Phagocytosis and the Interactions of Various Phagocytes

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Phagocytosis and the Interactionsof Various
Phagocytes
Medical Microbiology

• BIOL 533
• Lecture 6

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Leukocyte Chemotaxins

• Types of chemotaxins
• C5a attracts neutrophils and monocytes
• Made by bacteria
• Peptide clipped off N-terminus (beginning with
  N-formylmethionine) during peptide maturation
  after protein synthesis
• Made by bacteria and nucleated blood cells
• Leucotrieneslipid products of cell membrane
  metabolism

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Leukocyte Chemotaxins

• Function of chemotaxins
• Enhance and direct motility of phagocytic cells
• To a limited extent, oxidative metabolism of
  phagocytic cells

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Opsonization and Opsonins

• General aspects
• Substances that enhance ability of phagocytes to
  ingest microbes
• Defend against presence of capsules and other
  microbial mechanisms that interfere with
  phagocytosis

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Opsonization and Opsonins

• Types of opsonins
• Antibodies
• C3b component of complement
• Binds covalently to bacterial surface and is
  recognized by receptors on neutrophils,
  monocytes, and macrophage
• Bacteria become bound to surface of phagocyte
  facilitating their uptake

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Opsonization and Opsonins

• Types of opsonins, continued
• Mechanism
• White blood cell receptors for C3b
• At least 3CR1, CR2, CR3 (complement receptor)
• Children deficient in CR3 very vulnerable to
  bacterial infections

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PhagocytesTypes of Cells

• Neutrophilscell origin
• Actively motile cells produced in bone marrow
• Differentiate from stem cells over about a
  two-week period
• Production of granules during this time
• Azurophil
• Produce specific granules later

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PhagocytesTypes of Cells

• Neutrophilscell origin, continued
• Upon maturation (in numbers of 1010 per day),
  they move into peripheral blood and circulate for
  about 6.5 hours
• Next move into capillary bed and marginate

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PhagocytesTypes of Cells

• Neutrophilscell origin, continued
• Margination caused by stickiness due to
  interleukin-1
• Summoned by chemotaxis, they move through
  endothelial cell junctions (diapdesis) into
  extravascular tissue spaces

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PhagocytesTypes of Cells

• Neutrophils are most active in gut
• Gut has enormous microbial population lying just
  one cell layer away from aseptic tissue
• Flora generates large amounts of chemotaxins that
  recruit most of bodys available leukocytes

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PhagocytesTypes of Cells

• As a result, submucosa of gut is in a constant
  state of inflammation
• Keep microbial flora down
• Synthesis of neutrophils inhibited by chemicals
  or radiation
• Infections in gut region

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PhagocytesTypes of Cells

• Monocytes and macrophage
• Compared to neutrophils
• Arrive at damaged tissue later in infection
• Days after neutrophils have been active in
  fighting intruders
• Eventually settle in tissues and become resident
  macrophage

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PhagocytesTypes of Cells

• Monocytes and macrophage
• Share progenitor cell type, but kinetics of
  maturation appearance are very different
• Monocytes continue cell differentiation after
  leaving bone marrow
• Monocytes and macrophage involved in both
  constititive and inducible mechanisms
• Interact with T cells and play important role in
  cell-mediated immunity

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PhagocytesTypes of Cells

• Tissue (resident) macrophage
• Exist throughout body
• Different names and functions in different
  tissues
• Kupffer cellsliver
• Alveolar macrophagelungs
• Osteoclastsbone
• Microgliabrain

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PhagocytesTypes of Cells

• Monocyte and macrophage functions
• Phagocytize invading microbes
• Contribute greatly to inflammatory response
• Releases
• IL-1enhances sticking of neutrophil to capillary
  endothelia
• TNFactivates newly arrived neutrophils

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Mechanism of Phagocyte Killing

• Neutrophils
• General steps
• Attach to microbes
• Ingest microbes
• Kill microbes
• Granulesconsidered as enlarged lysosomes
  containing hydrolytic enzymes

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Mechanism of Phagocyte Killing

• Neutrophil granule types
• Azurophil (primary granule)
• Contains
• Lysozyme
• Elastase
• A chymotryptic-like protease
• Myeloperoxidase
• Several antibacterial cationic proteins

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Mechanism of Phagocyte Killing

• Neutrophil granule types
• Specific (secondary granule)
• Contains
• Cytochrome
• Lysozyme
• Lactoferin (iron-binding protein)
• Vitamin B12 binding protein
• Collagenase

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Mechanism of Phagocyte Killing

• The neutrophil membrane
• Contains receptors for chemotaxin and opsonins
• After binding chemotaxins, receptors are
  internalized and replaced with new ones

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Mechanism of Phagocyte Killing

• Effectiveness of chemotaxis very effective
• Neutrophils are very motile
• Move by rearranging cytoplasmic microfilaments
  and microtubules
• Actin and myosin in microfilaments are affected
  by protein gelsolin
• Portions that face upstream in chemotactic
  gradient form structure called lamellipodium
• Cytoplasm is densely packed with microfilaments
• Portions face downstream form knob-like uropod

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Mechanism of Phagocyte Killing

• Process of phagocytosis
• General aspects
• Differs from pinocytosis in that particles, not
  liquids, taken up

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Mechanism of Phagocyte Killing

