Elements of the Immune System

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Elements of the Immune System

• Cells of the Immune System
• Chemical Mediators
• Cytokines, Chemokines, Complement, Eicosanoids
• Apoptosis
• Acute Phase Response
• Inflammation
• Fever
• Innate/Specific Immunity

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Cells of the Immune System

• Cells of the Immune System
  (white blood cells WBCs,
  leukocyte)
• Hematopoietic stem cells to
• Myeloid cell line
• Monocytes/macrophages, neutrophils, eosinophils,
  basophils, megakaryocytes,
• erythrocytes (red blood cells RBCs)
• Lymphoid cell line
• B lyphocytes , T lymphocytes, natural killer (NK)
  cells

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Cells of the Immune SystemHematopoietic cell
differentiation Fig 11-1
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Cells of the Immune System

• White blood cells distinguished by
• Morphology/stain
• Function
• Cell surface markers
• Clusters of differentiation (CD)
• Major histocompatibility complex (MHC) antigens
• MHC I (CD8) on all nucleated cells
• MHC II (CD4) on antigen presenting cells

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Cells of the Immune SystemLymphoid Organs

• Primary lymphoid organs (sites of hematopoiesis)
• Bone marrow
• Thymus T cell maturation
• Secondary lymphoid organs (sites of T cell
  activation)
• Lymph nodes
• Peyers patches
• Tonsils
• Spleen

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Cells of the Immune System

• Polymorphonucleocytes (PMNs) or granulocytes
  refers to neutrophils, eosinophils and basophils.
  Howerver, term PMN generally refers to the
  most abundant of these, neutrophils
• Neutrophils PMNs segmented neutrophils
  (segs)
• Take up hemotoxylin (basic) and eosin (acid)
  (HE) stains pink
• multilobed
• 50-70 of circulating WBCs
• Professional phagocytic cell
• (Immature) band (unsegmented) forms to mature
  segmented neutrophils
• Bacterial infection mobilization increased
  number of neuts with some bands shift to the
  left
• In blood 7h, in tissues 3 days
• 1st line defense against bacteria but short lived

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Cells of the Immune System

• Neutrophils continued
• Granules lysosomes
• Primary (azurophilic)
• O2-independent Elastase, lysozyme, defensins,
• O2-dependent myeloperoxidase
• Secondary (specific)
• O2-independent lactoferrin, lysozyme
• O2-dependent NADPH oxidase cofactors

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Cells of the Immune System

• Neutrophils continued
• Receptors for recognition of microbes by
  neutrophils
• Receptors for opsonins
• Receptors of complement (C) proteins
• Receptors for Fc portion of Abs
• Toll-like receptors (TLRs)
• Seven-transmembrane alpha helical receptors
  stimulate migration
• for bacterial peptides
• for chemokines

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Cells of the Immune System

• Mononuclear phagocytes
• Monocytes in blood (3-8 of WBC) differentiate
  into macrophages in tissues
• Macrophages (Macs)
• Professional phagocytic cells like neutrophils
• Terminology Kupffer cells (liver), histiocytes
  (CT), microglial cells (brain), osteoclasts,
  alveolar Macs
• Reticular Endothelial System (RES) old term
  referring to all monocytes and macrophages

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Cells of the Immune System

• Macrophages continued
• Contribute to both innate and adaptive immunity
• Monocytes in blood for 1 day Macs live
  months/years in tissues
• Unlike neutrophils, macs proliferate
• Antigen-presenting cell (APC)
• other APCs B cells and dendritic cells (DCs)
• Respond slower than PMNs but live longer in
  tissues
• Secrete many cytokines shock
• Activated (angry) Macs better killers than
  neutrophils
• Phagocytize more vigorously, use more O2, more
  enzymes,

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Cells of the Immune System

• Macrophages continued
• Recognition of microbes by macrophages
• Receptors for opsonins
• Receptors for C proteins
• Receptor for Fc portion of Abs
• Toll-like receptors
• Mannose receptors
• Bind terminal mannose fucose on bacterial
  glycolipids and gl-proteins
• Seven-transmembrane alpha helical receptors
  stimulate migration
• for bacterial peptides
• for chemokines

