ANSC3104 Lecture 4

Innate Immunity

Learning Outcomes

- Understand the roles and characteristics of the surface membrane

barriers that form the first line of immune defence
- Describe the non-specific cellular responses to antigen, with a focus on
phagocytosis, complement, inflammation and NK cells
- Explain how complement brings together aspects of innate (natural)
and adaptive (acquired) immunity to enhance the immune response

Three lines of immune defence

Infection: start and spread

- Infection occurs only after the organism or infectious agent has gained entry
into the body
o 1. List the ways in which an organism or infectious agent can gain entry
into the body?
Ingestion: eating/drinking (food poisoning)
Inhalation: respiratory pathogens
Lesion: wound, surgical lesion
Mammary glands (e.g. mastitis in cows)
Urinary tract
Ear, nose, eye, etc.
Insect bite
o 2. List the ways in which an organism or infectious agent can then spread
internally in an animal?
Skin is a really good barrier and there are other aspects of the
barrier system that are quite protective and good at stopping
infection infectious agents have ways of getting around this:
 Virulence factors (e.g. to allow
for adhesion)
Produce toxins at site of infection
(e.g. enterotoxogenic E. coli)
Target particular cells (some
pathogens will target
macrophages or T cells to hide
from the immune response)
Form biofilms (surface covering,
no longer exposed to the
immune response)
Extension from original site
Target tissues
o E.g. sheep with cheesy
gland, the pathogens
target the lymph nodes
and then cause necrosis at
that site through the
production of toxins
Blood stream: if there is a lesion or wound that goes directly
into the vasculature, or particular pathogens like anthrax can
target the blood stream

Innate immunity main players

- Barriers
- Innate immune cells
o Granulocytes, monocyte/macrophages, mast cells, NK cells, dendritic cells
- Mediators (soluble)
o Antimicrobial secreted substances, inflammatory mediators, complement,
cytokines/chemokines
- Actions
o Phagocytosis, direct cell killing, opsinisation, inflammation, cell migration
(chemotaxis getting other cells in to help), initiation of downstream
immune responses

Innate immune barriers

- Ski
n : has
epithelia
(great
barrier),
has tight

junctions between each cell, at the site of the skin

also have production of antimicrobial substances that can prevent bacteria from
getting a foothold at that site; also have a cutaneous microflora
 - Respiratory tract: mucous, ciliated epithelial cells can beat upwards to
transport foreign material out; also production/secretions of antimicrobials
- Intestinal tract: epithelial cells are the main protective ones, goblet cells
mucous producing, peristalsis (moving everything through), antimicrobial
secretions

Epithelial cells

- Barrier
o Tight junctions
- Antimicrobial peptides: prevent the
bacteria from getting a foothold
- Sense pathogens
- Activation
o Epithelial cells can be activated to
produce chemokines (interleukin A (a
cytokine that is chemotactic) tries
to attract other cells in)
- Image:
o (3) Produce toxins that kill epithelial
cells
o (4) Destroy the tight junctions
o (7) Some have adhesive molecules
that allow them to gain entry in to the cells

Other surface membrane defences

- Mucus (respiratory tract, intestinal lining)

- Hairs in the nose
- Stomach acid and acidic pH of the vagina
- Enzymes (soluble mediators) and antimicrobial products
- Expulsion: coughing, sneezing, vomiting, etc.
- Commensal bacteria

Enzymes and antimicrobial products

- Very important as they are the front line (ready to go, no time lag in the defence
they provide)
- Provide protection early in infection
- Anti-microbial proteins/peptides
o Produced by epithelial cells and phagocytes
o Direct and immediate no time lag in defence
o Detect early infection
o Include:
Lysozyme: digests and breaks specific bonds in bacterial cell walls
Defensins: ancient, evolutionarily conserved antimicrobial peptides
that disrupt bacteria, fungi cell membranes and some viral
envelopes
Cathelicidins: made as inactive propeptides and cleaved to form the
active peptide when needed (can be targeted to where they are
needed)
Histatins: produced in the oral cavity
- Having the right diversity in your microbiome (commensals within the gut) may
act to be protective in a lot of different disease conditions
 o Interactions between the microbiome and innate immune system: if you
have had a course of antibiotics and then are exposed to a viral pathogen,
there can be lack of responsiveness of the system, has suppressed that
interaction as the commensals have been wiped out as well as the
bacterial pathogen, is dampening down the interaction between the
commensal bacteria and the innate immune system at the site of the gut
this can have systemic effects. Its tempering the immune response so
that you have a level of responsiveness of your cells that you can instantly
respond to other pathogens

