Chapter 3

Innate Immunity-
Induced
Possible
immune
responses
Innate cell
receptors
Distinguish between microbial and human
carbohydrates, lipids, proteins and nucleic
acids (self vs pathogen)
Bind pathogens and infected human cells,
but not uninfected human cells
Receptors have cross reactivity, meaning
they can recognize pathogens that share a
specific cell surface structure
100+ innate immune receptors
Different cells express different receptors
or combination of receptors
Macrophage
Receptors
Many ligands are at high
density which allows
numerous receptor
molecules to bind in
cooperative fashion resulting
in irreversible attachment
and ultimate phagocytosis
Receptor-mediated endocytosis
Endosome/Phagosome
Lysosome
Phagolysosome-
Toll-like receptors (TLRs)
Transmembrane protein
◦ Variable region
◦ Conserved region
Signaling domain is a Toll Interleukin -1 Receptor
(TIR)
Pattern recognition domain of TLR is a repeated
sequence motif of 20-29 aa residues rich
in leucine -leucine-rich repeats (LRR)
Different TLR specificities is a result of a variation
in the number of LRR and in the amino acid
sequences of these repeats
Either heterodimer or homodimer
TLR-4
Gram-negative bacteria
release lipopolysaccharide (LPS)

Lipopolysacharide binding
protein (LBP) picks up soluble
LPS and delivers it to CD14

CD14 acts as co-receptor to TLR4

TLR4 interacts with MD2 and

forms a complex with CD14 and
LPS

This activates TIR domain

Adaptor protein- protein that has no enzymatic or other activity itself
but brings together two other components so they can interact. (MyD88
and TRAF6)
X-linked hypohidrotic ectodermal dysplasia and immunodeficiency
(NEMO deficiency)

Lack subunit of IKK (IKKγ or NFκB

essential modulator- NEMO)
On X chromosome so more
common in boys
Results in susceptibility to
bacterial infections - low activation
of NFκB through macrophage
TLR4
NFκB also important in
development of tissue derived
from embryonic ectoderm: skin
teeth hair
Conical or missing teeth
Visible symptoms help diagnose disease
Cytokines
Used as a means of communication between cells

Short lived

Exert influence within short distance

Sometimes make direct contact with other cell

Secreted by one cell and binds receptor on another cell, which results in cell
signaling that changes behavior of second cell
Inflammatory
Cytokines

Inflammation caused by
immune response not by
infection or wound itself

Chemokine- cytokine
involved in directing
WBCs to sites where
their functions are
needed
NOD-like receptors (NLRs)
nucleotide-
binding oligomerization domain
In cytoplasm

Detect degraded phagocytized pathogens

NOD1 and NOD2- recognize components of cell walls

Receptors form oligomers

CARD domain of NOD binds CARD domain on RIPK2

RIPK2 phosphorylates kinase TAK1

TAK1 phosphorylates and activates IKK

IKK phosphorylates IκB, which
disassociates from NFκB leading to cellular activation
IL-1β
Important in chronic inflammation
(rheumatoid arthritis)

Destructive potential so highly

regulated

Synthesized on ribosomes and in

cytoplasm and kept there as an
inactive precursor protein proIL-1β

Caspase 1 cleavage results in

activation
Production of IL-1β and Inflammasome
NLRP3

adaptor protein
CARD domain

procaspase 1

Oligomerization of NLRP3
High concentration of procaspase

autoproteolysis of procaspase 1

Caspase 1 proIL-1β yielding active IL-

1β
 Mutation in
NLRP3 gene
chronic inflammatory conditions.​​
Treatment: injection IL-1 receptor
antagonist (IL-1RA) (anakinra)​​
Fx: binds IL-1 receptor and
prevents IL-1 from sending signal
to cell​
 Short lived dedicated killers circulating in the blood
waiting for a call from the resident tissue macrophage

Historically called “microphage”

Most abundant WBC-(~50 billion in circulation)

Neutrophils
Stored in bone marrow for 5 days before released
are first
responders
Excluded from healthy tissue

Dominant phagocytic cell at infection site

Form pus
Adhesion
molecules
IMPORTANT F OR MOV E ME NT
OF LE UKOCYTE S BE TWE E N
BLOOD AND TISSUE
 Neutrophils
in Blood
No Infection
Infection
Blood vessels dilate
Endothelial cells express
selectins
Neutrophiles contact vascular
endothelium in capillary venules
Interaction between selectin
molecules and sialyl-Lewisx
carbohydrate groups on
neutrophil
Neutrophils begin to roll
Diapedesis/
Extravasation
TNF-α induces vascular
endothelium to
produce ICAM-
1 and ICAM-
2 adhesion molecules that
bind CR3 and LFA-
1 (integrin molecules) on
neutrophils
Initial interactions are
weak, but get stronger
when CXCL8 chemokine
binds receptors on
neutrophil
Phagocytosis by
neutrophils
Range of phagocytic receptors and complement
receptors

