2. • Epithelia may be disrupted by wounds, abrasions,
and insect bites that may transmit pathogens.
• Once pathogens penetrate through the epithelial
barrier layers into the tissue spaces of the body,
– an array of cellular membrane receptors and soluble
proteins recognize microbial components and play the
essential roles of detecting the pathogen and
triggering effective defenses against it.
• Phagocytic cells make up the next line of defense
against pathogens that have penetrated the
epithelial cell barriers.
3. • The main cell types that carry out phagocytosis—
– Macrophages, neutrophils and dendritic cells in
tissues and
– Monocytes in the blood
• Elie Metchnikoff initially described the process of
phagocytosis in the 1880s using cells from
starfish (echinoderm invertebrates) similar to
vertebrate white blood cells.
• He ascribed to phagocytosis a major role in
immunity; we now know that defects in
phagocytosis lead to severe immunodeficiency.
4. • Most tissues contain resident populations of
MACROPHAGES that function as sentinels for the innate
immune system.
• These macrophages recognize microbes such as bacteria
through various cell surface receptors, extend their
plasma membrane to engulf them, and internalize them
in phagosomes (endosomes resulting from phagocytosis,
Figure).
• Lysosomes then fuse with the phagosomes, delivering
agents that kill and degrade the microbes.
7. • NEUTROPHILS are a second major type of phagocyte,
usually recruited to sites of infection.
• Finally, DENDRITIC CELLS also can bind and
phagocytose microbes uptake and degradation of
microbes by dendritic cells play key roles in the
initiation of adaptive immune responses.
• In addition to triggering phagocytosis, various
receptors on phagocytes recognize microbes and
activate the production of a variety of molecules that
contribute in other ways to eliminating infection.
• A phagocyte’s recognition of microbes and the
responses that result are shown in Figure.
8.
9. Overview figure:
Microbial invasion brings many effectors of innate immunity into play.
Entry of microbial invaders through lesions in epithelial barriers generates
inflammatory signals and exposes the invaders to attack by different effector
molecules and cells.
Microbes with surface components recognized by C-reactive protein (CRP), mannose
binding lectin (MBL), or surfactant proteins A or D (SP-A and SP-D) are bound by
these opsonizing molecules, marking the microbes for phagocytosis by neutrophils
and macrophages.
Some bacteria and fungi can activate complement directly, or via bound CRP or MBL,
leading to further opsonization or direct lysis.
Inflammatory signals cause phagocytes such as monocytes and neutrophils to bind to
the walls of blood vessels, extravasate, and move to the site of infection, where
they phagocytose and kill infecting microorganisms.
Binding of microbes to receptors on phagocytes activates phagocytosis and production
of additional antimicrobial and proinflammatory molecules that intensify the
response, in part by recruiting more phagocytes and soluble mediators (CRP, MBL,
and complement) from the bloodstream to the site of infection.
Inset: Dendritic cells bind microbes via receptors and are activated to mature; they also
internalize and degrade microbes. These dendritic cells migrate through lymphatic
vessels to nearby lymph nodes, where they present antigen-derived peptides on
their MHC proteins to T cells. Antigen-activated T cells then initiate adaptive
immune responses against the pathogen. Cytokines produced during innate
immune responses also support and direct the adaptive immune responses to
infection.
10. How does a phagocytic cell recognize microbes, triggering
their phagocytosis?
• Phagocytes express on their surfaces a variety of receptors
• Some of the receptors directly recognize specific conserved
molecular components on the surfaces of microbes, such as
cell wall components of bacteria and fungi.
• These conserved motifs, usually present in many copies on the
surface of a bacterium, fungal cell, parasite, or virus particle,
are called pathogen-associated molecular patterns (PAMPs).
• Most PAMPs that induce phagocytosis are cell wall
components, including complex carbohydrates such as
mannans and –glucans, lipopolysaccharides (LPS), other lipid-
containing molecules, peptidoglycans, and surface proteins.
11. • Note: these cell wall components can be
expressed by microbes whether or not the
microbes are pathogenic (cause disease); hence
some researchers have started to use the more
general term microbe-associated molecular
patterns (MAMPs).
• The receptors that recognize PAMPs are called
pattern recognition receptors (PRRs).
• There are other PRRs that, after PAMP binding,
do not activate phagocytosis but trigger other
types of responses.
12. • Activation of phagocytosis can also occur indirectly
(shown in Figure) by phagocyte recognition of soluble
proteins that have bound to microbial surfaces, thus
enhancing phagocytosis, a process called opsonization
(from the Greek word for “to make tasty”).
