Inflammation 3

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  • After traversing the endothelium PMNs secrete collagenases to dissolve basement membrane
    Then they enter the interstitium and reach the site of injury by travelling along the concentration gradient created by the chemokines - Chemotaxis
  • Towards & along: this is unidirectional movement
  • This is a diagram showing the effect of chemokine concentration gradient on chemotaxis direction. The attracted cell moves through the gradient toward the higher concentration of chemokine.
  • CD44 is on the leucocytes which binds with heparin sulfate on the endothelium / fibronectin in the interstitium.
    CD44 also can bind to ICAM1 or VCAM present on endothelium (belong to Ig family)
  • Details of Leukocyte Chemotaxis:
    Neutrophil adheres via integrin binding
    Integrin binds to FN in ECM
    Actually recognizes RGD sequence (arg-gly-asp) in FN (not shown in figure)
    RGD is also found in other ECM proteins
    Integrin is recycled
  • Nitric oxide, which is released from endothelial cells and macrophages and produces vasodilatation and cytotoxicity.
  • Phagocytic cells are either microphages (neutrophils) or macrophages (monocytes from blood or histiocytes from tissues). The process of phagocytosis involves
    a.Adhesion of opsonized particles to Fc fragment receptors on the surface of the phagocyte.
    b. Engulfment by extending pseudopodia around the particle. Fusion of these pseudopodia will create a heterophagic vacuole formed by internalized plasmalemma.
    c. Fusion of the lysosomes with the phagocytic vacuole.
    d. Intracellular microbial killing
  • Phagocytic cells are either microphages (neutrophils) or macrophages (monocytes from blood or histiocytes from tissues). The process of phagocytosis involves
    a.Adhesion: attachement of particles to cell surface. This is aided by opsonins. Fc fragment receptors on the surface of the phagocyte.
    b. Engulfment by extending pseudopodia around the particle. Fusion of these pseudopodia will create a heterophagic vacuole formed by internalized plasmalemma.
    c. Fusion of the lysosomes with the phagocytic vacuole.i.e. phagolysosome formation (pH=4)
    d. Degradation / Intracellular microbial killing: results in residual body or extrusion.
  • Phagocytic cells are either microphages (neutrophils) or macrophages (monocytes from blood or histiocytes from tissues). The process of phagocytosis involves
    a.Adhesion: attachement of particles to cell surface. This is aided by opsonins. Fc fragment receptors on the surface of the phagocyte.
    b. Engulfment by extending pseudopodia around the particle. Fusion of these pseudopodia will create a heterophagic vacuole formed by internalized plasmalemma.
    c. Fusion of the lysosomes with the phagocytic vacuole.i.e. phagolysosome formation (pH=4)
    d. Degradation / Intracellular microbial killing: results in residual body or extrusion.
  • Phagocytic cells are either microphages (neutrophils) or macrophages (monocytes from blood or histiocytes from tissues). The process of phagocytosis involves
    a.Adhesion: attachement of particles to cell surface. This is aided by opsonins. Fc fragment receptors on the surface of the phagocyte. Attachemnt immobilizes particles and facilitates engulfment.
    b. Engulfment by extending pseudopodia around the particle. Fusion of these pseudopodia will create a heterophagic vacuole formed by internalized plasmalemma.
    c. Fusion of the lysosomes with the phagocytic vacuole.i.e. phagolysosome formation (pH=4)
    d. Degradation / Intracellular microbial killing: results in residual body or extrusion.
