate the offending agent Serves to get rid of both initial cause & consequences of injury

4. HISTORICAL HIGHLIGHTS Dates back to Egyptian papyrus around 300B.C Celsus 1 st century A.D- 4 cardinal signs Rubor Calor Dolor Tumor 5 th sign- functio lasae - Rudolf Virchow(19 th century A.D)

5. Elie Metchnikoff (1845-1916) is considered to be the father of natural immunity , together with Paul Ehrlich- pioneers of cellular and humoral immunology. recognised for the biological significance of leukocyte recruitment and phagocytosis of microbes in host defence against infection Galen (130-201 AD) viewed inflammation, particularly pus, as part of the beneficial reactive response to injury

6. Louis Pasteur Paul Ehrlich proposed “horror autotoxicus ” as immune reactivity against self, which is now called autoimmunity Louis Pasteur (1822-1895) discovered that tiny single cell organisms caused putrefaction & called them bacteria or microbes

7. Julius Cohnheim von Recklinghausen Julius Friedrich Cohnheim (1839-1884) observed the dilatation of the arteries and veins, adhesion of colourless cells to the endothelial cells transmigration Friedrich Daniel von Recklinghausen (1833-1910) characterized the pus cells in acute inflammation

8. CLASSIFICATION OF INFLAMMATION -Per-acute inflammation : - - very short course and the animal die soon (few hours) after exposure to the causative agent. Acute inflammation : - Sub-acute inflammation : - -caused by mild irritant with less prominent circulatory and cellular changes (neutrophils decrease and macrophages increase). Chronic inflammation According to duration:-

9. CAUSES OF INFLAMMATION INFECTIONS TISSUE NECROSIS-ischemia, trauma, physical & chemical injury etc FOREIGN BODY IMMUNE REACTIONS

10. STEPS OF TYPICAL INFLAMMATION

11. STEPS OF TYPICAL INFLAMMATION

12. ACUTE & CHRONIC INFLAMMATION Initial rapid response to infections & tissue damage is acute inflammation Develops within minutes or hours Short duration(lasts few hours to days) Less specific If acute inflammation fails to acheive its goal, it progress into a protracted phase called chronic inflammation

13. CELLS OF ACUTE INFLAMMATION

14. CELLS OF ACUTE INFLAMMATION

15. CELLS OF ACUTE INFLAMMATION NEUTROPHIL BASOPHIL EOSINOPHIL

16. CELLS OF ACUTE INFLAMMATION MACROPHAGES stationed at strategic points where microbial invasion or accumulation of foreign particles is likely to occur. These cells together as a group are known as the mononuclear phagocyte system previously known as the reticuloendothelial system Named after their location

17. RESIDENT MACROPHAGES

18. RESIDENT MACROPHAGES , CLASSICALLY ACTIVATED MACROPHAGES/ M1 "KILLER" MACROPHAGES ALTERNATIVELY ACTIVATED MACROPHAGES/ WOUND-HEALING MACROPHAGES / M2 REPAIR MACROPHAGE T Y P E S

19. RESIDENT MACROPHAGES ,

20. RESIDENT MACROPHAGES ,

21. RESIDENT MACROPHAGES

22. NEUTROPHILS Neutrophils are the most common type of leucocyte in blood and constitute 40-75% of circulating leucocytes highly motile and phagocytic secrete enzymes that degrade tissue components, ingest and destroy damaged tissue and kill invading microorganisms

23. NEUTROPHIL GRANULES primary granules purplish killing and degradation of engulfed microorganisms specific granules smaller than primary granules tertiary granules secrete enzymes to degrade tissue reservoir of membrane-associated receptors needed for neutrophil function

24. NEUTROPHIL

25. EOSINOPHILS Eosinophils account for 1-6% of leucocytes in circulating blood; The eosinophil (12-17 μm in diameter) is larger than the neutrophil and is easily recognised by its large specific granules Contains a histaminase major basic protein , other basic protein hydrolytic lysosomal enzymes and a peroxidase (different from the myeloperoxidase of neutrophils) termed eosinophil peroxidase (EPO) . Smaller granules are also present in mature eosinophils and contain aryl sulphatase and acid phosphatase

26. EOSINOPHILS . All eosinophils have receptors for IgE FUNCTIONS: central role in the induction and maintenance of inflammatory responses due to allergy , act as pro-inflammatory leucocytes defence against helminthic parasites minor role as antigen presenting cells

27. EOSINOPHILS

28. BASOPHIL S Basophils are the least common leucocyte and constitute less than 1% of leucocytes in circulating blood. Characterised by large intensely basophilic cytoplasmic granules

29. BASOPHIL S metachromatic staining property. The granules contain PROTEOGLYCANS- mix of heparin and chondroitin sulphate histamine slow reacting substance of anaphylaxis (SRS-A), eosinophil chemotactic factor of anaphylaxis (ECF-A) tryptase .

30. BASOPHIL S

31. DENDRITIC CELL S antigen -presenting cells (APCs) characterized by a unique capacity to stimulate naive T cells

32. DENDRITIC CELL S Immature DCs -PRRs, Toll-like receptor (TLR) mannose receptors, PRR against inflammatory compounds released by damaged tissues Upon recognizing a pathogen release large amounts of proinflammatory or antiviral cytokines activation of innate immune cells, thereby limiting the spread of infection DCs acquire a “mature” phenotype

33. DENDRITIC CELL S hematopoietic stem cell- HSC MDP-macrophage/DC progenitors CDP-common DC progenitor FLT-3L- FMS-like tyrosine kinase 3 ligand GM-CSF- Granulocyte-macrophage colony-stimulating factor ( GM - CSF ), M-CSF- macrophage colony-stimulating factor ( GM - CSF ),

34. DENDRITIC CELL S more common major stimulator of T cells express all TLR except TLR9 extremely rare fighting wound infection lack TLR 4,5,7 and TLR9 mDC -   mDCs are efficient in uptake, processing, and presentation of foreign antigens pDC - ability to quickly secrete large amounts of Type I interferons (IFN) in response to a viral

35. DENDRITIC CELL S

36. INNATE LYMPHOID CELLS-ILC group of lymphocytes that do not express antigen-specific receptors classified on the basis of their transcriptional and functional profile ILC HELPER ILC CYTOTOXIC ILC ILC 1 ILC -2 ILC -3

37. INNATE LYMPHOID CELLS-ILC

38. γδ T CELLS Subset of T-cell with specific TCR-has γ and δ chains Intra epithelial lymphocytes Antigen it recogonises is unknown- thought to recogonise Lipid antigen HSP Do not require MHC activation Rapid cytokine release Recently- phagocytic function Memory cell subset BRIDGE BETWEEN INNATE & ADAPTIVE IMMUNITY

39. CELLULAR VASCUAR CHANGE CHANGE 3 MAJOR COMPONENTS ACUTE INFLAMMATION

40. TRANSIENT VASOCONSTRICTION VASODILATION INCREASED BLOOD FLOW ERYTHEMA, HEAT STASIS INCREASED PERMEABILITY MIGRATION LEUKOCYTE ADHESION CAM EXPRESSION LEUKOCYTE ACCUMULATE ALONG ENDOTHELIUM

41. CHANGES IN VASCULAR FLOW & CALIBER CHANGE IN VASCULAR PERMEABILITY VASCULAR CHANGE

42. VASCULAR CHANGE ( Reactions Of Blood Vessels In Acute Inflammation) CHANGES IN VASCULAR FLOW & CALIBER

43. REACTIONS OF BLOOD VESSELS IN ACUTE INFLAMMATION- CHANGES IN VASCULAR FLOW & CALIBER Vasodilation earliest manifestations sometimes it follows a transient constriction of arterioles, lasting a few seconds. first involves the arterioles and then leads to opening of new capillary beds in the area. The result is increased blood flow , heat and redness Vasodilation is induced by the action of several mediators, histamine and nitric oxide (NO) .