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Cells of the Immune System

• Toll-like receptors (TLRs) from Drysophila
  toll protein family of membrane proteins on
  PMNs, Macs, and dendritic cells that recognize
  microbial molecules.
• Stimulate innate immune responses by these cells
• TLR receptors on humans cells (TLR-1 TLR-13)
• Recognize pathogen-associated molecular patterns
  (PAMPs)
• LPS, peptidoglycan, teichoic acid,
  lipoarabinomannan, flagellin, fungal glycans,
  unmethylated cytosine-guanosine DNA, RNA2, other
  
• Some TLRs need second signal TLR-4 recognized
  LPS-CD 14

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Cells of the Immune System

• Receptors and responses of phagocytes

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Cells of the Immune System

• Phagocytosis/killing of microbes by neutrophils
  and Macs
• Phagocytosis
• Phagocytosis gt0.5 um pinocytosis for smaller
  particles
• Microbes adhere to surface receptors which
  deliver signals
• Cytoskeleton-dependent process of engulfment
• Cup-shaped membrane projects around microbe
• If projected membranes dont meet properly, get
  coiling phagocytosis?
• Vacuole zips-up and pinches off phagosome
• Phagocytosed microbes (hopefully) killed by neuts
  and Macs
• Phagocytosed microbial peptides presented in
  surface MHC II molecules of Macs (not
  neutrophils)

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Cells of the Immune System

• Coiling phagocytosis

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Cells of the Immune System

• Phagocytosis/killing of microbes by neutrophils
  and Macs
• Killing
• Phagocytes must be activated to kill
• Neuts activated by surface receptors TLR, Fc, C
  receptor
• Macs activated by those, mannose receptor, and
  receptor for IFN-g
• Phagosome fuses with lysosome phagolysosome
  killing site
• Oxygen-dependent killing by neutrophils and Macs
• Phagocyte oxidase in phagolysosome membrane
  reduces O2 into reactive oxygen intermediates
  (ROIs) such as superoxide with NADPH
• Superoxide dismuted to H2O2
• Neuts - myeloperoxidase converts H2O2 Cl ions
  to hypochlorous ions
• Macs inducible nitric oxide synthase (iNOS)
  catalyses conversion of arginine to citrulline,
  releasing diffusible NO gas. NO H2O2 or O2-
  toxic radicals
• Oxygen-independent killing by neutrophils
• enzymes from granules kill Gram negative
  organisms by disrupting membranes and other
  mechanisms

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Cells of the Immune System

• Neutrophils vs Macrophages
• Both are professional phagocytic cells
• Neutrophils move to insult first
• Macrophages (but not neutrophils) proliferate
• Macrophages (but not neutrophils) contribute to
  adaptive immunity
• Macrophages (but not neutrophils) are APCs
• Neutrophils (but not macrophages) produce
  myeloperoxidase
• Macrophages (but not neutrophils) produce nitric
  oxide
• Macrophages are major producers of cytokines
• Over-production of Mac cytokines shock

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Cells of the Immune System

• Mast Cells
• Derived from marrow
• Relatively large (10-13 um), tissue-residents
  cells
• Connective tissue skin, lung alveoli, GI mucosa,
  nasal mucosa
• Receptors (1) IgE antibody (2) TLRs (3)
  complement
• 100 densely packed metachromatic granules
  contain Histamine, TNFa, other preformed
  inflammatory mediators
• Activate receptors degranulation and release of
  mediators
• Inflammation
• Immediate hypersensitivity with cross-linking of
  IgE

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Cells of the Immune System

• Basophils
• Similar to mast cells but circulating, different
  lineage, and small (5-7 um)
• Least common circulating WBC - lt0.3
• Takes up hematoxylin (basic) stain blue
• High affinity IgE receptors triggered when Ag
  binds to IgE
• Late phase of IgE-associated allergic reactions
  in tissues

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Cells of the Immune System

• Eosinophils
• Take up eosin red
• 1-6 of circulating WBCs
• Granules with phosphatase, peroxidase, basic
  proteins
• Receptors for IgE, IgG, and IgA
• Allergic reactions and parasitic infections
• Release reactive proteins some toxic to parasitic
  worms
• Phagocytic cell ???