  • Defects in leucocyte function: Defects inleucocyte function, both fenetic and acquired, lead to increased vulnerability to infections:
    --- Defects in leucocyte adheshion
    --- Defects in phagolysosome function. One such disorder is Chediak-Higashi syndrome, an AR condition characterized by neutropenia, defective degranulation and delayed microbial killing
    --- Defects in microbicidal activity: The importance of exygen dependent bactericidal mechanism is shown by the exstence of a group of congenital disrders with defects in bacterial killing called CGD
  • Defects in leucocyte function: Defects inleucocyte function, both genetic and acquired, lead to increased vulnerability to infections:
    GENETIC
    --- LAD1 beta chain of CD11/CD18 integrins
    --- LAD2 fucosyl tranferase required for synthesis of sialyated oligosccharide (receptor for selectin)
    --- CGD (decreased oxidative burst)
    X-linked (NADPH oxidase – membrane component)
    Autosomal recessive (NADPH oxidase - cytoplasmic)
    MPO deficiency (absent MPO-H2O2 system)
    --- Chediak-Higashi syndrome (protein involved in organelle membrane fusion)
  • Defects in leucocyte function: Defects inleucocyte function, both genetic and acquired, lead to increased vulnerability to infections:
    ACQUIRED
    -- thermal injury, diabetes, malignancy, sepsis, immunodeficiencies (chemotaxis)
    -- Hemodialysis, diabetes mellitus (Adhesion)
    -- Leukemia, anemia, sepsis, diabetes, neonates, malnutrition (phagocytosis and microbicidal activity)
  • Adhesion cascade in leukocyte adhesion deficiency (LAD) syndromes.
    <emedicine.medscape.com/article/886248-media>
  • Introduction to Chemokine Families and the Cells They Affect :
    Chemokines (Greek -kinos, movement) are a family of small cytokines, or proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. Proteins are classified as chemokines according to shared structural characteristics such as small size (they are all approximately 8-10 kilodaltons in size), and the presence of four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. However, these proteins have historically been known under several other names including the SIS family of cytokines, SIG family of cytokines, SCY family of cytokines, Platelet factor-4 superfamily or intercrines. Some chemokines are considered pro-inflammatory and can be induced during an immune response to promote cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines are found in all vertebrates, some viruses and some bacteria, but none have been described for other invertebrates. These proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.
  • In biochemistry and pharmacology, a ligand (Latin ligare = to bind) is a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose. In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein.
    The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. In contrast to the meaning in metalorganic and inorganic chemistry, it is irrelevant whether the ligand actually binds at a metal site, as it is the case in hemoglobin.
    Ligand binding to a receptor alters the chemical conformation, that is the three dimensional shape of the receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. Ligands include substrates, inhibitors, activators, and neurotransmitters. The tendency or strength of binding is called affinity.
    Radioligands are radioisotope labeled compounds and used in vivo as tracers in PET studies and for in vitro .
  • This is a diagram showing the effect of chemokine concentration gradient on chemotaxis direction. The attracted cell moves through the gradient toward the higher concentration of chemokine.
  • Inflammation 3

    1. 1. Inflammation and Repair - 3 Dr.CSBR.Prasad, M.D. May-2015-CSBRP
    2. 2. DIAPEDESISDIAPEDESIS (Transmigration)(Transmigration) Migration of Leukocytes through the interendothelial gaps May-2015-CSBRP
    3. 3. DiapedesisDiapedesis • It occurs mostly thru post capillary venules • Chemokines stimulate the PMNs to emigrate into interstitium • PMNs leave the vessel thru inter endothelial gaps • In the interstitium they travel towards the site of injury May-2015-CSBRP
