Lecture 6 BIOL 533 1
Phagocytosis and the Interactions
of Various Phagocytes
BIOL 533
Lecture 6
Medical Microbiology
Lecture 6 BIOL 533 2
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
? Leucotrienes—lipid products of cell membrane
metabolism
Lecture 6 BIOL 533 3
Leukocyte Chemotaxins
? Function of chemotaxins
– Enhance and direct motility of phagocytic cells
– To a limited extent,oxidative metabolism of
phagocytic cells
Lecture 6 BIOL 533 4
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
Lecture 6 BIOL 533 5
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
Lecture 6 BIOL 533 6
Opsonization and Opsonins
? Types of opsonins,continued
– Mechanism
? White blood cell receptors for C3b
? At least 3—CR1,CR2,CR3 (complement receptor)
? Children deficient in CR3 very vulnerable to
bacterial infections
Lecture 6 BIOL 533 7
Phagocytes— Types of Cells
? Neutrophils—cell 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
Lecture 6 BIOL 533 8
Phagocytes— Types of Cells
? Neutrophils—cell 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
Lecture 6 BIOL 533 9
Phagocytes— Types of Cells
? Neutrophils—cell origin,continued
– Margination caused by stickiness due to
interleukin-1
– Summoned by chemotaxis,they move
through endothelial cell junctions (diapdesis)
into extravascular tissue spaces
Lecture 6 BIOL 533 10
Phagocytes— Types 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 body’s available leukocytes
Lecture 6 BIOL 533 11
Phagocytes— Types 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
Lecture 6 BIOL 533 12
Phagocytes— Types 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
Lecture 6 BIOL 533 13
Phagocytes— Types 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
Lecture 6 BIOL 533 14
Phagocytes— Types of Cells
? Tissue (resident) macrophage
– Exist throughout body
– Different names and functions in different
tissues
? Kupffer cells—liver
? Alveolar macrophage—lungs
? Osteoclasts—bone
? Microglia—brain
Lecture 6 BIOL 533 15
Phagocytes— Types of Cells
? Monocyte and macrophage functions
– Phagocytize invading microbes
– Contribute greatly to inflammatory response
? Releases
– IL-1—enhances sticking of neutrophil to capillary
endothelia
– TNF—activates newly arrived neutrophils
Lecture 6 BIOL 533 16
Mechanism of Phagocyte Killing
? Neutrophils
– General steps
? Attach to microbes
? Ingest microbes
? Kill microbes
– Granules—considered as enlarged lysosomes
containing hydrolytic enzymes
Lecture 6 BIOL 533 17
Mechanism of Phagocyte Killing
? Neutrophil granule types
– Azurophil (primary granule)
? Contains
– Lysozyme
– Elastase
– A chymotryptic-like protease
– Myeloperoxidase
– Several antibacterial cationic proteins
Lecture 6 BIOL 533 18
Mechanism of Phagocyte Killing
? Neutrophil granule types
– Specific (secondary granule)
? Contains
– Cytochrome
– Lysozyme
– Lactoferin (iron-binding protein)
– Vitamin B12 binding protein
– Collagenase
Lecture 6 BIOL 533 19
Mechanism of Phagocyte Killing
? The neutrophil membrane
– Contains receptors for chemotaxin and
opsonins
– After binding chemotaxins,receptors are
internalized and replaced with new ones
Lecture 6 BIOL 533 20
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
Lecture 6 BIOL 533 21
Mechanism of Phagocyte Killing
? Process of phagocytosis
– General aspects
? Differs from pinocytosis in that particles,not
liquids,taken up
Lecture 6 BIOL 533 22
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)
Lecture 6 BIOL 533 23
Mechanism of Phagocyte Killing
? Process of phagocytosis,continued
– Form phagosome—pouch-like structure that
invaginates,displacing the nucleus and
granules toward uropod
– Form phagolysosome—membrane of granules
and phagosome fuse,releasing toxic
substances
? Forms separate pinched-off organelle
– Bacteria coated with antibacterial proteins
Lecture 6 BIOL 533 24
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
Lecture 6 BIOL 533 25
Oxygen-Dependent Killing
? Reaction
– O2 ? O2—
(reduces oxygen to superoxide radical)
– 2O2— + H2O ? H2O2 + O2
(superoxide dismutase)
Lecture 6 BIOL 533 26
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)