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Cells of the Immune System

• Megakaryocyte
• Large (50-100 um diam) WBC with lg nucleus
• Neutrophils have a diameter of 12-15 um
• In bone marrow
• Produces 3000 platelets per cell
• Platelets are anucleate cytoplasmic fragments
• Smallest circulating blood cells (2 um diam)
• Erythrocyes 6 um diam with 2 um thickness
• 10 day life span in circulation
• Form blood clots and release mediators from
  granules

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Cells of the Immune System

• Dendritic Cells (DCs) an antigen presenting
  cell (APC)
• Myeloid origin primarily
• Terminology
• Precursor DCs in circulation
• Immature DCs in tissues capture Ag but do not
  present to nodes
• Have sticky, octopus-like arms (dendrites)
• Fc receptors, mannose receptors, Toll-Like
  Receptors (TLRs)
• Langerhans cells in skin
• Mature DCs in T-cell zones of lymph nodes
  present Ag
• Fewer receptors but more MHC-I and II molecules
  on surface

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Cells of the Immune System

• Lymphocytes smaller than myeloid leukocytes lg
  nucleus and agranular cytoplasm
• B cells APCs antibody production (plasma
  cells) memory cells
• Surface with Igs, MHC II, C receptors
• T cells helper T cells and cytotoxic T cells
• 60-80 of blood lymphocytes
• Surface with TCR, CD3, CD4, CD8
• NK (natural killer) cells lg granular
• Fc receptors for ADCC
• Recognize and kill virally infected cells

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Cells of the Immune System

• Natural Killer (NK) cells
• Large granular lymphocyte (not B or T cells no
  surface Ig or TCR)
• 5-20 of monocytes in blood
• Stimulated by INFa, INFb, from virally infected
  cells
• Stimulated by TNF and ILs from TH1 cells and
  activated Macs and DCs
• Granules with perforin, granulysin (creates
  pores) and granzyme (induces apoptosis)
• Like cytotoxic T cells (but cytotoxic T cells
  adaptive immunity)
• Kills cells with reduced MHC I expression
• Activates Macs by secreting INFg
• No specific immunity or memory
• Similar to neutrophils and Macs in this respect
• Also IgG Fc receptors for antibody dependent cell
  cytotoxicity (ADCC)
• adaptive immunity (induced apoptosis)

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Cells of the Immune System

• Activation of responses How PMNs, Macs, NKs
  recognize bugs
• Macs and PMNs
• Opsonin receptors
• Receptors for bacterial carbohydrates (mannose) -
  Macs
• Toll-like receptors (TLRs)
• Natural Killer cells
• Altered surface of virally infected cells
• Reduced expression of MHC I antigen
• - MHC I on cells inhibits NK cell activation
• Receptor for IgG Fc component
• For ADCC

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Cytokines / Chemokines

• Cytokines proteins secreted by immune cells in
  response to microbes or other antigens (Ags) or
  other cytokines that mediate inflammatory and
  immune reactions. Major communicators between
  immune cells
• Chemokines family of structurally homologous
  low-molecular weight cytokines that stimulate
  movement of leukocytes to tissues

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Cytokines

• Interferons (INFs)
• Type I mediate innate response to viral
  infections
• INF-a family of related peptides produced by
  monocytes
• INF-b single protein produced by fibroblasts
  and other cells
• Although structurally different, bind to same
  receptor, same responses
• paracrine action virally infected cells secrete
  to non-infected cells
• induce enzymes that interfere with transcription
  of viral RNA/DNA
• increase expression MHC I molecules
• Type II / INF-g innate/adaptive response to
  intracellular infections
• Produced by T cells, NK cells
• Activates macrophages

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Cytokines

• Tumor necrosis factor alpha (TNF-a)
• - sometimes referred to as cachectin (induces
  weight loss)
• - produced mainly by activated monocytes /
  macrophages
• - NK cells, mast cells, activated T cells also
• - Recruit and activate neutrophils and
  monocytes/macrophages to sites
• - Induce macrophages to secrete chemokines
• - Stimulate vascular endothelial cells to express
  adhesion molecules
• Low conc local inflammation
• Large conc to circulation induce fever and
  promote acute phase response
• TNF-b like TNF-a but produced by T cells