• Many of these soluble phagocytosis-enhancing proteins
(called opsonins) also bind to conserved, repeating
components on the surfaces of microbes such as
carbohydrate structures, lipopolysaccharides, and viral
proteins (PAMPs).
• Once bound to microbe surfaces, opsonins are
recognized by membrane opsonin receptors on
phagocytes, activating phagocytosis (see Table 5.3
bottom).
13.
14. • A variety of soluble proteins function as
opsonins; many play other roles as well in innate
immunity. For example;
1. The two surfactant collectin proteins, SP-A and
SP-D, found in the blood as well as in mucosal
secretions in the lungs and elsewhere, where
they function as opsonins.
– After binding to microbes they are recognized by the
CD91 opsonin receptor (see Table 5-3) and promote
phagocytosis by alveolar macrophages and other
macrophage populations.
– This function of SP-A and SP-D contributes to
clearance of the fungal respiratory pathogen
Pneumocystis carinii, a major cause of pneumonia in
individuals with AIDS.
15. 2. Mannose-binding lectin (MBL), a third collectin with
opsonizing activity, is found in the blood and respiratory fluids.
3. L-ficolin, a member of the ficolin family that is related to MBL
and other collectins, is found in the blood, where it binds to
acetylated sugars on microbes, including some streptococcal
bacteria.
4. Complement component C1q also functions as an opsonin,
binding bacterial cell wall components such as
lipopolysaccharides and some viral proteins.
[As a result of their structural similarities, all (MBL and other
collectins, ficolins, and C1q) are bound by the CD91 opsonin
receptor (see Table 5-3) and activate pathogen phagocytosis]
16. 5. Some antibodies-
• Opsonisation is promoted by antibody.
• Membrane of phagocytes (macrophages, neutrophils)
has receptors for certain classes of antibodies (IgA
antibodies and some IgG antibody subclasses) called Fc
receptors (FcRs) which also are important for the
opsonizing activity of these antibodies.
• After binding of antibodies specifically to antigens on
microbe surfaces, the Fc regions of these antibodies can
be recognized by specific FcRs, triggering phagocytosis of
the antigen-antibody complex.
• An important mechanism by which the adaptive
immune response clears antigens, opsonization by
antibodies.
17. 6. C-reactive protein (CRP)- another opsonin,
recognizes phosphorylcholine and
carbohydrates on bacteria, fungi, and
parasites, and is then bound by Fc receptors
(FcRs) for IgG found on most phagocytes
7. Several components of the complement
system are among the most effective
opsonins.
18. • Among the most effective opsonins are several components of
the complement system, which will be described later
• Present in both invertebrates and vertebrates, complement
straddles both the innate and adaptive immune systems,
indicating that it is ancient and important.
• In vertebrates, complement consists of more than 30 binding
proteins and enzymes that function in a cascade of sequential
activation steps.
• It can be triggered by several innate soluble pattern-recognition
proteins (including the first complement component, C1q, and
the structurally related lectins MBL and ficolins, C-reactive
protein, and properdin), as well as by microbe-bound antibodies
generated by the adaptive immune response.
• Phagocytosis is one of many important antimicrobial effects
resulting from complement activation.
19. Phagocytosed Microbes are Killed by Multiple
Mechanisms
• The binding of microbes (bacteria, fungi, protozoan
parasites, and viruses) to phagocytes (via pattern
recognition receptors / opsonins and opsonin receptors)
activates signaling pathways.
• These signaling pathways trigger actin polymerization,
resulting in membrane extensions around the microbe
particles and their internalization, forming phagosomes.
• The phagosomes then fuse with lysosomes (in
neutrophils, with preformed primary and secondary
granules).
• The resulting phagolysosomes contain an arsenal of
antimicrobial agents that then kill and degrade the
internalized microbes.
20. • These agents include
– antimicrobial proteins and peptides (including
defensins and cathelicidins),
– low pH,
– acid-activated hydrolytic enzymes (including
lysozyme and proteases), and
– specialized molecules that mediate oxidative attack.
• Oxidative attack on the phagocytosed microbes,
which occurs in neutrophils, macrophages, and
dendritic cells, employs highly toxic reactive
oxygen species (ROS) and reactive nitrogen
species (RNS), which damage intracellular
components.
22. • The reactive oxygen species are generated by the
phagocytes’ unique NADPH oxidase enzyme
complex (also called phagosome oxidase), which
is activated when microbes bind to the phagocytic
receptors.
– The oxygen consumed by phagocytes to support ROS
production by NADPH oxidase is provided by a
metabolic process known as the respiratory burst,
during which oxygen uptake by the cell increases
severalfold.
