Chapter 3

Innate Immunity – The induced Response

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Three Lines of Defense

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 Induced Innate Immune Response

• Once the pathogen has breached the physical

barriers and outrun the initial response the induced
innate response takes place.
– Inflammation
– Leukocytes called into the site of infection
– Soluble molecules make and secreted into the blood and
brought the site of infection

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 Innate Immune Cell Receptors = Recognition of
Pathogen or Damaged/Infected Host Cells
• Two examples of Cells that use receptors to recognize
self from non-self are Macrophages and NK cells
• Both cell types need to be able to differentiate between
host and pathogen.
• There are limited types of innate receptors for recognition
but because many pathogens have the same types of
surface molecules this limited diversity works well.
– Ex: Gram negative bacteria have LPS in their outer membrane
so it doesn’t matter what genus it is it will still be recognized by
an LPS receptor on a macrophage.

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Receptors

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Phagocytosis by macrophages provides a first line of
cellular defense against invading microorganisms

• Pathogen invades 1st effector cells (resident

macrophages)
– Resident in tissues
– Prevalent in connective tissues
• Linings of GI, respiratory tract, lung alveoli, liver
– Long-lived phagocytic cells
– Involved in both innate and adaptive immunity
• Enhance phagocytosis more efficient using
macrophage surface receptors
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 Enhance phagocytosis more efficient using
macrophage surface receptors

• C3b is ligand for Macrophage CR1

• Pathogen C3b surface fragments + degraded by
factor I iC3b now a ligand for macrophages CR3
and CR4 receptors
• Combination of opsonization by complement and
phagocytosis by macrophages
– Pathogens recognized and destroyed at beginning of
infection
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 Phagocytosis by macrophages is aided by receptors of
innate immunity that bind directly to microbial surface
components
Phagocytic Receptors
• Surface components are characteristic of pathogens
– But absent from human cells
• CR3 and CR4 recognize iC3b and other ligands such as:
– LPS, lipopolysaccharide (Gram negative bacteria)
– Lipophosphoglycan
– Filamentous hemagglutinin
– Cell-surface structures on yeast
• Carbohydrate-binding proteins = lectins
– Bind to particular carbohydrates (not found on human cells)
– Mannose and glucan receptors are examples
• Scavenger receptors = preference for molecules that are negatively
charged.
– Nucleic acids, phosphate-containing lipoteichoic acids (Gram positive
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bacteria) and LPS (Gram negative bacteria)
Macrophage Receptors (PRR’s) for Phagocytosis
and Cytokine Signaling are Different

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 Binding of Microbe to Phagocytic Receptors
Initiates the Phagocytosis

• Binding of the phagocytic receptors initiates

receptor mediated endocytosis (phagocytosis).
• The microbe is enveloped into a vesicle
(phagosome)
• The phagosome is joined to a lysosome =
phagolysosome
• The lysosome contains degradative enzymes and
toxic substances to destroy the pathogen.
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 Endocytosis - Phagocytosis
Macrophages have several types of surface receptor that bind to constituents of
microbial surfaces and promote phagocytosis –

PRR (pattern recognition recpetors) on leukocytes bind to

PAMPs (pathogen associated molecular patterns) on pathogens

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 Receptors that detect microbial products signal
macrophage activation and cytokine secretion

• Macrophage have receptors or sensors for pathogen components

that signal macrophage to make and secrete cytokines
– Toll-like receptors (TLR)
• 10 receptors
• Specificities for different microbial products
• TLR-4 senses ligand LPS
– TLR-4 expressed on Macrophages
– TLR-4 detects LPS and sends a signal to the nucleus of the
macrophage to make and secrete inflammatory cytokines as well as
cytokines that activate innate immune responses
– LPS is a major gram-negative bacteria component
– LPS is an endotoxin responsible for septic shock
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The 4 Lineages of TLR’s and Their Ligands
Fig. 3.29

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Some TLR’s need to be on the outside and
some on the inside.