Cells of the immune system

- Leukocyte is overarching term for all types of white blood cells; is not limited to
just the innate or adaptive cells (more broad term), can then break down into
subtypes:
o Neutrophils: front line defence, are phagocytic, have bactericidal
mechanisms (they produce a lot of antimicrobial substances), they set the
scene for further aspects of the inflammatory response
o Lymphocytes: subset of broader categorisation of leukocytes, are involved
in adaptive immunity
Are the B and T cells that give the memory response
 o NK cells: important front line defence, they are surveyors, keep a constant
watch to detect anything abnormal, particularly important for tumour
responses, also important in viral responses, look like lymphocytes but are
part of the innate immune response (not the adaptive)
o Monocytes: differentiate into macrophages (in tissue) monocytes are in
blood, phagocytic, bactericidal, important in cell recruitment
- Mast cell: present within tissues not the blood, part of the innate immune
response
- Dendritic cell: present within tissues not the blood, part of the innate immune
response
- The definition of innate vs. adaptive is based on the specificity of the receptor
o The monocytes dont have those specific B or T cell receptors, but they
show the B or T cells what they need to be doing how they are
connected to the adaptive immune response

- Have barriers, cells

- Timeline:
o Innate immunity: early responses, occurring over a time period of 0-12
hours, important before the challenge can take hold
o Adaptive immunity: occurring over a period of 1-7 days

Recognition of foreign agents

- How do innate cells distinguish between self and non-self?

o There are a limited number of receptors these cells express that help
them identify what is self and what is non-self
Germ-line encoded: no modification, they are a set series of
receptors that every animal makes
o Recognise common features of pathogens
o Pattern recognition receptors (PRR) recognising microbial patterns
These recognise
Pathogen-associated molecular patterns (PAMPs)
Damage-associated molecular patterns (DAMPs)
o Damage to own cells, body sends out signals in
response
o These can also initiate some of the innate immune
responses
 - Really large range of things these cells can recognise as foreign (shown in table
above)
o From RNA, to flagellin on the bacteria, lipopolysaccharides
(carbohydrates), proteins, etc.
- PRR: there are a few different types of PRRs
o Toll-like receptors 1-10
Family of receptors with a similar structure, there are around 11 of
them (1-9 are in all species and 10 and 11 are only in some species)
o Range of other cell surface and intracellular receptors

Toll-like receptors

- TLR signalling induces cytokines

- PRR receptors not all on the cell surface
- Image below (toll-like receptors)
o TLR5: recognised in flagellin
o TLR4: recognises LPS which is a fairly major pathway for gram negative
bacteria
o There are some of these TLRs that are expressed within endosomes; once
you have a pathogen or foreign substance taken up within a cell, then it
goes into an endosomal structure and some of those TLRs are in the
endosome so they can see it (e.g. the ones that recognise DNA and RNA
are within endosomes)
o They have extensive signalling pathways that lead to the production of
things that will stimulate or somehow modify the immune response
- Image below:
o TLRs: on the surface and in the endosomal space
o NOD-like receptors (NLRs), RIG-like receptors (RLRs), cytosolic DNA
sensors: are within the cytosolic space, looking for signals within a cell
Are PRRs, not just on the surface of the cell, are looking everywhere
in the cell as there are viruses that can go into the cytosol, bacteria
that can go into the endosome, need to be aware of all the different
modes of infection

- An example (dont need to remember this): the risk of developing TB is 26 to 31

times higher in people living with HIV than among those without HIV
 o HIV causes deficiencies in the immune
response
o More susceptible to TB
o There is a link back to HIV if you are
infected with TB, you may be more
susceptible to HIV
o Looked at TLR2: TB, as one of its
survival strategies, signals through
TLR2 in order to downregulate/depress
the immune system in the cells it
attacks (macrophages); some of the
virulence factors (lipoproteins) of TB that signal via TLR2, enhance the
infectivity of HIV CD4+ T cells

Non-specific cellular responses

- 1. Phagocytosis
o One of the main processes of the innate immune
system for clearing/getting rid of foreign
agents/bacteria
o The three main cell types involved are:
neutrophils, macrophages and dendritic cells
Macrophages are called different things
depending on which tissue they are in but
are all similar in action:
Liver: Kupffer cells