– NADPH oxidase converts oxygen to superoxide ion
(•O2
-); other ROS generated by the action of additional
enzymes are hydrogen peroxide (H2O2), and
hypochlorous acid (HClO).
23. • The generation of RNS requires the transcriptional
activation of the gene for the enzyme inducible nitric
oxide synthase (iNOS, or NOS2)
– Expression of iNOS is activated by microbial components
binding to various PRRs.
– iNOS oxidizes L-arginine to yield L-citrulline and nitric
oxide (NO), a potent antimicrobial agent.
– In combination with superoxide ion (•O2
- ) generated by
NADPH oxidase, NO produces an additional reactive
nitrogen species, peroxynitrite (ONOO-) and toxic S-
nitrosothiols.
24. Generation of antimicrobial reactive oxygen and nitrogen species.
In the cytoplasm of neutrophils, macrophages, and dendritic cells, several enzymes,
including phagosome NADPH oxidase, transform molecular oxygen into highly reactive
oxygen species (ROS) that have antimicrobial activity. One of the products of this pathway,
superoxide anion, can interact with a reactive nitrogen species (RNS), generated by
inducible nitric oxide synthase (iNOS) to produce peroxynitrite, another RNS. NO can also
undergo oxidation to generate the RNS nitrogen dioxide.
25. • Collectively the ROS and RNS are highly toxic to phagocytosed
microbes
• Alter microbial molecules through
– oxidation,
– hydroxylation,
– chlorination,
– nitration, and
– S-nitrosylation,
– formation of sulfonic acids and destruction of iron-sulfur clusters in
proteins.
• One example of how these oxidative species may be toxic to
pathogens is
– the oxidation by ROS of cysteine sulfhydryls that are present in the
active sites of many enzymes, inactivating the enzymes.
• ROS and RNS also can be released from activated neutrophils and
macrophages and kill extracellular pathogens.
26. Phagocytosis Contributes to Cell Turnover and the Clearance of
Dead Cells
• As the body’s main scavenger cells, macrophages also utilize their
phagocytic receptors to take up and clear
– cellular debris,
– cells that have died from damage or toxic stimuli (necrotic cell death)
or from apoptosis (programmed cell death), and
– aging red blood cells.
• Collectively the components of dead/dying cells and damaged
tissues that are recognized by PRRs leading to their clearance are
sometimes referred to as damage-associated molecular patterns
(DAMPs).
• Phagocytosis is the major mode of clearance of cells that have
undergone apoptosis as part of developmental remodeling of
tissues, normal cell turnover or killing of pathogen-infected or
tumor cells by innate or adaptive immune responses.
27. • Apoptotic cells attract phagocytes by releasing the lipid
mediator lysophosphatidic acid, which functions as a
chemoattractant.
• These dying cells facilitate their own phagocytosis by
expressing on their surfaces an array of molecules not
expressed on healthy cells, including
– phospholipids (such as phosphatidyl serine and
lysophosphatidyl choline),
– proteins (annexin I), and
– altered carbohydrates.
• These DAMPs are recognized directly by phagocytic
receptors such as the phosphatidyl serine receptor and
scavenger receptor SR-A1.
• Other DAMPs are recognized by soluble pattern
recognition molecules that function as opsonins, including
– the collectins MBL, SP-A, and SP-D;
– Various complement components; and
– the pentraxins C-reactive protein and serum amyloid protein.
28. • These opsonins are then recognized by opsonin
receptors, activating phagocytosis and degradation of
the apoptotic cells.
• An important additional activity of macrophages in the
spleen and those in the liver (known as Kupffer cells) is
to recognize, phagocytose, and degrade aging and
damaged red blood cells.
• As these cells age, novel molecules that are recognized
by phagocytes accumulate in their plasma membrane.
– Phosphatidyl serine flips from the inner to the outer leaflet
of the lipid bilayer and is recognized by phosphatidyl serine
receptors on phagocytes.
• Modifications of erythrocyte membrane proteins have
also been detected that may promote phagocytosis.
29. • Obviously it is important for normal cells not to be phagocytosed.
• Accumulating evidence indicates that whether or not a cell is
phagocytosed is controlled by sets of
– “eat me” signals—the altered membrane components (DAMPs) —and
– “don’t eat me” signals expressed by normal cells.
• Young, healthy erythrocytes avoid being phagocytosed by not
expressing “eat me” signals, such as phosphatidyl serine, and also
by expressing a “don’t eat me” signal, the protein CD47.
• CD47, expressed on many celltypes throughout the body, is
recognized by the SIRP-receptor on macrophages, which transmits
signals that inhibit phagocytosis.
• Recent studies have shown that tumors use elevated CD47
expression to evade tumor surveillance and phagocytic elimination
by the immune system.