    4. 4. Adhesion molecules (AM) involved in diapedesis • AM present in between the endothelial cells and in the interstitium facilitate this process • These include: • PECAM-1 or CD31 • Several junctional AMs May-2015-CSBRP
    5. 5. DiapedesisDiapedesis • Must then cross basement membrane – Collagenases • PMNs secrete collagenases to dissolve basement membrane • Then they reach the site of injury by Chemotaxis May-2015-CSBRP
    6. 6. Summary of DIAPEDESIS Insertion of pseudopodia which widens the intercellular gaps Passage through the gaps by ameboid movement Passage through the basement membrane either by mechanical disruption or possibly by enzyme (collagenase) effect May-2015-CSBRP
    7. 7. May-2015-CSBRP WOW! PECAM-1 or CD 31
    8. 8. Neutrophil Transendothelial Migration (Diapedesis) May-2015-CSBRP
    9. 9. May-2015-CSBRP
    10. 10. May-2015-CSBRP
    11. 11. Chemotaxis of LeukocytesChemotaxis of Leukocytes May-2015-CSBRP
    12. 12. Chemotaxis In the intestitium leucocytes reach the site of injury by traveling along the concentration gradient created by the chemokines - Chemotaxis May-2015-CSBRP
    13. 13. Chemotaxis Def: Chemotaxis is a process by which the leucocytes travel towards and along the chemical concentration gradient Concentration gradient is created by chemokines Highest concentration occurs at the center of injury May-2015-CSBRP
    14. 14. This is a diagram showing the effect of chemokine concentration gradient on chemotaxis direction. The attracted cell moves through the gradient toward the higher concentration of chemokine. May-2015-CSBRP Neutrophil Bacteria
    15. 15. Chemotaxis • Leukocytes follow chemical gradient to the site of injury • Chemotactic agents: include – Soluble bacterial products – Complement components (C5a) – Cytokines (chemokine family e.g., IL-8) – LTB4 (AA metabolite) • Chemotactic agents bind surface receptors inducing calcium mobilization and assembly of cytoskeletal contractile elements May-2015-CSBRP
    16. 16. May-2015-CSBRP
    17. 17. Chemotactic substances will bind to leukocyte receptors, initiating a stimulus – receptor interaction that leads to activation of intracellular contractile proteins May-2015-CSBRP
    18. 18. Leukocytes: –Extend pseudopods with overlying surface adhesion molecules (integrins) that bind ECM during chemotaxis –Leucocytes walk towards the injury May-2015-CSBRP
    19. 19. Chemotaxis May-2015-CSBRP
    20. 20. When they reach the site… • PMNs able to adhere to the intercellular matrix by binding of integrins to CD44 • By this mechanism they are retained at the site where they are needed most May-2015-CSBRP
    21. 21. Details of Leukocyte Chemotaxis: Neutrophil adheres via integrin binding Integrin binds to FN in ECM Actually recognizes RGD sequence (arg-gly-asp) in FN (not shown in figure) RGD is also found in other ECM proteins Integrin is recycled May-2015-CSBRP
    22. 22. PHAGOCYTOSISPHAGOCYTOSIS May-2015-CSBRP
    23. 23. PHAGOCYTOSISPHAGOCYTOSIS Ingestion and processing of particulate material by phagocytic cells –Particulate matter can be: • Tissue debris, bacteria, other foreign cells –Phagocytic cells: • PMNs • MØ May-2015-CSBRP
    24. 24. PHAGOCYTOSISPHAGOCYTOSIS Phagocytic cells are two types: 1.Microphages (neutrophils) or 2.Macrophages (monocytes / histiocytes) May-2015-CSBRP
    25. 25. PHAGOCYTOSISPHAGOCYTOSIS The process of phagocytosis involves: 1. Adhesion – Immobilizes the particles 2. Engulfment by extending pseudopodia 3. Fusion - Phagolysosome formation of the lysosomes with the phagocytic vacuole 4. Degradation / Intracellular microbial killing May-2015-CSBRP
    26. 26. PhagocytosisPhagocytosis Recognition and attachment of the particle to be ingested Engulfment formation of a phagocytic vacuole Killing degradation May-2015-CSBRP
    27. 27. Process of Phagocytosis May-2015-CSBRP
    28. 28. May-2015-CSBRP