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Cytokines

• Interleukins made by leukocytes and act on
  leukocytes
• Interleukin-1 (IL-1) similar to TNF
• Made by activated Macs, neutrophils, endothelial
  cells, epithelial cells
• IL-2 - produced by activated T cells act in
  autocrine manner on T cells
• IL-3 - multilineage colony stimulating factor
  Produced by T cells and promotes expansion of
  marrow progenitor cells of blood cells.
• IL-4 produced by T cells Stimulate IgE
  production by B cells
• IL-5 - Produced by T cells stimulate growth and
  activation of eosinophils
• IL-7 - Produced by bone-marrow stromal cells
  stimulate B and T cells
• IL-10 - Produced by activated Macs inhibits
  Macs homeostatic control
• IL-12 produced by activated monocytes
  stimulates interferon gamma production by T and
  NK cells mediator of innate response

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Chemokines

• Chemokines (chemotactic cytokines) lg family of
  structurally homologous cytokines produced by
  various cells in response to inflam mediators
  that stimulate leukocyte movement and regulate
  leukocyte migration from blood to tissues.
• Alpha chemokines C-X-C chemokine
• Two cysteines separated by one amino acid
• Beta chemokines C-C chemokine
• Receptor seven-transmembrane alpha helical
  receptor

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Complement

• Complement (C) 35 proteins, primaily in plasma
• Nomenclature numbers and letters
• a usually designates small soluble peptide
• b usually designates peptide that binds to cell
  surface
• 3 pathways to produce C3 convertase (C3 into C3a
  and C3b)
• Classical discovered first activated by Ag/Ab
  complexes
• IgM, IgG1, and IgG3 are isotypes that fix C
• Alternative activated by bacterial surface
  compents (LPS)
• Lectin activated by mannose binding lectin (MBL)
• C3b C3 convertase C5 convertase which cleaves
  C5
• C5b modifies C6, C7, C8, C9 to membrane attack
  complex (MAC)

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Complement

• Classical pathway
• IgMgtIgG fix C1q which activates C1r to cleave
  C1s
• Activated C1s cleaves C2 (C2a, C2b) and C4 (C4a,
  C4b)
• C4bC2b C3 convertase
• C4bC2b binds to C3b C5 convertase
• Lectin pathway
• Mannose-binding protein (MBP) binds to mannose on
  cell surface polysaccharides
• MBP then binds to MBP associated protein -1
  MASP -2
• MASP-1 and MASP-2 cleave C2 and C4 C4bC2b C3
  convertase
• Alternative (properdin) pathway
• C3 in plasma spontaneously splits into C3a and
  C3b become inactive
• If C3b binds to bacterial surface component
  (LPS), becomes stable
• C3b binds factor B C3bB
• Factor D cleaves B from C3bB to C3bBb C3
  convertase
• Factor P (properdin) stabilizes C3bBb
• C3bBb binds to another C3b C3b3bBb C5
  convertase

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Complement

• Complement pathways

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Complement

• C products perform multiple functions
• Chemotactic factors C5a recuit leukocytes
• Anaphylatoxins C3a, C5a promote acute
  inflammation by causing release of vasoactive
  mediators from mast cells.
• Opsonins C3b adheres to bacterial cells
• Complement receptor CR1 on PMNs, Macs, and B
  cells
• Facilitates phagocygtosis
• Membrane Attack Complex C5b,6,7,8,9
  (C5b,6,7,8)1 (C9)n
• C7 is hydrophobic and inserts into lipid bilayers
• Gram negative bacteria have lipid outer membrane
• C9 polymerizes at C5-C8 to form pores
• Water enters cell swelling and rupture
• Calcium also enters cells which induces apoptosis

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Eicosanoids

• Eicosanoids arachidonic acid (AA) metabolites
• AA derived from 20 carbon FA in membrane
  phospholipids
• AA produced from membrane with phospholipase A2
• Action of enzyme inhibited by glucocorticoids
• Prostaglandins, leukotrienes, thromboxanes
• Local hormones produced by mast cells and other
  cells
• Mediator in inflammation after infection or
  injury
• Cyclooxygenase pathway - prostoglandins and
  thromboxanes
• Inhibited by aspirin and other NSAIDS
• Lipoxygenase pathway leukotrienes, lipoxins