44. REACTIONS OF BLOOD VESSELS IN ACUTE INFLAMMATION- CHANGES IN VASCULAR FLOW & CALIBER Vasodilation is quickly followed by increased permeability loss of fluid and increased vessel diameter lead to slower blood flow , increased viscosity of the blood. result in dilation of small vessels that are packed with slowly moving red cells, a condition termed stasis , which is seen as vascular congestion

45. REACTIONS OF BLOOD VESSELS IN ACUTE INFLAMMATION- CHANGES IN VASCULAR FLOW & CALIBER As stasis develops, blood leukocytes, principally neutrophils, accumulate along the vascular endothelium . At the same time endothelial cells are activated by mediators produced at sites of infection and tissue damage, and express increased levels of adhesion molecules . Leukocytes then adhere to the endothelium, and soon afterward they migrate through the vascular wall into the interstitial tissue

46. MECHANISM OF INCREASED VASCULAR PERMEABILITY Endothelial injury, resulting in endothelial cell necrosis and detachment . [

47. CONTRACTION OF ENDOTHELIAL CELLS RESULTING IN INCREASED INTERENDOTHELIAL SPACES most common mechanism of vascular leakage elicited by Histamine bradykinin Leukotrienes neuropeptide substance P many other chemical mediators called the immediate transient response because it occurs rapidly after exposure to the mediator and is usually short-lived (15–30 minutes )

48. In some forms of mild injury (e.g. after burns, x-irradiation or ultraviolet radiation, and exposure to certain bacterial toxins), vascular leakage begins after a delay of 2 to 12 hours, and lasts for several hours or even days ; this delayed prolonged leakage may be caused by contraction of endothelial cells or mild endothelial damage Eg : Late-appearing sunburn

49. ENDOTHELIAL INJURY , RESULTING IN ENDOTHELIAL CELL NECROSIS AND DETACHMENT Direct damage In severe injury-burns, microbes Neutrophils that adhere to the endothelium leakage starts immediately after injury and is sustained for several hours until the damaged vessels are thrombosed or repaired.

50. INCREASED TRANSPORT OF FLUIDS AND PROTEINS, CALLED TRANSCYTOSIS , THROUGH THE ENDOTHELIAL CELL involve channels consisting of interconnected, uncoated vesicles and vacuoles called the vesiculovacuolar organelle , many of which are located close to intercellular junctions Certain factors, such as VEGF seem to promote vascular leakage in part by increasing the number and size of these channels.

51. What is vesiculovacuolar organelle? The vesiculo-vacuolar organelle (VVO) is an organelle found in the cytoplasm of endothelial cells that line tumor microvessels and normal venules . VVOs are grape-like clusters of interconnecting uncoated vesicles and vacuoles , span the entire thickness of vascular endothelium, provides a potential trans-endothelial connection between the vascular lumen and the extravascular space.

52. What is vesiculovacuolar organelle? individual VVO vesicles and vacuoles communicated with each other and with the endothelial cells VVOs provide a major pathway for the extravasation of circulating macromolecules across endothelia upregulated VVO function accounts for the well known hyperpermeability of tumor blood vessels.

53. RESPONSES OF LYMPHATIC VESSELS Lymphatic vessels proliferate during inflammatory reactions to handle the increased load lymphatics and lymph nodes filters the extravascular fluids. lymph flow is increased and helps drain edema fluid that accumulates leukocytes and cell debris, as well as microbes, may find their way into lymph.

54. The lymphatics may become secondarily inflamed ( lymphangitis ) , as may the draining lymph nodes (lymphadenitis ) . Inflamed lymph nodes are often enlarged because of hyperplasia of the lymphoid follicles and increased numbers of lymphocytes and macrophages. - reactive , or inflammatory , lymphadenitis - red streaks near a skin wound

55. TRANSUDATE Vs. EXUDATE

56. CELLULAR CHANGES (Reactions Of Leukocytes In Acute Inflammation)-

57. Critical function of inflammation is to deliver leukocytes to the site of injury and to activate the leukocytes to eliminate the offending agents leukocytes ingest and kill bacteria and other microbes, and eliminate necrotic tissue and foreign substances. Leukocytes also produce growth factors that aid in repair

58. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS

59. MARGINATION TETHERING & ROLLING ADHESION INTRAVASCULAR CRAWLING DOCKING STRUCTURE FORMATION RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. Extravasation: The journey of leukocytes from the vessel lumen to the interstitial tissu e STEPS IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS

60. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MARGINATION IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS ADHESION TETHERING &ROLLING ENDOTHELIAL CRAWLING ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE

61. MARGINATION process in which free-flowing leucocytes exit the central blood stream, and initiate leukocyte and endothelial cell interactions by close mechanical contact. Mechanisms involve the interaction of leukocytes with erythrocytes flowing in the same microvessel ,.

62. increased permeability hemoconcentration slows blood flow locally blood flow shear rate drops below a critical value, Leukocytes move to periphery Allows the leukocyte-endothelial cell interactions MARGINATION

63. MARGINATION The leukocyte and RBCs flowed in the center of the channel When the channel diameter was increased to 12  μ m , RBCs followed a multifile motion and undergoes parachute-type deformity Because the motion of multifiled RBCs was unsteady, the leukocyte position slightly shifted toward the wall.

64. DO ALL THE LEUCOCYTES THAT ARE MARGINATED UNDERGO ADHESION???

65. thin layer of glycoproteins and proteoglycans lining the vascular lumen that forms an interface between the blood and the endothelium. dynamically interact with plasma proteins and become highly hydrated, forming a thick endothelial surface layer (ESL) serve as an interface between circulating leukocytes and the endothelium ENDOTHELIAL GLYCOCALYX

66. the thickness of the ESL greatly exceeds the length of selectins , glycocalyx forms a “cloak ” that conceals endothelial adhesion sites from passing neutrophils . Hence neutrophils can only access buried endothelial adhesion molecules if the velocity of blood flow is sufficiently slow to allow time for penetration of neutrophil microvilli into the ESL ENDOTHELIAL GLYCOCALYX

67. ENDOTHELIAL GLYCOCALYX

68. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MARGINATION IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS ADHESION TETHERING &ROLLING ENDOTHELIAL CRAWLING ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE

69. TETHERING AND ROLLING leukocytes must first attach to the endothelium lining the blood vessel wall before they can pass through into the inflammed tissues. The hemoconcentration in early phase of the inflammatory response slows blood flow sufficiently so that leukocytes can make functional contacts with the endothelium. These initial contacts “tether” the white cells to the endothelium. These initial capturing interactions are generally mediated by the selectin family of adhesion molecules

70. ROLLING individual and then rows of leukocytes adhere transiently to the endothelium, detach and bind again, thus rolling on the vessel wall . Histamine release from mast cells is responsible for the “baseline rolling” interactions are mediated by the selectin family of adhesion molecules

71. SELECTIN P-SELECTIN (platelets & endothelium) E-SELECTIN (endothelium) L-SELECTIN (leucocytes) expressed on all granulocytes and monocytes and on most lymphocytes stored in a-granules of platelets and in Weibel –Palade bodies of endothelial cells, and is translocated to the cell surface of activated endothelial cells and platelets. not expressed under baseline conditions, except in skin microvessels , but is rapidly induced by inflammatory cytokines endothelial cells express E- selectin and the ligands for L- selectin Leukocytes express L- selectin at the tips of their microvilli and also express ligands for E- and P- selectins , all of which bind to the complementary molecules on the endothelial cells.