    29. 29. PHAGOCYTOSISPHAGOCYTOSIS The process of phagocytosis: Adhesion • Collectins, C3b, Fc portion of Ig Opsonization: Coating the particle by OPSONINS • Complement C3b • Immunoglobulins IgG Opsonization facilitates phagocytosis May-2015-CSBRP
    30. 30. What isWhat is C3bC3b andand FcFc ?? May-2015-CSBRP
    31. 31. May-2015-CSBRP
    32. 32. May-2015-CSBRP
    33. 33. PHAGOCYTOSISPHAGOCYTOSIS The process of phagocytosis: Opsonins: Like sauce making the bread palatable, opsonins make particles palatable for phagocytes Opsonization facilitates phagocytosis PMNs and MØ has receptors for C3b & Fc May-2015-CSBRP
    34. 34. ActivationActivation May-2015-CSBRP
    35. 35. ActivationActivation • When the leucocytes gets stimulated by the cytokines, they get activated • With activation: – AM are modulated / potentiated – Chemotaxis is potentiated – Elaborate AA metabolites – Secretion / Degranulation – Oxidative out burst May-2015-CSBRP
    36. 36. Activation The biologic activities resulting from leukocyte activation include Chemotaxis Modulation of AM Elaboration of AA Metabolites Secretion / Degranulation Oxidative burst May-2015-CSBRP
    37. 37. Defects in Leucocyte Function Can be genetic / acquired Results in increased vulnerability to infections • Defects in leucocyte adheshion • Defects in phagolysosome function – Chediak - Higashi syndrome • Defects in microbicidal activity – Chronic Granulomatous Disease May-2015-CSBRP
    38. 38. Defects in Leucocyte Function GENETIC • LAD1 beta chain of CD11/CD18 integrins • LAD2 fucosyl tranferase required for synthesis of sialyated oligosccharide (receptor for selectin) • CGD (decreased oxidative burst) – X-linked (NADPH oxidase – membrane component) – Autosomal recessive (NADPH oxidase - cytoplasmic) – MPO deficiency (absent MPO-H2O2 system) • Chediak-Higashi syndrome (protein involved in organelle membrane fusion) May-2015-CSBRP
    39. 39. Defects in Leucocyte Function ACQUIRED • Thermal injury, diabetes, malignancy, sepsis, immunodeficiencies • Chemotaxis • Hemodialysis, diabetes mellitus • Adhesion • Leukemia, anemia, sepsis, diabetes, neonates, malnutrition • Phagocytosis and Microbicidal activity May-2015-CSBRP
    40. 40. E N D May-2015-CSBRP
    41. 41. Laboratory FindingsLaboratory Findings in Inflammationin Inflammation May-2015-CSBRP
    42. 42. Complete Blood Count (CBC) Total RBC Total WBC Hemoglobing(HB) Hematocrit (HCT) RBC Indices: MCV,MCH,MCHC WBC DifferentialMay-2015-CSBRP
    43. 43. Laboratory Findings in Inflammation Leukocytosis :15,000-20,000 cells/ul (4,000-10,000 cells/ul) Leukemoid reaction:40,000-100,000 cells/ul May-2015-CSBRP
    44. 44. Laboratory Findings in Inflammation “Left Shift”: an increase in the number of immature neutrophils Immature neutrophils: Bands or stabs Meta or Juvenile Myleocyte May-2015-CSBRP
    45. 45. “Left Shift” Baso Eos Meta Stabs Segs Lymph Mono 70 20 3321 1 Normal 75 8 11210 3 Left Shift May-2015-CSBRP
    46. 46. Laboratory Findings in Inflammation Neutrophilia : bacterial Lymphocytosis : viral Leukopenia:many viruses Eosinophilia: parasites & allergies May-2015-CSBRP
    47. 47. Laboratory Findings in Inflammation Erythrocyte Sedimentation Rate (ESR) will be increased May-2015-CSBRP
    48. 48. Erythrocyte Sedimentation Rate (ESR) 0 10 20 30 40 50 60 70 80 90 100 mm 1hr The distance, in mm, the RBC fall in 1 hr is the Sed Rate 0 10 20 30 40 50 60 70 80 90 100 mm May-2015-CSBRP
    49. 49. Acute Phase Proteins During an inflammatory response a number of interleukins(IL) are produced IL-6 stimulates the hepatic production of a number of proteins ,called acute phase proteins May-2015-CSBRP
    50. 50. Acute Phase Proteins Fibrinogen C-Reactive Protein (CRP) C3 C4 Ceruloplasmin May-2015-CSBRP
    51. 51. Acute Phase Proteins Acute Phase Proteins are normally found in the blood at low concentrations, but following hepatic stimulation by IL-6 their concentration increases Detection of elevated levels of acute phase proteins is an indication of an inflammatory response May-2015-CSBRP
    52. 52. May-2015-CSBRP
    53. 53. May-2015-CSBRP
    54. 54. May-2015-CSBRP
    55. 55. May-2015-CSBRP