Lecture 6 BIOL 533 27
Oxygen-Dependent Killing
– Children having chronic CGD,cont’d,
? Neutrophils can phagocytize normally,but do not
efficiently oxidize NADPH and kill via oxidative
pathway
? Usually don’t survive into adulthood
Lecture 6 BIOL 533 28
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
Lecture 6 BIOL 533 29
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
Lecture 6 BIOL 533 30
Oxygen-Independent Killing
? Azurophil granules discharge antimicrobial
cationic proteins
– Some are amphipathic and resemble other
cationic surface proteins such as polymyxin B
Lecture 6 BIOL 533 31
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
Lecture 6 BIOL 533 32
Oxygen-Independent Killing
? Efficiency
– Bacterial killing under highly anaerobic
conditions of deep abscesses
? Patients lacking genes
– Coding for cationic proteins
? None found,maybe lethal
Lecture 6 BIOL 533 33
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
Lecture 6 BIOL 533 34
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
Lecture 6 BIOL 533 35
Mechanism of Phagocyte Killing
? Eosinophils
? Much like neutrophils,but indicative of
parasitic infection
Lecture 6 BIOL 533 36
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
Lecture 6 BIOL 533 37
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
Lecture 6 BIOL 533 38
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)
Lecture 6 BIOL 533 39
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
Lecture 6 BIOL 533 40
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
Lecture 6 BIOL 533 41
Phagocytotic Killing
? Macrophages/neutrophils/mast cells
stimulated by
– TNF
– interferon
? Produce reactive nitrogen intermediates
– Nitric oxide
– Nitrite (NO2—)
– Nitrate (NO3—)
Lecture 6 BIOL 533 42
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
Lecture 6 BIOL 533 43
Macrophage Killing
? Herpes simplex
? Toxoplasma gondii
? Leishmania major
? Cryptococcus neoformans
? Schistosoma mansoni
Lecture 6 BIOL 533 44
Lecture 6
? Questions?
? Comments?
? Assignments..,
Phagocytosis and the Interactions
of Various Phagocytes
BIOL 533
Lecture 6
Medical Microbiology
Lecture 6 BIOL 533 2
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
? Leucotrienes—lipid products of cell membrane
metabolism
Lecture 6 BIOL 533 3
Leukocyte Chemotaxins
? Function of chemotaxins
– Enhance and direct motility of phagocytic cells
– To a limited extent,oxidative metabolism of
phagocytic cells
Lecture 6 BIOL 533 4
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
Lecture 6 BIOL 533 5
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
Lecture 6 BIOL 533 6
Opsonization and Opsonins
? Types of opsonins,continued
– Mechanism
? White blood cell receptors for C3b
? At least 3—CR1,CR2,CR3 (complement receptor)
? Children deficient in CR3 very vulnerable to
bacterial infections
Lecture 6 BIOL 533 7
Phagocytes— Types of Cells
? Neutrophils—cell 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
Lecture 6 BIOL 533 8
Phagocytes— Types of Cells
? Neutrophils—cell 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
Lecture 6 BIOL 533 9
Phagocytes— Types of Cells
? Neutrophils—cell origin,continued
– Margination caused by stickiness due to
interleukin-1
– Summoned by chemotaxis,they move
through endothelial cell junctions (diapdesis)
into extravascular tissue spaces
Lecture 6 BIOL 533 10
Phagocytes— Types 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 body’s available leukocytes
Lecture 6 BIOL 533 11
Phagocytes— Types 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
Lecture 6 BIOL 533 12
Phagocytes— Types 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
Lecture 6 BIOL 533 13
Phagocytes— Types 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
Lecture 6 BIOL 533 14
Phagocytes— Types of Cells
? Tissue (resident) macrophage
– Exist throughout body
– Different names and functions in different
tissues
? Kupffer cells—liver
? Alveolar macrophage—lungs
? Osteoclasts—bone
? Microglia—brain
Lecture 6 BIOL 533 15
Phagocytes— Types of Cells
? Monocyte and macrophage functions