Fig. 3.30

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• TLR trigger a common pathway of intracellular signaling
• Adaptor protein MyD88
• Interleukin-1 receptor associated kinase (IRAK)
• Translocation of TF nuclear factor B (NF B) from cytoplasm to the nucleus
• Directs the transcription of genes for inflammatory cytokines

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 Activation of resident macrophages induces
inflammation at sites of infection

• Development of inflammation in tissue leads to

– Local accumulation of fluid accompanied by
• swelling, redness, heat and pain
• Changes induced in blood capillaries
– increase diameter (dilation) reduction in the rate of blood flow increased
permeability to blood vessel wall increased supply of blood = redness and
heat
– increased permeability of blood vessels allows movement of fluid, plasma
proteins and WBC (neutrophils primarily) from the blood capillaries into the
tissue = swelling and pain
– Translocation of NF B to macrophage nucleus initiates transcription of
proinflammatory cytokines IL-1, IL-6, CXCL8, IL-12 and TNF-
• Look at fig. 3.9 with these cytokines and their functions.
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 Chemokines CXCL8 = IL-8

• IL-8 attracts leukocytes (neutrophils) to site of tissue damage or

infection
– bind to CXCR1 and CXCR2 on the neutrophil
• Direct traffic of leukocytes during their development
– Small, (60-140 aa)
• Two major families
– Cysteine residues
– CC or CXC
• Cells are attracted from blood to infection site by following a
concentration gradient of chemokine produced by cells at infection
site
• Chemokines interact with target cells by binding specific cell surface
receptors signal through associated GTP-binding proteins

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 TNF- released by macrophages induces
protection at the local level

• Causes and consequences of the release of TNF-

within a local area
– Endothelium (venules)
• Increased blood flow
• Increased permeability
• Endothelial adhesiveness for wbc and platelets
– Causes blood in venules to clot
– Prevents spread of infection to the blood (sepsis or
septicemia)
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 IL-1b
b, IL-12 and IL-6

• IL-6 – acts on local muscle to increase temperature

– Signal to liver to make acute phase reactants
• Mannose binding lectin and C-reactive protein
• IL-1b – works with TNF-a to activate endothelial cells and
induce inflammation
• IL-12 activates NK cells
– Lymphocyte of the innate immune system
– NK cells secrete cytokines to maintain the macrophages activity
– Protect against viral infections

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 NOD-Like Receptors

• Cytoplasmic receptors for

recognition of pathogens
• NOD-1 - recognizes a
degradation product of Gram (–)
peptidoglycan
• NOD-2 – recognizes a
degradation product of most
bacteria – muramyl dipeptide

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 Innate Immunity

• Innate immunity works immediately on pathogen

confrontation
– Chemical weapons include reactive ions, peptides
• kill pathogens directly
– Protease inhibitors, clotting cascade and kinin reactions
• Inhibit pathogen colonizing tissues
• Spread infections
– Complement
• Tags microbial surface proteins
– Specific receptors
• Bind chemical parts of microbial macromolecules that are not a part of self
• All of these components work to assist phagocytes in
destruction of invading pathogens 0
 Distinct but complementary properties of
Macrophages and Neutrophils

Macrophages Neutrophils
• Long-lived • Short-lived dedicated
• Reside in tissues killers
• Work as infection • Circulate in blood
begins • Wait for macrophage
– Raise alarm alarm
– To enter tissue

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 Neutrophils are dedicated phagocytes that are
summoned to sites of infection

• Granulocytes
– Granules containing antimicrobials
• Primary (azurophilic), secondary (specific) granules and tertiary (gelatinase)
granules
– Polymorphonuclear leukocytes
• Variable and irregular shapes of their nuclei
• Microphages
• Smaller sized than macrophages
• Most abundant wbc (50 billion in circulation)
• Life span < 2 days
• 60% of hematopoietic activity of bone marrow
• Large reserve kept in bone marrow

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 Neutrophils are excluded from healthy
tissue

• Neutrophils are excluded from healthy tissue

– Release of inflammatory mediators at infection
sites is what attracts neutrophils
– Become dominant phagocytic cells
• 3 X 109 neutrophils mouth and throat each day
• Arrival of neutrophils is the 1st of a series of rxns
– Inflammatory response