72. Selectins are composed of an N-terminal lectin domain an epidermal growth factor (EGF) domain complement regulatory (CR) proteins(two L selectin ), six E- selectin ) nine P- selectin ) a transmembrane domain cytoplasmic domain

73. One of the safeguards preventing unwanted inflammation is the regulated expression of cell adhesion molecules and/or their ligands. For example, E- selectin is not expressed by resting endothelium . Its expression is induced after activation of the endothelium by inflammatory cytokines and other stimuli P- selectin is stored preformed in the α granules of platelets and within the Weibel -Palade bodies of endothelial cells. Under conditions of chronic inflammatory stimulation, P- selectin can be stably expressed on the cell surface.

74. L-SELECTIN E-SELECTIN P-SELECTIN SIALYL LEWIS X GLYCAM-1 GLYCOPROTEIN PSGL ENDOTHELIUM NEUTROPHIL MONOCYTE T CELL NEUTROPHIL MONOCYTE T CELL NEUTROPHIL MONOCYTE T CELL ENDOTHELIUM ENDOTHELIUM

75. LEUKOCYTE ADHESION DEFICIENCY TYPE II (LAD II). This is a rare (four patients reported) defect in mutations in the gene which encodes the GDP- fucose transporter 1 (FUCT1) lack fucosylated glycoconjugates . These glycoconjugates include the selectin ligand(the sialyl Lewis  X  antigen ) As a result, selectin ligands are severely under fucosylated and there is marked deficiency in the ability of leukocytes from these patients to roll on endothelium Recurrent bacterial infections

76. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MARGINATION IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS ADHESION TETHERING &ROLLING ENDOTHELIAL CRAWLING ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE

77. ARREST AND ADHESION The processes of tethering and rolling are reversible. To stop rolling, the low affinity transient interactions of rolling must be replaced by high affinity adhesion between the leukocyte and the endothelial surface.

78. The weak rolling interactions slow down the leukocytes and give them the opportunity to bind more firmly to the endothelium. Firm adhesion is mediated by a family of heterodimeric leukocyte surface proteins called integrin

79. INTEGRINS Integrins are transmembrane cell surface proteins that bind to cytoskeletal proteins and communicate extracellular signals. INTEGRINS taking part in firm adhesion of leucocytes include LFA-1 MAC-1 VLA-4

80. VLA-Very Late Antigen-4 LFA-Lymphocyte Function-associated Antigen-1 MAC - MACROPHAGE ANTIGEN-1 VCAM-vascular cell adhesion molecule ICAM-intercellular adhesion molecule-1

81. LFA-1 INTEGRIN

82. Consisting of two subunits α subunit β subunit More than 24 different integrins formed from combinations of 18 α subunits 8 β subunits Extracellular domain(N terminus) Transmembrane region Intracellular/cytoplasmic tail ( C terminus) INTEGRINS

83. Neutrophils – predominantly β 2 -integrins, but also low amounts of β 1- and β 3-integrins. Monocytes - - β 1- and β 2 -integins Lymphocyte --pattern of β 2 -, and β 7-integrins varying with subtype and state of activation A common feature is that each leukocyte subtype expresses one or more members of the β 2 integrin family . -“ leukocyte” integrins ( CD18) β subunit

84. Of the four identified α chains that pair with CD18( β 2 subunit), α L (CD11a) α M (CD11b) are the most important for the adhesion of leukocytes .

85. The integrins formed , α L β2 – LFA-1 leukocyte function associated antigen1 ( CD11a/CD18 ) α M β2 - Mac-1 Macrophage Antigen-1 (CD11b/CD18 ) α L - CD11a α M- CD11b β 2 - CD18

86. Integrins exist in a low and high affinity binding state low affinity state favoured activated to favor the high affinity state efficiently bind their ligands. This can be done in two ways: Avidity modulation clusters the integrins in the plane of the membrane. Affinity modulation involves a conformational change in the integrin chains that favors their assumption of the active state. Activation of integrins

87. A hallmark of integrins is their ability to fine-tune their affinity for their ligands by switching their extracellular domain between several different conformations. The conformational change that increases the affinity of integrins for their ligands is believed to be controlled by proteins ( talins , kindilins ) that bind to the short cytoplasmic tails of integrins .

88. On resting cells, integrins are displayed in their closed or “bent” conformation- low binding affinity for ligands-inactive form Upon activation, the conformation of the molecule erects to an extended shape-a high-affinity conformation for ligands

89. Bidirectional signalling of integrins There are two directions of integrin signalling, which have different biological consequences INSIDE-OUT SIGNALLING OUTSIDE-IN SIGNALLING

90. Bidirectional signalling of integrins - INSIDE OUT SIGNLLING enables sufficiently strong interactions between integrins and extracellular matrix (ECM) proteins to allow integrins to transmit the forces required for cell migration and adhesion ECM remodelling and assembly

91. Bidirectional signalling of integrins - OUTSIDE –IN SIGNALLING control cell polarity cytoskeletal structure gene expression cell survival and proliferation. traditional signalling receptors transmitting information into cells by ‘outside–in’ signalling.

92. Bidirectional signalling of integrins - Although we conceptually separate the two processes, they are often closely linked; for example,

93. L- selectin on leukocytes is enriched on microvillous projections of the cell membrane, while integrin molecules that mediate the next step of emigration are restricted to the body of the leukocyte between microvilli In this way, the molecules mediating the initial tethering of leukocytes contact the endothelium first. Soon after this initial contact, the microvilli retract, allowing contact of the integrins with their counter-receptors on the endothelium. WHY SELECTINS INTERACT WITH THE RECEPTORS FIRST AND NOT INTEGRINS???

94. LEUKOCYTE ADHESION DEFICIENCY TYPE I (LAD I) Patients with LAD I do not express significant levels of β 2 integrins on the surfaces of their leukocytes. inherent defect in the β chain, CD18 . An incomplete β chain is degraded in the Golgi apparatus These individuals have a profound neutrophilia , but the neutrophils cannot bind to the endothelial cells die with bacterial overgrowth in their tissues in the absence of any tissue neutrophils.

95. LEUKOCYTE ADHESION DEFICIENCY TYPE III (LAD III) integrin expression and structure is intact but integrin activation (and thus binding) is defective.  Mutations in Kindlin (an integrin adaptor protein) have been reported in patients with LAD 3 Features similar to LADI-die with bacterial overgrowth in their tissues in the absence of any tissue neutrophils. This diagnosis should be considered in patients who have a LAD 1 phenotype but have normal CD18 surface expression.  Genetic analysis for KINDLIN mutations should be performed for all suspected cases. 

96. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MARGINATION IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS ADHESION TETHERING &ROLLING ENDOTHELIAL CRAWLING ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE

97. Intravascular crawling(locomotion) Many of the leukocytes that are tightly bound to endothelium next crawl over the luminal surface towards intercellular junction (preferred site of diapedesis )

98. Intravascular crawling(locomotion) Neutrophils blocking ICAM-1 completely abolished neutrophil crawling. blockade of LFA-1 -did not affect neutrophil crawling. blocking Mac-1 Ab significantly decreased the crawling cells Hence,Mac-1, and not LFA-1, is the central integrin mediating neutrophils crawling

99. Intravascular crawling(locomotion) Monocytes were found to actively move to cellular junctions to start transmigration within seconds. blocking of CD18, ICAM-1, or ICAM-2 -monocytes unable to locate endothelial cell junctions and caused the cells to wander injection of a blocking LFA-1 , but not Mac-1 Ab completely and rapidly detached crawling monocytes in vivo, indicating a predominant role of LFA-1

100. Intravascular crawling(locomotion) Lymphocyte crawling is LFA-1 dependent multiple scattered traction sites form by engagement of high-affinity LFA-1 with endothelial ICAM-1 , and these form in a rapid turnover fashion “millipede-like ” translocation over the endothelium

101. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MARGINATION IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS ADHESION TETHERING &ROLLING ENDOTHELIAL CRAWLING ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE

102. Docking Structures or Transmigratory Cups Function -has a large accumulation of adhesion receptors. --prevents leukocytes from becoming unbound.- final arrest of leucocyte

103. Docking Structures or Transmigratory Cups

104. MARGINATION ROLLING ADHESION INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. Extravasation: The journey of leukocytes from the vessel lumen to the interstitial tissu e STEPS IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS

105. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. DIAPEDESIS/TEM process whereby the leukocyte squeezes in ameboid fashion across the endothelial cells. happens at endothelial cell borders TEM is arguably the point of no return in the inflammatory response

106. Postcapillary venules are surrounded by their own basal lamina and loosely invested by a discontinuous layer of pericytes the leukocyte must still traverse these as well as the surrounding interstitial tissue to get to the site of inflammation

107. TRANS ENDOTHELIAL MIGRATION-TEM/ DIAPEDESIS TRANSCELLULAR PARACELLULAR

108. Molecules Regulating TEM Several endothelial molecules have been implicated in the control of diapedesis , including Of these , PECAM and CD99 have been the most extensively studied for their role in transmigration and are known to have no role in the preceding step Icam-1, Vcam-1, Junctional Adhesion Molecules A and C (JAM-A And JAM-C) Endothelial Cell-selective Adhesion Molecule, PECAM, CD99, And CD99L2.

109. PECAM-1 (CD31 ) Ig superfamily member concentrated at the borders of endothelial cells as well as expressed diffusely on platelets and leukocytes. Homophilic interaction of leukocyte PECAM with endothelial PECAM is required for TEM. When PECAM–PECAM interactions are blocked, leukocytes are able to get to the postcapillary venules at the site of TEM but are unable to transmigrate efficiently leukocytes are arrested tightly adherent to the apical surface of the cell

110. Similar to PECAM, homophilic interaction between CD99 at the endothelial cell border and CD99 on monocytes and neutrophils is required for transmigration. However, CD99 regulates a later step in transmigration than PECAM . CD99

111. . confocal images of leukocytes blocked in the act of transmigration by anti-CD99 show their leading edge under the endothelial cytoplasm, their cell body lodged at the border between endothelial cells, and the trailing uropod on the apical surface

112. Loosening the Junctions important for efficient transmigration. During trans-endothelial migration, neutrophils disrupt these structures by using a variety of mechanisms including by induction of endothelial signaling pathways .  actin-myosin contraction physical disruption ( “muscling ” their way through) Cadherin phosphorylation ROS production

113. Loosening the Junctions -BY SIGNALLING

114. Loosening the Junctions-by disruption Stimulation of ICAM-1 leads to phosphorylation of VE-cadherin, which is a prerequisite for adherens junction disassembly. P ICAM Mac-1 integrin Laf-1 integrin VE CADHERIN VE PTP WEAKENING OF JUNCTION VE PTP-vascular endothelial protein tyrosine phosphatase

115. Loosening the Junctions-by disruption

116. Recent in vitro studies VE-cadherin is transiently ,but completely removed from the site of transmigration Increase in concentration of adhesion molecules at the site of migration

117. In resting endothelial cell, VE -cadherin antibodies showed the expected staining along the endothelial border but unlike VE-cadherin, PECAM was also observed in a subjunctional grape-like structure of interconnected 50 nm vesicles and tubules PECAM from this compartment was observed to exchange and recycle with the surface membrane and, consequently, termed the Lateral Border Recycling Compartment or LBRC . THE LATERAL BORDER RECYCLING COMPARTMENT (LBRC)

118. LBRC compartment is a complex invagination of the junctional membrane. Under resting conditions roughly 30% of the total PECAM is in the LBRC [also contains JAM-A and CD99 ] Homophilic interactions between PECAM on the leucocyte and PECAM on the endothelial recruitment of the LBRC membrane to the site of TEM specific enrichment around the transmigrating leukocyte (“targeted recycling” of the LBRC) THE LATERAL BORDER RECYCLING COMPARTMENT (LBRC)

119. Events relevant to weakening of adherens junctions and those relevant to the LBRC are taking place simultaneously cells . These pathways intersect and both lead to increase in actin–myosin tension and phosphorylation of VE-cadherin, which facilitates its clearance for the adherens junction.

120. Once the leukocyte has moved across the junction, the LBRC may be pulled back into the cell, allowing the other components to diffuse back into place, reestablishing the endothelial junction

121. TRANSCELLULAR MIGRATION WHY TRANSCELLULAR MIGRATION?????

122. TRANSCELLULAR MIGRATION Transcellular leukocyte migration is generally much less common than paracellular emigration, appears to be favored in regional circulations that have very tight cell-cell junctions, e.g., the blood-brain barrier lymphocytes pursue this route of emigration more frequently than neutrophil

123. TRANSCELLULAR MIGRATION

124. TRANSCELLULAR MIGRATION generally interpreted as a mechanism of paracellular diapedesis . also precedes transcellular diapedesis EM studies suggest that transcellular diapedesis occurs preferentially in close juxtaposition to intact intercellular junctions ( perijunctionally ) leukocytes therefore must somehow ‘seek out these regions’ --- HOW???? LATERAL MIGRATION

125. TRANSCELLULAR MIGRATION ILP ‘ invadosome -like protrusions’

126. TRANSCELLULAR MIGRATION Invadosomes are actin-dependent adhesive and/or protrusive structures (~500 nm in both diameter and depth) that form specifically on the ventral surface of highly migratory and invasive cells, such as leukocytes, endothelial cells and tumor cells PROBING BY ILPs

127. TRANSCELLULAR MIGRATION Probing by ILPs dynamically protrude and retract (with half-lives of ~20 seconds) many ILPs (between 10 and 100) into the surface of the endothelium as they migrate laterally These protrusions cause the formation of endothelial cell-surface invaginations (termed ‘ podoprints ’ )

128. TRANSCELLULAR MIGRATION some of these structures spanned nearly the entire endothelial-cell depth, placing the apical and basal membranes in close apposition Probing serves as a means of identifying locations of relatively low endothelial resistance , into which protrusions can then extend progressively to promote transcellular pore podosomes can serve as ‘dynamic mechanosensors ’

129. TRANSCELLULAR MIGRATION ‘ transmigratory cup’ consists of vertical endothelial microvillus-like projections rich in actin, ICAM1, VCAM1, PECAM1 and JAM1 that surround the periphery of adherent leukocytes. This structure seems to guide diapedesis ENDOTHELIAL MIGRATORY CUP FORMATION