– Phagocytize invading microbes
– Contribute greatly to inflammatory response
? Releases
– IL-1—enhances sticking of neutrophil to capillary
endothelia
– TNF—activates newly arrived neutrophils
Lecture 6 BIOL 533 16
Mechanism of Phagocyte Killing
? Neutrophils
– General steps
? Attach to microbes
? Ingest microbes
? Kill microbes
– Granules—considered as enlarged lysosomes
containing hydrolytic enzymes
Lecture 6 BIOL 533 17
Mechanism of Phagocyte Killing
? Neutrophil granule types
– Azurophil (primary granule)
? Contains
– Lysozyme
– Elastase
– A chymotryptic-like protease
– Myeloperoxidase
– Several antibacterial cationic proteins
Lecture 6 BIOL 533 18
Mechanism of Phagocyte Killing
? Neutrophil granule types
– Specific (secondary granule)
? Contains
– Cytochrome
– Lysozyme
– Lactoferin (iron-binding protein)
– Vitamin B12 binding protein
– Collagenase
Lecture 6 BIOL 533 19
Mechanism of Phagocyte Killing
? The neutrophil membrane
– Contains receptors for chemotaxin and
opsonins
– After binding chemotaxins,receptors are
internalized and replaced with new ones
Lecture 6 BIOL 533 20
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
Lecture 6 BIOL 533 21
Mechanism of Phagocyte Killing
? Process of phagocytosis
– General aspects
? Differs from pinocytosis in that particles,not
liquids,taken up
Lecture 6 BIOL 533 22
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)
Lecture 6 BIOL 533 23
Mechanism of Phagocyte Killing
? Process of phagocytosis,continued
– Form phagosome—pouch-like structure that
invaginates,displacing the nucleus and
granules toward uropod
– Form phagolysosome—membrane of granules
and phagosome fuse,releasing toxic
substances
? Forms separate pinched-off organelle
– Bacteria coated with antibacterial proteins
Lecture 6 BIOL 533 24
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
Lecture 6 BIOL 533 25
Oxygen-Dependent Killing
? Reaction
– O2 ? O2—
(reduces oxygen to superoxide radical)
– 2O2— + H2O ? H2O2 + O2
(superoxide dismutase)
Lecture 6 BIOL 533 26
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)
Lecture 6 BIOL 533 27
Oxygen-Dependent Killing
– Children having chronic CGD,cont’d,
? Neutrophils can phagocytize normally,but do not
efficiently oxidize NADPH and kill via oxidative
pathway
? Usually don’t survive into adulthood
Lecture 6 BIOL 533 28
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
Lecture 6 BIOL 533 29
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
Lecture 6 BIOL 533 30
Oxygen-Independent Killing
? Azurophil granules discharge antimicrobial
cationic proteins
– Some are amphipathic and resemble other
cationic surface proteins such as polymyxin B
Lecture 6 BIOL 533 31
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
Lecture 6 BIOL 533 32
Oxygen-Independent Killing
? Efficiency
– Bacterial killing under highly anaerobic
conditions of deep abscesses
? Patients lacking genes
– Coding for cationic proteins
? None found,maybe lethal
Lecture 6 BIOL 533 33
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
Lecture 6 BIOL 533 34
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
Lecture 6 BIOL 533 35
Mechanism of Phagocyte Killing
? Eosinophils
? Much like neutrophils,but indicative of
parasitic infection
Lecture 6 BIOL 533 36
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
Lecture 6 BIOL 533 37
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
Lecture 6 BIOL 533 38
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)
Lecture 6 BIOL 533 39
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
Lecture 6 BIOL 533 40
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
Lecture 6 BIOL 533 41
Phagocytotic Killing
? Macrophages/neutrophils/mast cells
stimulated by
– TNF
– interferon
? Produce reactive nitrogen intermediates
– Nitric oxide
– Nitrite (NO2—)
– Nitrate (NO3—)
Lecture 6 BIOL 533 42
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
Lecture 6 BIOL 533 43
Macrophage Killing
? Herpes simplex
? Toxoplasma gondii
? Leishmania major
? Cryptococcus neoformans
? Schistosoma mansoni
Lecture 6 BIOL 533 44
Lecture 6
? Questions?
? Comments?
? Assignments..,