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 Inflammatory Response

• Involves recruitment of cells and molecules of

innate immunity into sites of infection
– Neutrophils pus
– Extracellular bacteria
• Ex: S. aureus superficial infections and abscesses that neutrophils tackle in large
numbers
– Ex: Pus-forming = pyogenic bacteria

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 Leukocyte adhesion molecules

• The four structural classes of adhesion molecule

present on white blood cells and the cells with
which they interact are:
• Selectins – are carbohydrate-binding lectins
• Vascular addressins - contain carbohydrate groups
to which selectins bind
• Integrins – typically bind to Ig superfamily proteins
• Immunoglobulin superfamily – e.g. ICAM-1
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Leukocyte adhesion molecules

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 The homing of neutrophils
to infected tissues is induced by
inflammatory mediators
• Neutrophil receptors
– Inflammatory mediators
• C5a cleaved during complement activation
• CXCL8
– secreted by activated macrophages
• Peptides containing N-formylmethionine (not human)
• Ligand binding to neutrophil surface receptor changes
in neutrophil adhesion molecules
– Assist neutrophils in migrating out of blood capillaries
• Extravasation

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 The homing of neutrophils
to infected tissues is induced by
inflammatory mediators
• Inflammatory mediators (Cytokines) secreted by
macrophages change the ligand expression for the
neutrophil receptors on the surface of endothelial cells
near the site of infection.
• This allows the neutrophils to exit the blood near the site of
infection = Extravasation
– 4 steps
• Note that the WBC’s at some point will do something analogous to this process, we
are just using neutrophil homing as our model.

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 Step 1 cytokines/inflammatory mediators induce
selectin expression on vascular endothelium to bind
neutrophils
Transient interaction between neutrophil (sialyl-Lewis X)
and selectin on the endothelium

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Step 2 Rolling adhesion tight binding migration to
infection site Interactions between LFA-1 to ICAM-1.
Strong interaction is induced by CXCL8 held on ECM
proteoglycans chemokine attraction neutrophil
squeezes between endothelial cells

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 Neutrophils are potent pathogen killers but are
themselves programmed to die
• Phagocytosis by neutrophils
– Fc receptors
– Complement receptors
– Phagocytosis of complement
opsonized pathogens
– On availability of specific Abs
• Opsonized with antibody and
complement
• Neutrophil process of phagocytosis is
similar to that of macrophages, but
– > range of particulate engulfed
– > microbicidals
– 3 types of granules
– devoted to storage & delivery of
antimicrobial weaponry
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Neutrophil Granules for Killing

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 Bacterial agents produced or released by phagocytic
cells on the ingestion of microorganisms (macrophage &
neutrophil)
Are toxins or bind
to essential nutrients

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 Neutrophil Granules

• Primary/Azurophilic granules
– Lysozyme, defensins, myeloperoxidase, neutral
proteases, elastase, proteinase 3…
• Secondary/Specific granules
– Lactorferrin – metal sequestering protease
– Lysozyme
– NADPH oxidase proteins
• Tertiary/Gelatinase granules
– Gelatinase – metal sequestering protease – restricts
bacteria from gaining access to iron

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 Respiratory Burst
• Pathogen engulfed by neutrophil degradative enzymes/toxins
fusion of phagosomes with neutrophil granules
– Granules = modified lysosomes of hydrolytic degradative enzymes, NADPH-
dependent oxidases & -defensins
• NADPH oxidase produces Superoxide radicals which are converted
to Hydrogen peroxide by superoxide dismutase.
– Reaction causes an increased consumption of hydrogen ions raised pH
activation of primary and secondary granule contents.
• Respiratory burst = Transient increase in oxygen consumption =
purpose is to raise the pH of the phagosome so the granule
contents can become active to kill pathogen
• Lysozymes release contents lower pH continue the destruction
of engulfed pathogens
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Respiratory Burst Kill pathogens

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 Toxic Oxygen species produced during the
respiratory burst can diffuse out and damage host
cells

• To limit damage by respiratory burst

– Phagocytes synthesize enzymes to inactivate toxic oxygen
species
– Ex: catalase
• Catalase degrades H2O2 H2O + O2
• What happens to these neutrophils?
– Neutrophils can’t replenish granule contents so the die.