130. TRANSCELLULAR MIGRATION Requires energy-dependent membrane-fusion events occur. The protrusive forces that are supplied by invadasome like protrusion (ILPs ) promote close apposition of apical and basal endothelial membranes and might also supply energy that is directly involved in the membrane fusion process. TRANSMEMBRANE PORE FORMATION

131. TRANSCELLULAR MIGRATION Another school of thought In vitro studies show enrichment of the caveolar marker caveolin-1, various vesicles and vesiculo-vacuolar organelles (VVOs) and fusogenic proteins in the endothelium at sites of transcellular pore formation “ the fusing of the vesicles may form a gradual trans-endothelial channel Leucocyte cross the endothelium ” TRANSMEMBRANE PORE FORMATION

132. WOULDN’T THE MIGRATION OF ONE CELL THROUGH THE BODY OF ANOTHER CELL CAUSE A MAJOR DISRUPTION OF BARRIER INTEGRITY????????? does not appear to affect vascular permeability to fluid and solutes the endothelial monolayer mobilizes internal membranes to seal off discontinuities. vascular permeability is minimally altered

133. Two recent publications demonstrated in vivo the presence of abnormal transendothelial migratory events characterized by the neutrophil partially migrating through the junction with oscillatory movements in the junction (i.e., hesitant migration ) returning back to the circulation in an abluminal -to-luminal direction ( reverse migration ) following leukotriene B4- (LTB4-) induced inflammation ATYPICAL DIAPEDESIS

134. Once the leucocyte has passed across the endothelial barrier, it still must cross the subendothelial basal lamina. This process generally requires far more time neutrophil (PMN) and monocytes move between the abluminal surface of the endothelial cell and the basal lamina searching for areas where collagen IV and laminin are deposited at low density. These sites, which have been termed low-expression regions (LERs) These correspond to areas where there is a gap in pericyte coverage. Crossing subendothelial regions

135. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. Extravasation: The journey of leukocytes from the vessel lumen to the interstitial tissu e STEPS IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS

136. RECRUITMENT FROM THE BLOOD INTO EXTRAVASCULAR TISSUES. MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS chemotaxis - defined as locomotion oriented along a chemical gradient. CHEMOATTRACTANTS ENDOGENEOUS EXOGENEOUS BACTERIAL PRODUCTS peptides that possess an N- formylmethionine terminal amino acid Lipids - CYTOKINES COMPLEMENT SYSTEM ARACHIDONIC ACID METABOLITES-LTB4

137. activation of second messengers chemoattractant binds specific seven- transmembrane G protein–coupled receptors on the surface of leukocytes The leukocyte moves by extending filopodia that pull the back of the cell in the direction of extension ↑cytosolic calcium activate small guanosine triphosphatases activate numerous kinases. induce polymerization of actin ↑polymerized actin at the leading edge of the cell localization of myosin filaments at the back

138. WHY NEUTROPHILS APPEAR FIRST IN INFLAMMATION?? more numerous in the blood, they respond more rapidly to chemokines attach more firmly to the adhesion molecules After entering tissues, neutrophils are short-lived; they undergo apoptosis and disappear after 24 to 48 hours

139. Monocytes not only survive longer but may proliferate in the tissues, and thus become the dominant population in chronic inflammatory reactions. Exceptions to this pattern of cellular infiltration Pseudomonas bacteria—the cellular infiltrate is dominated by continuously recruited neutrophils for several days; in viral infections, lymphocytes may be the first cells to arrive; in some hypersensitivity reactions, eosinophils may be the main cell type

140. ROBBINS AND COTRAN PATHOLOGIC BASIS OF DISEASE, 8/E Janeway's Immunobiology © 2012 by Garland Science, Taylor & Francis Group, LLC Molecular Basis of Leukocyte-Endothelium Interactions During the Inflammatory Response Olga Barreiro  a , Francisco Sánchez-Madrid  a Klaus Ley, The role of selectins in inflammation and disease Cardiovascular Research Center and Departments of Biomedical Engineering, Molecular Physiology and Biological Physics, Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game.  Nature reviews Molecular cell biology . 2010;11(4):288-300. doi:10.1038/nrm2871. Alexandre Chigaev * and  Larry A. Sklar * Aspects of VLA-4 and LFA-1 regulation that may contribute to rolling and firm adhesion Front. Immunol ., 02 August 2012 REFERENCES

141. On, Around, and Through: Neutrophil-Endothelial Interactions in Innate Immunity Eric P. Schmidt, Warren L. Lee, Rachel L. Zemans , Cory Yamashita, Gregory P. Downey Biology and structure of leukocyte β integrins and their role in inflammation[version 1; referees: 3 approved] M. Amin Arnaout Leukocyte Biology & Inflammation Program, Structural Biology Program, Nephrology, Center for Regenerative Medicine, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA   Qin J, Vinogradova O, Plow EF (2004) Integrin Bidirectional Signaling : A Molecular View. PLoS Biol 2(6): e169. https://doi.org/10.1371/journal.pbio.0020169

142. Integrin Regulation during Leukocyte Jan Herter and Alexander Zarbock http://www.jimmunol.org/content/190/9/4451 doi : 10.4049/jimmunol.1203179 J Immunol 2013; 190:4451-4457; ; Monocyte trafficking across the vessel wall Teresa Gerhardt Klaus Ley Cardiovasc Res (2015) 107 (3): 321-330. DOI: https://doi.org/10.1093/cvr/cvv147 Published: 20 May 2015

143. THANK YOU

144. ACUTE INFLAMMATION-3 BY SHINU KOSHY 1YR M.D.S

145. CONTENTS INTRODUCTION HISTORICAL HIGHLIGHTS CLASSIFICATION CAUSES & STEPS OF INLAMMATION ACUTE AND CHRONIC INFLAMMATION CELLS OF ACUTE INFLAMMATION COMPONENTS OF ACUTE INFLAMMATION VASCULAR CHANGES CELLULAR CHANGES CHANGES IN EXTRACELLULAR MATRIX RECOGNITION OF MICROBES KILLING MECHANISM NET LEUCOCYTE MEDIATED TISSUE INJURY TERMINATION OF ACUTE INFLAMMATION FATE OF ACUTE INFLAMMATION MORPHOLOGICAL PATTERNS OF ACUTE INFLAMMATION REFERENCES

146. IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS MARGINATION ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE VASCULAR CHANGES CELLULAR CHANGES

147. IN THE LUMEN MIGRATION ACROSS THE ENDOTHELIUM AND VESSEL WALL MIGRATION IN THE TISSUES TOWARD A CHEMOTACTIC STIMULUS MARGINATION ADHESION TETHERING &ROLLING INTRAVASCULAR CRAWLING FORMATION OF DOCKING STRUCTURE VASCULAR CHANGES CELLULAR CHANGES

148. RECOGNITION OF MICROBES AND DEAD TISSUES Recognition of offending agents is the first step in all inflammatory reactions.