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Eicosanoids

• Eicosanoid metabolism

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Eicosanoids

• Eicosanoids arachidonic acid (AA) metabolites
• Prostaglandins
• Prostaglandin D2 (PGD2)
• binds to receptors on smooth muscle cells and
  acts as a vasodilator and bronchoconstrictor
• Chemotaxis of neutrophils in inflammation
• Leukotrienes
• Leukotriene C4 (LTC4), LTD4, LTE4 bind to
  receptors on smooth muscle, causing prolonged
  bronchoconstriction asthma
• Lipoxins A4 (LXA4) and LXB4 are anti-inflammatory
  mediators

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Apoptosis

• Apoptosis DNA cleavage, membrane blebbing,
  changes in membrane lipid distribution,
  detachment of cells
• Apoptosed cells express molecules recognized by
  neutrophils
• Damaged cells removed cleanly as apposed to
  necrosis where damaged cells release contents in
  environment.
• Important for eliminating unwanted lymphocytes
• Induction by activation/cleavage of series of 14
  caspases which cleave cellular elements.
  Caspases activated by
• Intrinsic mitochondrial membrane of inactive
  lymphocytes leak products activate caspase
  programmed cell death or death by neglect
• Extrinsic repeatedly activated T cells
  up-regulate FasL reacts with Fas on same or
  adjacent cell
• NK cell and cytotoxic T cell release perforin,
  granulysin and granzyme

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Acute Phase Response

• Acute Phase Response production of proteins by
  liver in response to infection, trauma,
  inflammation, malignancies
• Within hrs of event but remain elevated during
  chronic insults
• From IL-1, TNF, INFg, etc by monocytes, Macs,
  endothelial cells
• Unique proteins
• C-reactive protein (CRP) reacts with C
  polysaccharide of pneumococci
• serum amyloid A (SAA)
• Elevated normal proteins C components,
  fibrinogen etc.
• Other liver protein synthesis reduced albumin
  anemia of chronic disease?
• Objective evidence for disease in occult
  infections or diseases
• Tests
• CRP measurement (ELISA)
• Erythrocyte sedimentation rate (ESR) increased

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Erythrocyte sedimentation rate

• The erythrocyte sedimentation rate (ESR), also
  called a sedimentation rate, is the rate at which
  RBCs precipitate in a period of 1 hour. It is a
  non-specific measure of inflammation. To perform
  the test, anticoagulated blood is placed in an
  upright tube, and the rate at which the RBCs fall
  is measured and reported in mm/h.
• The ESR is governed by the balance between
  pro-sedimentation factors, mainly fibrinogen, and
  those factors resisting sedimentation, namely the
  negative charge of the erythrocytes (zeta
  potential). When an inflammatory process is
  present, the high proportion of fibrinogen in the
  blood causes red blood cells to stick to each
  other. The red cells form stacks called
  'rouleaux' which settle faster

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Inflammation

• Inflammation complex reaction to microbes
  necrosis
• Vascular responses, migration/activation of
  leukocytes, systemic
• Destroy, dilute, wall off injury
• Injured tissue replaced by fibrous tissue
  scarring and repair
• Mediated by chemical factors
• Terminated when insult eliminated and by
  anti-inflam mechanisms
• Types of inflammation
• Acute rapid onset (seconds or minutes), short
  duration (hrs, days) with exudate and neutrophils
• Chronic slow onset with long duration and
  characterized by macrophages and lymphocytes, new
  blood vessels, fibrosis, (granulation tissue),
  tissue necrosis

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Inflammation

• Acute Inflammation
• Vessel dilation, leaky microvasculature,
  emigration of cells fluid
• Fluid migration from circulation to tissues
• Transudate normal, low protein content, sp
  gravity lt 1.012
• Exudate with inflammation high protein, cells
• Edema fluid but few cells
• Purulent (pus) lost of cells, mostly
  neutrophils
• Stimuli
• Infections
• Trauma, tissue necrosis, foreign bodies
• Chemical agents
• Hypersensitivity reactions

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Inflammation

• Acute inflammation continued
• Vasodilation induced by histamine, NO on vascular
  sm muscle
• Leaks due to endothelial gaps in venules
• Histamine, leukotrienes
• Primarily from mast cells
• rapid contraction of cytoskeletal proteins
• IL-l, TNF, INFg delayed, endothelial cell
  retraction
• Extravasation of leukocytes
• Margination of leukocytes with slowing of blood
  flow
• Rolling leukocyte transient adherence to
  endothelial cells
• Adherence due to up-regulation of selectins,
  integrins, other
• Mediated by histamine, platelet activating factor
  (PAF), TNF, IL-1,
• Pavementing leukocytes firmly adhere to
  endothelial cells
• Diapedesis movement of leukocytes thru vascular
  wall to tissues