Range of material neutrophils engulf is greater than

macrophages

Greater diversity of antimicrobial substances stored

in granules
Programmed to die young so they devote more
resources to storage and delivery of antimicrobial
substances
 1° (azurophillic) granules- proteins and peptides that
Arsenal of disrupt and digest microbes
◦ Myeloperoxidase- lysozyme, defensins
the 2° (specific) granules
neutrophils ◦ Lactoferrin- competes with pathogens for metal ions
◦ Components of NADPH oxidase

3° (gelatinase) granules
◦ Gelatinase- metal containing protease- binds iron
 NADPH
Oxidase
• Assembled in phagosome after
fusion with the three types
of granules.​

• Increase pH of the phagosome to

7.8-8.0 because of consumption of
hydrogen ion​

• pH activates antimicrobial peptides

and proteins resulting in attack
of pathogen​

• pH slowly returns to neutral

• Lysosomes fuse
with phagosome and pH
goes down to complete the
degradation of the pathogen
Respiratory burst
Metabolic change accompanied by
transient increase in oxygen
consumption. Leads to generation of
toxic oxygen metabolites and other
antibacterial substances that attack the
phagocytosed material
Some toxic oxygen species diffuse out
and attack neighboring cells
To offset this damage there is an
increase in catalase and similar enzymes
which degrade hydrogen peroxide
Death of
neutrophil
Apoptosis - phagocytized by
macrophage

Netosis- Neutrophil extracellular traps

(nets)
◦ Nucleus swells, bursts
◦ Chromatin dissolves and extrudes
from cell along with proteins
from granules
◦ Even after death neutrophils
continue to trap and kill pathogens
◦ Upon successful neutralization of
the pathogen,
resident macrophages begin
the cleanup and repair
Chronic
granulomatous
disease
Localized nodules of infection

Mutation in one of the genes

that make up the
NADPH oxidase complex

No respiratory burst

No increase in pH
 Inflammation at a
localized infection
can result in
systemic effects
◦ Fever-
◦ helps fight infection
because bacteria and
viruses replicate and
grow better at temp
lower than body temp
◦ Lethargy, somnolence
and anorexia keep body
from wasting energy
that can be used to fight
infection
Pyrogens- molecules that induce fever
Exogenous pyrogens: bacterial products
Endogenous pyrogens: cytokines
Acute Plasma proteins quickly made in large amounts
by liver in response to infection

phase
proteins
 C reactive protein

C reactive protein used as a test for infection, inflammation, tissue

damage

Named for ability to bind C polysaccharide of Strep. pneumoniae

Binds fungi, yeast and some parasites

Acts as opsonin to activate the classical complement pathway

Binds phagocytes, suggests ability to deliver pathogens to phagocytes

Acute phase C reactive protein is a strategy to overcome population of pathogens

proteins
Serum amyloid A

Protein interacts with cell surface receptors TLRs and CD36 scavenger
receptor

Activates cells to produce cytokines

Amplifies state of inflammation triggered by pathogen

Importance
of acute-
phase
proteins
Additional
Complement
pathways
LE CTIN PATHWAY
CLASSICAL PATHWAY
 Lectin
Pathway
 Mannose Binding Lectin
• C-type lectin (requires calcium to work)​
• Binds mannose-containing carbohydrates of
bacteria, fungi, protozoa and viruses​
• Acute phase protein​
• Three identical polypeptides per flower
• Each MBL molecule has 5-6 flowers so 15-18
potential binding sites to pathogen​
• Act as opsonins
• Activate complement resulting in phagocytosis and
direct lysis by terminal complement proteins​
C4bC2a (Classical C3 convertase)
Classical
Pathway
 C-reactive protein
•C-reactive protein bound to
bacterium can interact with
C1 (similar to MBL with MASP-
1 and MASP-2)

•18 C1q molecules

•2 C1r molecules

•2 C1s molecules

•C-reactive protein binds to

C1q stalks causes C1r to cut
itself and other C1r and both
C1s molecules
C4bC2a (Classical C3 convertase)
Convergence of all
three complement pathways
C3 is present in higher concentration in
plasma then is C4 so contribution
of alternative convertase much greater
than classical convertase
Frequency of nonfunctional variants of MBL
greater than 10% in population
deficiency of MBL common increased
susceptibility to infection
Neisseria meningitidis infection greater in
people with two nonfunctional alleles of MBL
and terminal complement proteins
Alternative pathway

Lectin pathway

Classical pathway
Toll-like receptors (TLRs)
Receptors on plasma membrane
◦ Carbohydrates
◦ Lipids
◦ proteins