    56. 56. May-2015-CSBRP
    57. 57. May-2015-CSBRP
    58. 58. May-2015-CSBRP
    59. 59. May-2015-CSBRP
    60. 60. Inflammatory Paracrines • What causes the characteristic sequence of events in acute inflammation? Various cells at the site of tissue damage or of a specific immune response release regulatory molecules that act locally as paracrines. • Macrophages and lymphocytes are important sources of inflammatory paracrines. As we have discussed, macrophages release IL-1 and TNF-alpha, which have powerful, widespread effects. • Also important are mast cells, which are found throughout the body, especially under epithelia. Mast cells are filled with large vesicles containing histamine and other inflammatory paracrines (They also release PG D2, several LTs and TNF-alpha, described below). Factors associated with tissue damage can trigger the exocytosis. But sometimes it is a specific immune response that triggers the release of the inflammatory paracrines. • Also, various arachidonic acid derivatives are important. Both prostaglandins (notably PG D2) and leukotrienes (LT) can be important, depending on the tissue. Note the effectiveness of aspirin and various NSAIDs in quieting inflammation. • Complement peptides, C3a and C5a • Various other molecules including nitric oxide, certain platelet products, kinins, and certain other substances we will not discuss (serotonin, etc) May-2015-CSBRP
    61. 61. What types of molecules trigger inflammation? • Inflammatory paracrines May-2015-CSBRP
    62. 62. Name some cells that release the paracrine molecules. • Mast cells • Macrophages • Almost every cell can release AA derivatives May-2015-CSBRP
    63. 63. What is complement C5a? When the complement system is activated, a small peptide C5a is cleaved from the protein C5. C5a readily diffuses, causing chemotaxis and inflammation in general May-2015-CSBRP
    64. 64. What change caused by inflammatory paracrines results in edema in the affected area? Increased capillary permeability May-2015-CSBRP
    65. 65. Suppose tissue damage triggers the release of a prostaglandin that causes inflammation in the area. What specific molecule does the tissue damage activate that starts the synthesis of the prostaglandin? Phospholipase A2 Other enzymes, including COX, then convert the arachidonic acid to the prostaglandin May-2015-CSBRP
    66. 66. May-2015-CSBRP
    67. 67. The Role of Chemokines in Homing to Inflammation May-2015-CSBRP
    68. 68. Tissue Distribution of Chemokines May-2015-CSBRP
    69. 69. Introduction to Chemokine Families and the Cells They Affect May-2015-CSBRP
    70. 70. May-2015-CSBRP
    71. 71. May-2015-CSBRP
    72. 72. Ligands • In biochemistry and pharmacology, a ligand (Latin ligare = to bind) is a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose. • In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein. • The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. In contrast to the meaning in metalorganic and inorganic chemistry, it is irrelevant whether the ligand actually binds at a metal site, as it is the case in hemoglobin. • Ligand binding to a receptor alters the chemical conformation, that is the three dimensional shape of the receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. Ligands include substrates, inhibitors, activators, and neurotransmitters. The tendency or strength of binding is called affinity. • Radioligands are radioisotope labeled compounds and used in vivo as tracers in PET studies and for in vitro . May-2015-CSBRP
    73. 73. This is a diagram showing the effect of chemokine concentration gradient on chemotaxis direction. The attracted cell moves through the gradient toward the higher concentration of chemokine. May-2015-CSBRP
    74. 74. WOW! The movement of leucocytes from out of the blood vessels into the tissues spaces is known as DIAPEDESIS May-2015-CSBRP
    75. 75. WOW! The movement of leucocytes from out of the blood vessels into the tissues spaces is known as DIAPEDESIS May-2015-CSBRP

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