Neutrophils apoptosis phagocytosed by macrophage

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 Neutrophil NET’s
flow Yuet 's

Netosis – a way neutrophils die that leads to explosion

lipid
• release
granular
t

/ bilayercontents
( )

capture and destruction of the pathogen

• NET’s – neutrophil extracellular traps - The
k

neutrophil’s nucleus swells and leads to the cell

bursting (netosis) leaving behind all the
antimicrobial components of the granules as well
as DNA and histones that serve to trap the
pathogen.
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 Chronic Granulomatous Disease
in

working or

NADPH oxidase is non-functional neutrophils granuloma

macrophages
Not cells )
Forms dead / dying
• ( group
of

– No oxidative burst
– pH isn’t raised in the phagolyosome no activation of
granular contents no killing of pathogen
– Infected neutrophils and macrophages are surrounded
by other leukocytes (walling them off) to stop the spread
of the pathogen granuloma formation

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 Macrophages produce cytokines:
TNF- x, IL-1 and IL-6 = Pyrogens
1-
to increased te p ( fever )
Cytokines Lead
-

• A spectrum of biological activity to coordinate the body’s

response to infection
– “Heat cytokines” alter energy mobilization to generate increased
temperature and they act on:
• hypothalamic temperature-control sites,
• muscle
• fat cells
• Result in body temperature increase fever

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 What’s the role of fever?

• Helps immune system fight infection

– Most bacterial and viral pathogens grow better at T’s lower than
system leukocytes ability
body temperature T te - p .
t rate
of reproduction
t t immune (

to
fight)
– Elevated T’s → bacteria, viral replication decreases
– Ag processing increases
– Human cells become more resistant to TNF-x deleterious
effects
• Systemic effect of TNF- x, IL-1 and IL-6 changes soluble
plasma proteins secreted by the hepatocytes acute →

phase response acute phase proteins

→

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Inflammatory cytokines raise body temperature
and activate hepatocytes to make the acute-
phase response
:
nm

All three are

pyrogen ←
-

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 Acute phase proteins
No
antibodies
if
Cnr
is

needed
present
• Acute-phase response proteins
– C-reactive protein
• activate classical complement cascade
– Serum Amyloid A
• Binds to some TLR’s leading to increased
inflammation through increased cytokine release
– Mannose-binding lectin binds
to mannose t
terminalsmannose residues

• Activate the Lectin complement cascade

• Enhance complement fixation to pathogen surfaces
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 Acute Phase Proteins

• Produced in response to inflammatory cytokines

• Produced by hepatocytes in liver

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 C-reactive protein
• Pentamer of identical subunits (pentraxin family)
– Binds to phosphorylcholine component of LPS of bacterial and
fungal cell walls
• But not to phosphorylcholine in human cells
• Acts as an opsonin
• Can bind C1q to initiate classical pathway of complement fixation in absence of Abs
• C-reactive protein binds with C1q stalks
• Abs binds with C1q globular heads
• but then…
• The same sequence of complement reactions occurs
whether C-reactive protein or Ab interacts with pathogen
and C1q binds
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 Mannose-binding lectin (MBL)

• Binds mannose-containing carbohydrates of

bacteria and yeast
– Calcium-dependent lectin
– MBL molecule is similar in structure to C1q
– 15 to 18 potential binding sites to attach to the pathogen
surface
• Even weak individual interactions with a carbohydrate structure can be developed
into a high-avidity using multipoint attachments
• Mannose containing carbohydrates on human cells do not bind MBL because their
geometry does not permit multipoint attachment to MBL

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-Acute-phase
response increases
the supply of the
recognition molecules
of innate immunity
-Shows opsonisation
of pathogen by MBL
and CRP