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Inflammation

• Leukocyte migration from blood vessels Robbins
  Fig 2-6 p 53

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Inflammation

• Acute inflammation continued
• Chemotaxis migration of leukocytes to site of
  injury
• MOA
• chemotactant binds to specific receptor on
  leukocyte
• Action of second messengers result in
  polymerization of actin
• Pseudopodia extension and fusion
• chemoattractants chemokines, C5a, leukotriene
  B4, bact. proteins
• Termination of response
• Stimulus ends, mediators short-lived
• Anti-inflammatory cytokines
• Lipoxins LXA4 and LXB4
• Transforming growth factor B (TGF-B) from Macs

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Inflammation

• Chronic inflammation simultaneous inflammation,
  tissue destruction and attempts to repair
• Causes
• Persistent infection - tuberculosis
• Prolonged exposure to toxic agent
• Exogenous silicosis
• Endogenous arthrosclerosis
• Autoimmunity
• Morphological features
• Infiltration with mononuclear cells (Macs,
  lymphocytes, plasma cells)
• Tissue destruction from agent and inflammatory
  cells
• Attempts to heal by connective tissue replacement
  

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Inflammation

• Chronic inflammation continued
• Mononuclear cell infiltration
• Macrophages are the major player
• blood monocytes live 1 day, tissue Macs live
  months to years
• Overtake neutrophils as predominant cell type by
  48h
• Macrophages proliferate (unlike neutrophils)
• Lymphocytes - recruited by IL-1, TNF, and
  chemokines from Macs
• Secrete INFg which activates Macs (bidirectional
  interaction)
• Eosinophils for IgE mediated reactions
  parasitic infections

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Inflammation

• Chronic inflammation continued
• Granulomatous inflammation granuloma formation
• Persistent infection or stimulus
• Aggregation of macrophages that are transformed
  into epithelium-like cells (epitheloid cells)
• Giant cells epitheloid cells that fuse together
• Focus surrounded by collar of lymphocytes (and
  some plasma cells)
• Older granulomas develop rim of fibroblasts and
  CT
• Note different from granulation tissue which is
  the tissue of early repair (24 h after injury)
  characterized by angiogenesis and fibroblast
  proliferation

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Fever

• Fever pyrexia gt 1oC
• Neural pathways for thermoregulation in
  hypothalmus
• Cytokines of innate immune system (TNF, IL-1
  endogenous pyrogens) bind to receptors on
  vascular endothelial cells in hypothalmus.
  Endothelial cells then produce prostagland PGE2,
  and other eicosanoids that reset hypothalmic
  thermoregulatory center.
• LPS, other products exogenous pyrogens
  (stimulate leukocytes to release cytokines)
• NSAIDS inhibit cyclooxygenase block
  prostaglandin synthesis
• Fever of unkown origin (FUO) gt101F for 3 weeks
• Caused by infections, rheumatic fever,
  sarcoidosis, neoplastic diseases, drugs
• Note pyogen pus pyrogen fever

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Innate Immunity

• Protection against infection that relies on
  mechanisms that exist before infection
• First line of defense
• Barriers
• Skin (epidermis and dermis)
• Mucous membranes respiratory, GI, genitourinary
  tracts
• Lacrimal apparatus tears
• Saliva
• Epiglottis
• Chemical
• Sebum acids
• Perspiration lysozyme
• Gastric juice
• Urine
• transferrin

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Innate Immunity

• Protection against infection that relies on
  mechanisms that exist before infection
• Second line of defense
• Phagocytosis
• Inflammation
• Fever
• Complement
• Intererron

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Adaptive Immunity

• Protection that develops after exposure to an
  agent and is specific for that agent
• Humoral immunity for intracellular and
  extracellular pathogens
• antibodies (Abs)
• Prevent reinfection and spread
• Eliminate agent
• opsonization ab interacts with both agent and
  host cell)
• Fix complement (C) (opsonization, bactericidal
  Ab)
• Large number (polyclonal) non-self
• Surface receptors on B cells
• Cell-mediated immunity (CMI)
• Up-regulated Macs
• Cytotoxic T cells