Phagocytes

- Three main types of phagocytic cells:

o Neutrophils: circulate in blood and migrate into tissues very quickly in
response to local invasion to the site where they are needed
o Monocytes of blood and macrophages of tissues: monocyte-macrophage
system
Sustained phagocytosis
Assist in tissue repair

o Dendritic cells: link the innate and adaptive immune responses, tissue
resident cells, involved (chiefly) in antigen presentation, can also
phagocytose pathogens
Adaptive immune response
 - Used intravital microscopy: can look at what is happening inside an animal in
real time
- Injured site is labelled with red dye
- Neutrophils are green: many cells migrate to the site of injury to repair the
damage
o Very fast response, this is why neutrophils are the first line of defence

Neutrophils NETs (Neutrophil Extracellular Traps)

- Neutrophils can make these NETS

 - Act like a net: when they are relocated to site of injury/pathogen/immune
response etc. they can spill their nuclear contents out into the extracellular
space
o Associated with these nuclear contents (sticky) are microbial granules
which can kill bacteria
o This net is poisonous
o Aside from ingesting the bacteria, this is another mechanism neutrophils
can use to trap and get rid of bacteria in tissues
Phagocytes

- Phagocytosis i.e. eating bacteria

- Involves:
o 1. Chemotaxis: attraction of cells to
a site along a chemical gradient to
invader
Examples of chemotactic
substances: bacterial
products, interleukin (IL)-8,
blood vessel damage,
complement factors
Along a chemical gradient
Neutrophils
See directional migration of
neutrophils
Can see in image, neutrophils migrating towards the chemotactic
substance (fMLP in this example)
o 2. Adherence: bind invader to their cell membrane
o 3. Ingestion: internalise the invader
Then ingest or internalise foreign material
o 4. Digestion: through generation of toxic radicals and enzymes (performed
in the lysozymes)
One of the mechanisms of breaking down the cell once it has been
phagocytosed is oxidative burst: produces oxygen radicals
Lysozyme: enzymes; generation of a phagolysosome a fusion of
the phagocytic parcel that has come in with the lysozymes
o 5. Release of bacterial waste

Summary

- Surface membrane barriers have a range of characteristics, roles and

mechanisms that act in the first line of host defence (not just a barrier, have
active roles)
- Innate immune cells recognise pathogens using a range of germ-line encoded
Pathogen Recognition Receptors (PRR e.g. TLRs)
- Phagocytosis (recognition, ingestion and killing of pathogens) is an innate
effector mechanism
- Three main cell types involved in phagocytosis: neutrophils (or PMNL
polymorphonuclear leukocytes), monocyte/macrophages, dendritic cell (DC)
phagocytes
 Lecture 5
Innate immune cells and molecules

- Phagocytosis
- Complement system
- Inflammation
- Efficiency of phagocytosis is increased by complement an antibody bind to
surface of organism making it more attractive to phagocytic cells

Non-specific cellular responses

- 2. Complement
o Originally used to describe the activity of molecules in serum which could
complement the ability of antibody to lyse bacteria
o If there was uncoated bacteria, they would be cleared but at a slower rate
o Then is antibody specific to the bacteria was added to the mixture
o And if a combination of antibody and these plasma proteins was added,
then the rate of killing of the bacteria was enhanced (augmented)
o This augmented aspect: now getting an interaction between both the
innate and acquired immune response; the antibodies are from the
acquired/adaptive immune response and the complement is part of the
innate immune response

Complement

- Complement is a series of around 30 plasma proteins

- Known as a complement cascade as these proteins work in concert; the
complement system is a highly complex molecular chain reaction or cascade
- Basic principle: products of one reaction catalyse a second reaction, whose
products catalyse a third reaction etc.
- Present in the serum as inactive proenzyme (zymogens),
with a trigger they become active and activate the next step and so on
- Requires a trigger to initiate chain reaction; important because you dont want
this to go off and damage cells it shouldn't (self-cells)
 - Amplification: steps in this where you can get self-amplification of this process
so you can get more and more complement being produced if required, or more
and more coding of the pathogens
- Check points: you cannot get complement forming on the host cells, it can only
form on pathogen cells

Complement cascade

- Simplified diagram of the complement cascade

- There are now three known pathways by which complement can be activated:
o Lectin pathway: based on binding of lectins (soluble carbohydrate binding
proteins e.g. MBL (mannose binding lectins))
o Classical pathway: can be triggered without antibody, can also be
triggered in the presence of antibody (involves complement proteins C1,
C2 and C4)
o Alternative pathway: direct activation of the first component (C3),
interacts with other proteins (factor B, factor D, etc.)
- All three pathways lead to this first step where you get breakdown of C3 into
C3a and C3b fragments and the C3b is deposited on the surface of the pathogen
this is the opsonisation step where the pathogen is now recognised even
better by phagocytes to be killed
o Breakdown product C3a is a trigger for other inflammatory processes (as
is C5a)
o There is no waste in the immune response, all components are used (as
signals in later immune steps etc.)