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 MBL activates a proteolytic enzyme complex:
MBL-associated serine protease = MASP
• MASP-2 (serine protease) cleaves
C4 and C2
• Initiates complement activation
– Lectin pathway of complement
activation
– MBL serves as an opsonin to facilitate
bacteria uptake by monocytes in the
blood
– Monocytes do not express the
macrophage mannose receptor but
have receptors that can bind to MBL
molecules coating a bacterial surface
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 C-reactive protein and MBL are present at low
levels in plasma

• Levels increase during acute-phase response

– C-reactive and MBL both bind structures that are common to
pathogens but absent on human cells
– Initiate complement activation
– Fixation by a pathway almost identical to classical pathway used
by Abs
• Abs of the adaptive immune response = similar role to that of C-reactive protein and
MBL in innate immunity
• Expand the range of pathogen-recognition molecules
• Complement components used in the classical and lectin pathway are structurally
and functionally related to components of the alternative pathway

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• C4 and C3 are similar in function,
structure and the thioester bond and T
→
-

£
C2 and factor B are similar. both
res .

on
"
3
0

g
• C3 convertases: canas
OP s
O

oaf platform
+ s

– Alternative – C3bBb
,

protease
complement
– Classical – C4b2a for
in

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 Classical Pathway Initiated by Ab’s
• Figure 9.28
• 1 IgM or 2 IgG’s bound by C1q C1r activation and cleavage of C1s
• C1s cleaves C4 and C4 similar to MASP2 in lectin pathway and just
like in the Classical started by CRP
• C3 convertase = C4b2a

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Initiation of the Classical Pathway

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 MASP and C1 – Similar Structure and Function
-

ons
109 protease
protease

po
Ho
Binding Binding

Both have binding portions and protease

action 0
Close structural relationships between components of
the alternative, lectin-mediated & classical pathways
of complement activation

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 Intracellular Infection:Type I interferons inhibit viral
replication and activate host defenses
• Human cell infected with virus → response is secretion of
type I interferons (interferon)
– Family of interferons different from IFN- y secreted from NK cells,
→

CD8 T cells and CD4 TH1 cells (type II interferon)

– Type I interferon t t B M an innate

Z
i

; n fer
• Interfere with viral replication by infected cells
-
on

• Alert immune system cells that infection is present gamma

• Make virus-infected cells more vulnerable to killer lymphocyte attack
– Almost all nucleated cells are susceptible to viral infections
– Almost all nucleated cells have the capacity to make type I
interferon as well as its cell-surface receptor t
Ferron
ter
" to B
y
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 Type I interferons inhibit viral replication and
Receptors
activate host defenses Recognition
viral recognition
Bacterial preceptors

• The type I interferon receptor is always present on cell

surfaces in
NODI
– Ready to bind interferon made in response to infection NOD Zeytoplasm
• Type I interferon is barely detectable in healthy individuals
blood outside
RIGI
– But at infection type I interferon becomes abundant MDA 5 ocyhfoplasm
• Many isotypes of type I interferons – main two are Alpha
and Beta
– Interferon- B (IFN- B ) = single form in humans
– Interferon- (IFN- ) = multiple forms
x x

– Interferon- , - - , - and - - FYI

S re X T w

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 Intracellular (viral) Infection
• RIG-1 and MDA-5 – cytoplasmic sensors for viral RNA’s → type 1
Interferon production Recognize
viral
in
• RIG-1 – retinoic-acid-inducible gene 1 components cytoplasm TLR 's inside
• MDA-5 – melanoma differentiation-associated protein 5 cytoplasm
• Use the fig. below 3.32 as the beginning pt for fig. 3.33

B made
nucleated
*
erythrocyte
'

t be
can NOT
an can
At B
by
" activate mauve
blown
nucleated
ca
BUT
primarily
cells make
Nn
-

can
cells
NR ::
±:* 0
 Virus-infected cells are stimulated to produce type 1
interferons
cell
Autocrine Rxu same
• Trigger signals → that lead to
:

Paracrine Rxu : diff cell

phosphorylation, dimerization and

.