• Process of phagocytosis, continued
• Receptors on phagocyte surface progressively
  attach to ligands on bacterial surface
• Stimulates mechanisms of killing
• Oxidative metabolism leading to production of
  hydrogen peroxide and compounds lethal to
  microbes (oxygen-dependent killing)
• Discharge of toxic compounds from granules into
  phagosome (oxygen-independent killing)

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Mechanism of Phagocyte Killing

• Process of phagocytosis, continued
• Form phagosomepouch-like structure that
  invaginates, displacing the nucleus and granules
  toward uropod
• Form phagolysosomemembrane of granules and
  phagosome fuse, releasing toxic substances
• Forms separate pinched-off organelle
• Bacteria coated with antibacterial proteins

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Oxygen-Dependent Killing

• Fusion of specific granules with phagosome
  membrane (derived from plasma membrane) brings
  together
• NADPH oxidase (oxidizes NADPH found in
  neutrophil plasma membrane)
• Unique cyt b (granule membrane)
• A quinone

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Oxygen-Dependent Killing

• Reaction
• O2 ? O2 (reduces oxygen to superoxide
  radical)
• 2O2 H2O ? H2O2 O2 (superoxide dismutase)

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Oxygen-Dependent Killing

• Patients lacking cytochrome components
• Children having chronic granulomatous disease
  (CGD)
• Failure to synthesize superoxide radical and
  therefore hydrogen peroxide
• Due to decreased amount of cytochrome b
• Gene for larger subunit is missing (90K, 20K)

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Oxygen-Dependent Killing

• Children having chronic CGD, contd.
• Neutrophils can phagocytize normally, but do not
  efficiently oxidize NADPH and kill via oxidative
  pathway
• Usually dont survive into adulthood

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Oxygen-Dependent Killing

• How does oxidative process kill?
• Interaction with myeloperoxidase supplied by
  fusion with azurophil
• Combines chloride ions and hydrogen peroxide to
  form hypochlorous ions (analogous to bleach)
• Bacteria lacking catalase produce hydrogen
  peroxide (pneumococci) basically commit suicide
• Pneumococci are not dangerous to CGD patients

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Oxygen-Independent Killing

• Process
• Triggered by binding opsonized bacteria to the
  plasma membrane of neutrophils
• Specific granules fuse first
• Deliver several bacteriodical proteins, including
  lysozyme and lactoferin

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Oxygen-Independent Killing

• Azurophil granules discharge antimicrobial
  cationic proteins
• Some are amphipathic and resemble other cationic
  surface proteins such as polymyxin B

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Oxygen-Independent Killing

• Azurophil granules, continued
• Disrupt outer membrane of Gram and kill by
  causing leakage of vital components
• Each of the proteins has unique antimicrobial
  spectrum, but tend to affect Gram more than
  Gram
• Proteins may account for survival of some CGD
  children

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Oxygen-Independent Killing

• Efficiency
• Bacterial killing under highly anaerobic
  conditions of deep abscesses
• Patients lacking genes
• Coding for cationic proteins
• None found, maybe lethal

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Oxygen-Independent Killing

• Chediak-Higashi syndrome (genetic disease)
• Premature fusion of neutrophil granules while
  cells in bone marrow
• When mature cells phagocytize, granules are
  already spent, substantially reducing killing
  power

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Comparison of Bacterial Sensitivity

• Gram rods in gut killed by oxygen-independent
• Gram bacteria on skin and upper respiratory
  epithelia are resistant to oxygen-independent and
  killed by oxygen-dependent

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Mechanism of Phagocyte Killing

• Eosinophils
• Much like neutrophils, but indicative of
  parasitic infection

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Killing by Monocytes and Macrophage

• General aspects
• Tend to take care of what is left after battle
  with neutrophils
• Mechanisms of chemotaxis, phagocytosis, and
  killing resemble mechanisms of neutrophils
• Not studied in same detail

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Killing by Monocytes and Macrophage

• Differences
• Continue to differentiate after leaving bone
  marrow and are activated
• Called angry macrophage
• Phagocytize more vigorously
• Take up more oxygen
• Secrete large quantity of hydrolytic enzymes
• In general, better prepared to kill

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Killing by Monocytes and Macrophage

• Activated by
• Elicited by substances made in response to
  presence of bacteria (C3b) or viruses
  (interferon)
• Endotoxin of Gram
• Tetrapeptide derived from immunoglobulins
  (tuftsin)

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Killing by Monocytes and Macrophage

• Microbial (bacterial, fungi, protozoa) growth
  within
• Some can grow until activated, then killed
• Participation in immune response
• Help rid body of not only microbial invaders, but
  also tumor and foreign cells

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Killing by Monocytes and Macrophage

• Immune response process
• Stimulate development of T lymphocytes
• Respond to signals from other lymphocytes that
  stimulate differentiation and activation of
  macrophage

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Phagocytotic Killing

• Macrophages/neutrophils/mast cells stimulated by
• TNF
• interferon
• Produce reactive nitrogen intermediates
• Nitric oxide
• Nitrite (NO2)
• Nitrate (NO3)

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Phagocytotic Killing

• Released from cells or contained within vacuoles
• Macrophages produce NO from arginine when
  stimulated by cytokines
• NO can block cellular respiration by complexing
  iron in electron transport proteins

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Macrophage Killing

• Herpes simplex
• Toxoplasma gondii
• Leishmania major
• Cryptococcus neoformans
• Schistosoma mansoni

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Lecture 6

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