Receptors on endosomal membrane

◦ Pathogen nucleic acids
Toll-like
receptors
(TLRs)
10 human TLR genes

Number of TLRs are limited, but

recognize characteristic features
of all four groups of pathogenic
microorganisms
 Human cells can detect viral nucleic acids
and produce type I interferons
◦ Interfere with viral replication of infected cell
◦ Warn neighboring cells to prepare for viral
Innate infection
◦ Alert immune system cells that an infection is
immunity occurring
◦ Make virus infected cells more vulnerable to attack
against viral by killer lymphocytes

infection All cells can be infected by virus so all cells have ability
to make type I interferons and their receptors
IFN receptor is always present on cell surface

Type I interferon barely detectable in healthy

individual. Infection greatly increases production
 RIG-1 like receptors recognize viral RNA

RLR- RIG 1 like receptor

RIG- retenoic acid inducible gene-1
MAVS- mitochondrial antiviral signaling protein
Virus
infected cells
produce type
I interferons
Interferon
Response
Several interferon-
induced proteins are
transcription factors that
turn on many genes,
including those for the
transcription
of interferons.
Feedback loop
Plasmacytoid Dendritic
cells
Professional interferon-producing
cells

Secrete up to 1000 fold more

interferon than other cells

Found in blood and lymphoid tissue

TLR7 and TLR9

in endosomal membrane detect
viral infection and signal
through MyD88 ultimately resulting
in mass production type I interferon

Interferon spreads systemically to

help prevent systemic spread of
infection
 Natural Killer
Cells (NK cells)
NK cells are large and active lymphocytes
Speedily induced to respond to infection,
cancer and other forms of stress
Two functions during viral infection
1. Kill virally infected cells
2. Maintain and increase state of
inflammation
◦ Secrete inflammatory cytokines that
act on resident macrophages to
increase their ability to secrete
cytokines and to
phagocytize microorganisms
Distinguish NK cell from T cell- lymphocyte that expresses CD56 protein and no CD3
protein
 CD56dim 90% of blood NK cells

Have fewer CD56 molecules

Better at killing cells

CD56bright Predominate in tissues other than blood

Two
subpopulations
80% of NK cells in lungs
NK cells

Uterine important in placenta formation

NK cells
(uNK) Enlarge maternal blood vessels to supply placenta
most
abundant Almost all uNK cells CD56bright

leukocyte
poor at killing, good at secreting growth factors and non-
in uterus inflammatory cytokines
NK
recirculation
Made in bone marrow
Enter blood and circulate to lymph
node
Return to blood via lymph
Can enter tissue and return to
circulation via draining lymph
NK safety features
100 million + NK cells circulating in healthy human

NK cells filled with granulocytes capable of killing

human cells

Can’t release granules until in very intimate contact

with target cell

Kill only one cell at a time

Decision to kill or not kill made from many receptor
ligand interactions (sum of inhibitory and activating
interactions)
Base line is active inhibition based on inhibitory
receptors
 Activation of NK
cells by type I IFN
•Resident NK cells are exposed to IFN-α
and IFN-β ​
•Induces mitosis and proliferation​
•Induce differentiation into cytotoxic
effector cells that kill virally infected cells
NK interaction with
target cell
Receptor-ligand interactions concentrate at
localized region and form immunological
synapse (NK-cell synapse)
◦ Holds cells together
◦ Exchange of information and material

Receptors are recruited into this synapse to

engage their ligand if it is present
 If majority of interactions are inhibitory then cell is
released
If majority of interactions are activating then cell is
killed
Death of Strengthens interaction

target cell Reorganization of network

Quick, precise delivery of toxic cargo
Disrupts membrane and apoptosis occurs
 TLRs on NK cells
◦TLR3 recognizes ds RNA
◦ TLR7 and TLR8 recognize ss RNA
NK cells and macrophages
 IFN-γ or type II interferon

Structurally and functionally different from type I

Cytokines interferon

secreted by IFN-γ further activates macrophage to be better at

phagocytosis and destroying pathogens and in
NK cells secreting inflammatory cytokines

IL-12 by macrophages and IFN-γ by NK cells create a

positive feedback loop increasing the innate response
 Dendritic cells found in all tissues
Sample environment to detect infection and use info
to stimulate the responses of lymphocytes against
pathogen

Innate: dendritic-NK
NK cells Adaptive: dendritic-T cells

and Take up pathogens and their products from

environment, can be infected. This changes receptors
Dendritic on dendritic cells surface which receptors on NK cells
monitor
cells When NK cell detects changes on dendritic cell surface
a synapse forms between the two cells

Dendritic cell expresses IL-15 which induces NK cell

Lab
Experiments
RELATION S H IP BETWEEN
DENDRITIC CELLS AND NK
CELLS