149. RECOGNITION OF MICROBES AND DEAD TISSUES PATHOGEN-ASSOCIATED MOLECULAR PATTERNS certain microbial components that are shared among related microbes and are often essential for infectivity (and thus cannot be mutated to allow the microbes to evade the defense mechanisms) DAMAGE-ASSOCIATED MOLECULAR PATTERNS molecules released by injured and necrotic cells PATTERN RECOGNITION RECEPTORS the cellular receptors that recognize these molecules

150. ENDOCYTIC PRR SIGNALING PRR found on the surface of phagocytes  and  promote the attachment of microorganisms to phagocytes leading to their subsequent engulfment and destruction receptors bind a number of microbial molecules:  LPS, peptidoglycan, teichoic acids, flagellin , viral RNA, and certain single-stranded viral RNAs. promotes the synthesis and secretion of intracellular regulatory molecules such as cytokines that are crucial to initiating innate immunity and adaptive immunity . PRR

151. SIGNALLING PRR ENDOCYTIC PRR

152. ENDOCYTIC PRR SIGNALING PRR MANNOSE RECEPTOR FOUND ON CELL SURFACES FOUND ON CYTOPLASM SECRETED SIGNALING PRRS FOUND IN PLASMA AND TISSUE FLUID FOUND ON MEMBRANES OF ENDOSOMES TLR 1/2, 2/6, 4, 5, 10 TLR 3,7,8,9 NLR CARD-containing proteins EX: RIG mannan-binding lectin N - formyl Met RECEPTOR OPSONIN RECEPTOR SCAVENGER RECEPTOR ENDOCYTIC PRR SIGNALING PRR

153. SIGNALING PRR

154. SIGNALING PRR

155. TOLL LIKE RECEPTORS TLRs are transmembrane -spanning proteins In mice and humans combined there are 13 paralogous TLRs; 10 in humans and 12 in mice. TLR10 (only present in humans), and TLR11–13 (only present in mice) Activation of the TLRs leads to induction of inflammatory responses development of antigen specific adaptive immunity

156. Many ‘non-immune’ cell types, including epithelial cells, neurons, astrocytes, and fibroblasts, also express TLRs and respond to their activation. INTRACELLULAR(ENDOSOME) TLR 3,7, 8, 9- CELL SURFACE TLR ½, 2/6, 4, 5, 10

157. INTRACELLULAR(ENDOSOME) TLR 3,7, 8, 9- CELL SURFACE TLR ½, 2/6, 4, 5, 10

158. STRUCTURE single-pass transmembrane proteins Extracellular region composed of 18-25 copies of a leucine -rich repeat (LRR ) horseshoe-shaped protein ligand binding Transmembrane region Mammalian TLRs are activated when binding of a ligand induces them to form dimers TIR ( Toll-IL-l receptor) domain(intracellular) in their cytoplasmic tail, which interacts with other TIR-type domains

159. ligand binding to the LRR(extracellular) domain induces formation of receptor homodimers or, in some cases, heterodimers. resulting TIR (intracellular) domain conformational change allows interactions between TIR domains of adjacent TLRs binding of additional adaptor proteins essential for triggering intracellular signaling MyD88- myeloiddifferentiation factor 88 Mal/TIRAP- MyD88 Adaptor-Like / TIR domain-containing Adaptor ProteinP ( TRIF/TICAM- TIR domain-containing adaptor protein Inducing interFeron - β / TIR- Containing Adaptor Molecule, TRAM TRIF-related Adaptor Molecule

160. TLR-4 recognizes bacterial lipopolysaccharide in association with the host accessory proteins MD-2 and CD14. In the case of TLR4, LPS first binds to the CD14 receptor, which then transfers it to TLR4. TLR4 homodimerizes and forms a complex with the protein MD2. Cells need both MD2 and TLR4 in order to recognize LPS.

161. 2 pathways of signal transduction: The cytoplasmic Toll/IL-1 receptor (TIR) domain mediates downstream signalling through adaptor recruitment. Based upon adaptor usage TLR signalling can be divided into two categories; those that signal through the protein myeloid differentiation factor 88 (MyD88) and those that don’t. MyD88-dependent pathway that is common to all TLRs, MyD88-independent pathway-( TRIF based ) that is peculiar to the TLR3- and TLR4 signaling pathways

162. MyD88 dependent pathway Common to all TLRs except TLR3 Many ligands Adapter protein-MyD88 Production of inflammatory cytokines MyD88 independent pathway TRIF Pathway TLR3 & TLR4 Mainly dsRNA Adapter protein-TRIF Production of interferons

163. MYDDOSOME MyD88 TRIF Pro inflammatory cytokines

164. MAPK NF Κ B I Κ B IRF 3

165. NOD LIKE RECEPTORS NLRs are intracellular sensors for microbial products and activate NFKB to initiate the same inflammatory responses as the TLRs. NLR proteins typically contain a C-terminal part consists of a receptor domain, which is characterized by a series of leucine -rich repeats (LRRs). central nucleotide-binding domain termed NACHT/NOD domain N-terminal effector domains PYRIN- ---→NLRP subfamily caspase activation& recruitment domain-CARD-- →NLRC subfamily

166. NACHT LRR NACHT N-effector PYRIN CARD NLRP NLRC LRR

167. NOD 1 OR NOD 2---NLRC NLRP-INFLAMMASOME FORMATION

168. NLR SIGNALLING upregulation of NF κ B and activation of pro-inflammatory genes; By NOD1 & 2 (NLRC) Inflammasome formation, caspase-1 activation and secretion of IL-1 β and IL-18 By NLRP

169. The inflammasome . - protein complex that recognizes products of dead cells and some microbes and induces the secretion of biologically active interleukin 1. The inflammasome consists of a sensor protein (a leucine -rich protein called NLRP3) an adapter, The enzyme caspase-1,.

170. Cytoplasmic NOD proteins reside In the cytoplasm In Inactive form Binding of bacterial ligands to NOD proteins induces recruitment of RIPK2, which activates TAK1, leading to NFKB activation

171. Cytoplasmic NOD proteins reside In the cytoplasm In Inactive form Binding of bacterial ligands to NOD proteins induces recruitment of RIPK2, which activates TAK1, leading to NFKB activation

172. RIG LIKE RECEPTORS The RIG-I-like receptors (RLRs) are a family of cytosolic pattern recognition receptors that are essential for detecting viral RNA The RLR family includes three members: Retinoic acid-inducible gene I (RIG-I) Melanoma differentiation-associated gene 5 (MDA5) Laboratory of genetics and physiology 2 (LGP2). These receptors are expressed in both immune and non-immune cell types and regulate signaling pathways that promote the expression of type I and type III interferons (IFNs) NF-kappa B-dependent expression of pro-inflammatory cytokines.