Membrane attack complex (MAC)

- The MAC is the effector arm of the complement cascade

- Effector mechanism of innate immunity
- Formed on the surface of pathogenic bacterial cells
 - Assembly of terminal components of the complement system
- Forms transmembrane channels that disrupt the cell membrane, leading to cell
lysis and death
- Is where a pore is formed on the surface of a pathogen only formed on
pathogenic bacterial cells and involves assembly of terminal components of a
complement system
o That pore is like a transmembrane channel, it punches a hole in the lipid
bilayer and can get influx of fluids from the outside and the cell bursts

Assembly and regulation of the MAC based on structures of C5b6 and sC5b9

- Looked at C5 and C5b to see these would fit together how the pore would be
formed
- It caused a conformational change when it broke down from C5 to C5b, if there
was no membrane for it to bind to, then it activates itself so it doesnt form a
pore
- If there are the right conditions, get formation of MAC
- Direct/effector pathway for destroying the bacteria, also have an indirect
pathway where its opsonised and then you get phagocytosis (two-pronged
attack)

Complement system

- Actions of the complement fragments

o No waste in the immune response
- Opsonisation (of the key aspects)
o Based on the classical pathway for complement activation
o Leading to phagocytosis
o Integration between adaptive and innate immune response: the two arms
of the immune response are integrating
o Image: have antibody and C3b present on the surface of bacteria they
signal to phagocytes that this needs to be removed, come and digest this
- Initiate local inflammation
o C5a>C3a>C4a
 o Increase the blood flow and vascular permeability
o Stimulate mast cells to release granules
Degranulate release their granules which contain the active
components
o Increase migration (chemotaxis) and activation of macrophages and
neutrophils

Non-specific cellular responses

- 3. Inflammation (where all these things

are tying together)
o Oldest pathophysical phenomena
o Roman philosopher Cornelius
Celsus, 1AD, great medical writer
formulated four cardinal signs of
inflammation calor, rubor, tumor,
dolor (heat, redness, swelling
and pain)
Identified it was important
to wash wounds with
antiseptic solutions
o Later Virchow (father of modern pathology) added fifth sign: fanctio laesa
(loss of function)

What is inflammation?

- Reaction of living tissues to local injury

- Agents that cause inflammation:

Agent Type
Physical Foreign object (e.g. splinter),
trauma, heat/cold, radiant energy
(UV and sunlight)
Infectious Microbes (bacteria, viruses, fungi),
protozoa, parasites
Chemical Harmful/corrosive/toxic chemicals,
biologicals (plant toxins, venoms,
microbial toxins)
Other Cancerous cells/tumours, immune
response (autoimmunity,
 hypersensitivity), ischaemia (lack
of blood supply and subsequent
death of the tissue)

- Inflammation is a protective response aimed at containing and eliminating the

cause of tissue injury as well as dead cells and tissues from the original insult
- Sets in motion the events that eventually heal and restore normal function (if
possible)

Comparison: acute vs. chronic

Feature Acute inflammation Chronic

inflammation
Onset Fast: minutes or hours Slow: days (even
months and years)
Cellular infiltrate Mainly neutrophils Monocytes/macrophag
es and lymphocytes
Tissue injury, fibrosis Usually mild and self- Often severe and
limited (fibrosis) progressive (excessive
fibrosis can be a
pathological form of
inflammation that has
negative impacts as
well as the positive
effects it should have)
Local and systemic Prominent (very Less prominent (may
signs obvious) be subtle)

- Acute = fast
- Chronic = long term
- Chronic: lymphocytes are the adaptive arm of the immune response

Main processes

- Vascular changes
o Leads to swelling as well as the influx of the mediators that are there to
clean up afterwards
- Chemotactic factors released
o Factors that attract cells into the site those are the cellular infiltrate and
effectors
Cellular infiltration and effector functions
- Fever
o Is triggered by certain cytokines, can help to control the proliferation of
certain bacteria that do not survive in hot environments
- Healing
- Many of these initiate and/or participate in the inflammatory process; not
separate events, all come together
Vascular changes