passage of TF interferon
response factor 3 (IRF3) into
nucleus
• TF NF KB and AP-1 mobilize and avigation
par
coordinate with IRF3 to turn on
transcription of the IFN- B gene
• Secreted IFN- p binds to the IFN
receptor on the infected cell
surface (autocrine)
• Autocrine mobilization of other
IFN-response factors to change
gene expression patterns to
Auto origami
or

produce the interferon response 0

 Virus-infected cells are stimulated to produce type 1
interferons

• IRF7 turning on transcription

of the IFN- gene
•

•
:
Without the need for AP-1 or
NF B
Secreted IFN- will also bind
to the interferon receptor 2
3
4

expressed by nearby cells

(paracrine) that are not
1
infected by the virus
• This paracrine action helps
these cells to resist infection 0
 Interferon Response
• Interferon response leads to transcription and translation
(expression) of enzymes
– that degrade viral RNA
– That stop protein production of viral particles
• IFN’s also induces cellular changes that make the infected
cell more likely to be attacked by killer lymphocytes
• NK cells of innate immunity secrete cytokines and kill
infected cells
– IFN- L or IFN- B binds to circulating NK’s cell IFN receptor
– NK cells become activated
– NK cells attack virus-infected cells one at a time

0
 Major functions of type I interferons

tRNA
reproduction
up
Cut
PNA "

programed sfavtofproet
*
no
so
Death
apoptosis
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,
* cel

0
 NK cells provide an early defense against
intracellular infections
• Natural killer cells (NK cell) – expresses CD56 and lacks
CD3
– 2nd type of cytotoxic lymphocyte (CTLs)
– 3rd type of lymphocyte that is distinct from B- and T-cells
– No rearrangement of B or T cell receptor genes
– Large lymphocytes
– Well developed cytoplasm with cytotoxic granules
– Provide innate immunity against intracellular infections
– Migrate to infection site in response to inflammatory cytokines
• Two effector functions
– Cell killing – induce apoptosis of infected cells
– Secretion of cytokines – activation of macrophages
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NK cells provide an early response to virus
infection
• NK cells can kill certain types of target cells
– Base level of cytotoxicity is 20-100 fold > on exposure to IFN-
and IFN- produced in response to viral infection
• Type I interferon induce proliferation of NK
– In addition to activation by type I interferons, NK cells can also
be activated by IL-12 (macrophages)
– Four key cytokines produce activated NK cells early in an
infection
– Stimulation of NK cells with IFN- and IFN- favors the
development of cytotoxic effector function
– Stimulation of NK cells IL-12 release favors the production of
cytokines
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NK Cell Cytotoxicity

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Cytokine Secreting NK cells

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Dendritic Cells and NK cell Interactions

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 Dendritic cells and NK cells

• When there are more NK cells than infected

dendritic cells the NK cells are cytotoxic to the
dendritic cells
• When there are more infected dendritic cells than
NK cells the NK cells signal the dendritic cells to
migrate to the nearest secondary lymphoid tissue
to activate the adaptive immune system
– This still needs more research but may be the break point
between the innate and activation of the adaptive immune
system 0
 Two major classes of NK-cell receptors - NK-cell
Immunoglobulin-like receptors -NK-cell Lectin-like
receptors

,
nucleated or "
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Af
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C "
C A
vs - '

co #
c a
'
µ * HE
age s i '

es d .

tr
ce
s
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-

0
 NK-cell Killing: A Balancing Act
• The one requirement of NK-cell receptors
– They must inhibit the NK cell from killing healthy self-
cells
• Balancing act between activating and inhibitory
signals
– When a cell is infected, malignant or has received
trauma the protein expression will be changed and the
amount of activating signals > inhibitory signals NK-
cell killing
– Example of activating ligands on surface of infected
cell are MIC-A and MIC-B 0
0
 ‘Missing-Self”

• Intracellular parasite changes

the expression or conformation
of MHC class I
• Interaction of the cell with
receptors on NK cell generates
a stimulatory signal
• NK cell allowed to kill the
infected cell
0