173. RIG LIKE RECEPTORS The RLR family includes three members: Retinoic acid-inducible gene I (RIG-I) Melanoma differentiation-associated gene 5 (MDA5) Laboratory of genetics and physiology 2 (LGP2). RIG-I binds preferentially to  short dsRNA MDA-5 recognizes preferentially long dsRNA

174. . .   ADAPTOR-MAVS/IPS (Mitochondrial Antiviral Signalling/Interferon-b Promoter Stimulator 1)

175. C-type lectin receptors (CLRs) comprise a large family of receptors that bind to carbohydrates in a calcium-dependent manner. . CLRs recognize carbohydrates on microorganisms such as viruses, bacteria, and fungi C – type LECTIN RECEPTORS Membrane bound

176. MANNOSE BINDING LECTIN MANNOSE BINDING LECTIN RECEPTORS also known as mannan -binding protein synthesized by the liver and released into the bloodstream as part of the acute phase response

177. INFLAMMATION KILLING OF MICROBE OPSONISATION

178. production of a variety of activated complement proteins trigger inflammation, Opsonisation- chemotactically attract phagocytes lysis of gram-negative bacteria and infected or transformed human cells bind to the carbohydrates on bacteria, yeast, some viruses, and some parasites) activates the lectin complement pathway

179. ENDOCYTIC PRR

180. members of the C-type lectin -like family MANNOSE RECEPTOR (CD206) expressed by macrophages and dendritic cells, recognizes various mannosylated ligands,including some present on fungi, bacteria, and viruses; The mannose receptor is an endocytic receptor; it constantly recycles between the plasma membrane and the early endocytic compartment. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

181. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

182. recognize various anionic polymers and acetylated low-density lipoproteins. six different molecular families. Class A scavenger receptors membrane proteins composed of trimers of collagen domains. They include SR-A I SR-A II, MARCO (macrophage receptor with a collagenous structure which all bind various bacterial cell-wall components and help to internalize bacteria. Class B scavenger receptors bind high -density lipoproteins, and they internalize lipids SCAVENGER RECEPTOR Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

183. crucial importance in macrophage and neutrophil phagocytosis bind complement-coated microbes The complement receptor CR3 also directly recognizes and phagocytoses microbes bearing β - glucans . COMPLEMENT RECEPTOR Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

184. G-PROTEIN-COUPLED RECEPTORS( fMLP receptor) The fMet-Leu-Phe ( fMLP ) receptor Bacterial Polypeptides receptor binding activate intracellular signaling pathways that direct the cell to move toward the most concentrated source of the ligand . induces the production of microbicidal reactive oxygen species i n the phagolysosome . Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

185. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

186. RECRUITMENT OF LEUCOCYTES TO THE SITE OF INFECTION REMOVAL OF THE OFFENDING AGENTS PHAGOCYTOSIS. recognition and attachment of the particle to be ingested by the leukocyte its engulfment , with subsequent formation of a phagocytic vacuole; and killing or degradation of the ingested material

187. PHAGOCYTOSIS.

188. PHAGOCYTOSIS. initiated when certain receptors on the surface of the phagocyte bind to components of a microbial surface. phagosome –( becomes acidified) phagolysosome Neutrophils also contain cytoplasmic granules called primary and secondary granules , which fuse with the phagosome and contain additional enzymes and antimicrobial peptides that attack the microbe

189. Mannose receptors Scavenger receptors receptors for various opsonins bind and ingest microbes PHAGOCYTOSIS . Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte

190. PHAGOCYTOSIS . Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte PHAGOCYTIC RECEPTOR- receptors that directly induce phagocytosis of bound particles Receptors that directly recogonises microbes Receptors that bind opsonised particles Mannose receptor Scavenger receptor SRA SRB MARCO Complement receptor Fc receptor

191. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte ATTACHMENT OF THE PHAGOCYTE TO THE MICROBE OR CELL Unenhanced attachment Enhanced attachment

192. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte ATTACHMENT OF THE PHAGOCYTE TO THE MICROBE OR CELL Unenhanced attachment the innate recognition of pathogen-associated molecular patterns or PAMPs – (common in microbial cell walls but not found on human cells ) -by means of endocytic pattern-recognition receptors , such as scavenger receptors and mannose receptors, on the surface of the phagocytes

193. Recognition And Attachment Of The Particle To Be Ingested By The Leukocyte ATTACHMENT OF THE PHAGOCYTE TO THE MICROBE OR CELL Enhanced attachment   attachment of microbes to phagocytes by way of an antibody molecule called IgG , the complement proteins C3b and C4b produced during the complement pathways  and acute phase proteins such as mannose-binding lectin (MBL) and C-reactive protein (CRP). Molecules such as, C3b, that promote enhanced attachment are called opsonins and the process is also known as opsonisation Enhanced attachment is much more specific and efficient than unenhanced.

194. Attachment: Role of Opsonins Attachment of foreign particles on the phagocytic surface is facilitated greatly by substances known as opsonins The best-known opsonins are immunoglobulin ( Ig ) G antibodies, complement components (or their activation products certain oligosaccharides terminating in mannose or galactose opsonins bind to the surface of the phagocyte by specific receptors: Fc receptors ( FcRs ) for the IgG ( FcγR ), complement receptors (CRs) for complement components, mannose receptor for the mannose-terminating oligosaccharides.

195. PHAGOCYTOSIS . its engulfment , with subsequent formation of a phagocytic vacuole Particle internalization requires activation of a number of signaling pathways that together orchestrate rearrangement of the actin cytoskeleton, extension of the plasma membrane, and fusion to form a phagolysosome . Phagocytosis is dependent on polymerization of actin filaments

196. Protein kinase C (PKC )- required at the earliest stages of particle internalization: it participates in the formation of actin filaments beneath the site of particle binding Phosphoinositide (PI)–3 kinase and phospholipase C (PLC) PI-3 kinase is required for membrane extension and fusion behind the particle Rho guanine triphosphate (GTP) ases . Rho family of GTPases coordinate actin dynamics during cell adhesion and motility and participate in a variety of signaling cascades, including activation of MAP kinases and activation of transcription factors

197. 2 MECHANISMS OF PHAGOCYTOSIS ZIPPER MECHANISM TRIGGER MECHANISM

198. Once the phagosome is formed, it is pulled to the interior of the cell by actin filaments and myosins As the phagosome is internalized, it matures, i.e., changes the composition of its membrane. During maturation, the phagosomes progressively acquire a variety of protein Early in the maturation process, an ATPase acidifies the phagosomal vacuole-2 SCHOOLS OF THOUGHTS “kiss and run” MODEL coalesce /exchange components by means of carrier vesicles

199. During phagocytosis the phagocytic cell undergoes an increase in glucose and oxygen consumption termed the respiratory burst. The respiratory burst generates several oxygen-containing compounds that kill the bacteria undergoing phagocytosis – oxygen-dependent intracellular killing. Bacteria can also be killed by pre-formed substances released from granules or lysosomes upon bacterial fusion with the phagosome – oxygen-independent intracellular killing .

200. PHAGOCYTOSIS . REACTIVE NITROGEN SPECIES

201. REACTIVE NITROGEN SPECIES(RNS) Formation of nitric oxide: By the breakdown of arginine in the presence of enzyme nitric oxide synthase Three tissue-specific NO synthases neuronal ( Nnos inducible ( iNOS ) –in cells of the immune system, macrophages – responsible for the overproduction of NO endothelial ( eNOS )

202. 1. Oxygen-dependent myeloperoxidase-independent intracellular killing . During phagocytosis, glucose is metabolized via the pentose monophosphate shunt, with formation of NADPH. Cytochrome B from the granulocyte-specific granule combines with and activates plasma membrane NADPH oxidase

203. 2.  Oxygen-dependent myeloperoxidase-dependent intracellular killing Fusion of azurophilic   granules  with the phagosome causes release of myeloperoxidase into the phagolysosome

204. 2.   Detoxification reactions   Granulocyte  self-protection

205. by pre-formed substances released from granules or lysosomes

206. Macrophages do not possess myeloperoxidase and so cannot make hypochlorite ion; they do produce hydrogen peroxide and superoxide by respiratory burst. Certain organisms such as the agents of tuberculosis, brucellosis, and toxoplasmosis are preferentially ingested by macrophages rather than PMNs and may remain viable and multiply within these cells; granulomas formed during these infections contain many of these macrophages

207. NO and superoxide radical combine to form strong oxidizing agent peroxynitrite : Peroxynitrous acid breaks down to form additional ROS/RNS (hydroxyl radical and nitrogen dioxide) L-Arginine L- Citrulline + NOS

208. NEUTROPHIL EXTRACELLULAR TRAPS (NET) extracellular fibrillar networks that provide a high concentration of antimicrobial substances at sites of infection and prevent the spread of the microbes by trapping them in the fibrils .

209. NEUTROPHIL EXTRACELLULAR TRAPS (NET) chromatin fibers with a diameter of 15–17 nm; DNA and histones additional proteins associated with NETs, including components from various types of granules. COMPOSITION fully hydrated NETs have a cloud-like appearance occupy a space that is 10–15-fold bigger than the volume of the cells they originate from Mesh like appearance, studded with globular proteins

210. NEUTROPHIL EXTRACELLULAR TRAPS (NET) INDUCERS OF NET FORMATION Infections with bacteria, fungi, and HIV parasites induce NETs. - microbial components such as lipopolysaccharide ROS like hydrogen peroxide antibody–antigen complexes platelets activated via Toll-like receptor

211. NEUTROPHIL EXTRACELLULAR TRAPS (NET) THE MECHANISM OF NET FORMATION NETs are the results of a unique form of cell death that morphologically is characterized by the loss of intracellular membranes before the integrity of the plasma membrane is compromised. “ NETosis ” - neutrophil cell death that leads to the formation of NETs

212. NEUTROPHIL EXTRACELLULAR TRAPS (NET) THE MECHANISM OF NET FORMATION

213. NEUTROPHIL EXTRACELLULAR TRAPS (NET) Recent studies- NETs can result from the release of nuclear fragments and then their chromatin without compromising the plasma membrane

214. NEUTROPHIL EXTRACELLULAR TRAPS (NET)

215. NETs control infection by “trapping,” preventing dissemination, inactivating virulence factors, and eliminating microbes. Trapping microbes prevents their dissemination from the initial infection site. Microbes stick to NETs through charge interactions pathogens mask themselves with a capsule or by changing their surface charge, thus preventing binding to NETs Bacteria also attach nucleases to their surfaces to disengage themselves from NETs NEUTROPHIL EXTRACELLULAR TRAPS (NET) ANTIMICROBIAL ACTIVITY

216. NEUTROPHIL EXTRACELLULAR TRAPS (NET) ANTIMICROBIAL ACTIVITY NETs can inactivate microbial proteins, called “virulence factors,” that modify the function of host cells NETs contain several proteins that kill or inhibit microbes. These include enzymes (lysozyme, proteases), antimicrobial peptides (BPI, defensins ), ion chelators ( calgranulin -high local concentration of antimicrobial components

217. Eventually, NETs are removed during the resolution of inflammation. NETs are susceptible to DNase1 It is not known what happens to the debris left by DNase1 but perhaps phagocytes, macrophages, and neutrophils newly recruited to the inflammatory site clean up the mess

218. NEUTROPHIL EXTRACELLULAR TRAPS (NET) THE DARK SIDE OF NET AUTOIMMUNE DISEASE - The nuclear chromatin in the NETs is a source of nuclear antigens in systemic autoimmune diseases, particularly lupus CYSTIS FIBROSIS -persistent neutrophil-rich inflammation that destroys the lungs-Treated with DNAses CLOTTING & ISCHAEMIA -scaffold for the stimulation of thrombus formation  PERIODONTITIS - P.gingivalis   recruit neutrophils into the gingival Histones-damage endothelium Association with atherosclerosis

219. LEUKOCYTE MEDIATED TISSUE INJURY In some infections that are difficult to eradicate, such as tuberculosis and certain Viral diseases, the prolonged host response contributes more to the pathology than does the microbe itself inflammatory response is inappropriately directed against host tissues , as in certain autoimmune diseases. host reacts excessively against usually harmless environmental substances, as in allergic diseases, including asthma.

220. ROS, NO, and lysosomal enzymes etc produced within the phagolysosome are also released into the extracellular space When phagocytes encounter materials that cannot be easily ingested , such as immune complexes deposited on immovable flat surfaces ( e.g ., glomerular basement membrane), the inability of the leukocytes to surround and ingest these substances ( frustrated phagocytosis ) triggers strong activation, and the release of large amounts of lysosomal enzymes into the extracellular environment. Some phagocytosed substances, such as urate crystals, may damage the membrane of the phagolysosome

221. TERMINATION OF ACUTE INFLAMMATORY RESPONSE offending agents are removed Mediators of inflammation are produced in rapid bursts , only as long as the stimulus persists, have short half-lives , and are degraded after their release

222. FATE OF ACUTE INFLAMMATION

223. MORPHOLOGIC PATTERNS OF ACUTE INFLAMMATION

224. SEROUS INFLAMMATION outpouring of a thin fluid that may be derived from the plasma or from the secretions of mesothelial cells lining the peritoneal, pleural, and pericardial cavities. Accumulation of fluid in these cavities is called an effusion . EX: skin blister resulting from a burn or viral infection

225. FIBRINOUS INFLAMMATION ↑VASCULAR PERMEABILITY (LARGE VASCULAR LEAKS) FIBRIN FORMED & DEPOSITED IN EXTRACELLULAR SPACE FIBRINOGEN LEAKS THROUGH VESSEL

226. characteristic of inflammation in the lining of body cavities , such as the meninges, pericardium Histologically, fibrin appears as an eosinophilic meshwork of threads or sometimes as an amorphous coagulum

227. Fibrinous exudates fibrinolysis and clearing of other debris by macrophages. not removed stimulate the ingrowth of fibroblasts and blood vessels scarring obliteration of the pericardial space

228. SUPPURATIVE/PURULENT INFLAMMATION characterized by the production of large amounts of pus or purulent exudate consisting of neutrophils, liquefactive necrosis, and edema fluid. pyogenic (pus-producing) bacteria Ex: acute appendicitis. Abscesses are localized collections of purulent inflammatory tissue caused by suppuration buried in a tissue, an organ, or a confined space. Central region-necrotic leucocyte, tissue cells Surrounded by- neutrophis Outermost-vascular dilatation, parenchymal&firoblastic proliferation

229. ULCER local defect, or excavation, of the surface of an organ or tissue that is produced by the sloughing (shedding) of inflamed necrotic tissue can occur only when tissue necrosis and resultant inflammation exist on or near a surface. It is most commonly encountered in (1) the mucosa of the mouth, stomach, intestines, or genitourinary tract; (2) the skin and subcutaneous tissue of the lower extremities in older persons who have circulatory disturbances that predispose to extensive ischemic necrosis. Ex: peptic ulcer (acute and chronic inflammation co-exist)

230. REFERENCES ROBBINS & COTRAN PATHOLOGIC BASIS OF DISEASES JANEWAY’S IMMUNOLOGY MIDDLETONS ALLERGY PRINCIPLES AND PRACTICE KUBY’S IMMUNOLOGY Kumar H, Kawai T, Akira S. Pathogen recognition in the innate immune response. Biochemical Journal. 2009 May 15;420(1):1-6. Jopeace AL, Howard CB, Murton BL, Edwards AD, Monie TP. Pattern recognition receptors and infectious diseases. InInsight and Control of Infectious Disease in Global Scenario 2012. InTech . Abdallah DS, Denkers EY. Neutrophils cast extracellular traps in response to protozoan parasites. Frontiers in immunology. 2012;3.