- When you have tissue damage, the first phase of inflammation is the fluidic
phase get fluid going into the site due to vascular changes like vasodilation
and increased vascular permeability
o Can also have direct effect if there is an injury to a blood vessel, this will
directly allow passage of fluids and other mediators into the site
- Image: bottom picture of blood vessel in an inflamed tissue get increase in
blood flow with dilation of the vessels, leakage of plasma proteins into the space
oedema, also get migration of neutrophils out of the vessels and into the
space which happens quite rapidly

- Histamines and bradykinin are called vasoactive amines: histamine is mainly

released by mast cells but can also be released by basophils and platelets
o Actions in vasodilation and mast cells are those cells that are located at
the tissue site
- Histamine: mast cells widely distributed in tissues close to blood vessels. Mast
cell activation granule release (degranulation). Basophils, platelets also a
source. Serotonin is a vasoactive mediator similar to histamine found in mast
cells and platelets in the gastrointestinal tract and CNS
- Bradykinin: released upon vascular and/or endothelial injury. Increases vascular
permeability and vasodilation. Bradykinin is also a major mediator involved in
the pain response (associated with the inflammation). Importantly, activates
phospholipase A2 (PLA2) to liberate arachidonic acid (AA). Arachidonic acid
yields active lipid called prostaglandins. Eicosanoids (e.g. prostaglandins,
leukotrienes) are both important mediators of vascular changes. Leukotrienes
are also strong chemoattractants have dual action
- Nitric oxide: can injure vascular endothelium leading to increased vascular
permeability as well as activating them to increase adhesion molecule
 expression. Important in the next phase (cellular). Produced by phagocytes,
endothelial cells
- Complement-derived peptides (C3a and C5a): increase vascular permeability,
cause smooth muscle contraction, activate leukocytes, and induce mast cell
degranulation. C5a is a potent chemotactic factor for neutrophils and
mononuclear phagocytes
- Endothelial cells lining the blood vessels are active not passive players (e.g.
produce nitric oxide, cytokines, express adhesion molecules)
- Prostoglandins and leukotrienes: dual action

Cellular response

- Margination: leukocytes move to the edge of the blood vessels rather than
central flow
- Rolling and adhesion: mediated by receptors on the leukocytes and
corresponding receptors on activated endothelial cells selectins and integrins
- Extravasation and migration: leukocytes pass between endothelial cells and
move to the site of inflammatory stimuli

- Image (above): if you have a stimulus (green), it could in its own right trigger
this or may have response to the bacteria that are part of that insult, releases
vasoactive and chemotactic factors which lead to extravasation of the cells
which migrate to the site. That occurs through a process that involves
margination, rolling and adhesion and extravasation and migration
- When the cells start to marginate they move to the edge of the blood vessels
they are associating more closely with the endothelial cells (cells that line the
blood vessels)
- Then they start rolling along these endothelial cells, that is mediated by
receptors on those cells called selectins and integrins
- These endothelial cells are not passive, are active participants in this process,
are expressing receptors that allow the neutrophils and macrophages to bind on
and go across the vascular wall
- Then the leukocytes can move between endothelial cells in order to move closer
to the site of inflammation stimuli (squeeze in between the endothelial cells)
 - Image: timing of this overall process is initially get oedema because of the
vascular changes that occur very quickly (swelling, entry of fluids into the space
fluidic phase), then get entry of neutrophils first responders, then get
monocytes and macrophages coming in later

Cellular response: phagocyte emigration

- Emigration from blood into tissue process of rolling, adherence and emigration
between endothelial cells (inflammation)
- Margination and rolling
o Margination where they start moving near the edge of the vascular wall
o They start rolling along until they get a firm adhesion because there is
formation of complementary binding between the receptors on the cells
and the receptors on the endothelial wall
This is a very important for how the lymphocytes and leukocytes get
around they need to know where to go this process of receptor
expression by cells and endothelial cells is how leukocytes know
where they need to go, if they dont see a complementary receptor
then they will stay in the blood stream, if they do, then they know
they need to go to that tissue site
o This process of squeezing between is called diapediesis
o Then get chemotactic movement along the gradient of the chemokines
This is where phagocytosis comes into play
Endothelial cells

- Cells in the blood stream that are involved in:

o PMNLs (neutrophils): elimination of microbes, dead tissue
- Mast cells: source of histamine which is mediating some of the vascular changes
- Macrophages: another kind of phagocytic cell as well as capable of producing
mediators of inflammation like cytokines
- Endothelial cells are capable of being a source of mediators as well
- Complement: dual actions of signalling to phagocytes as well as being involved
in direct elimination of microbes

Inflammation: five cardinal signs

 - First step: tissue damage which leads to chemotactic factor and vasoactive
factor release
- Vasoactive factors have two actions: vasodilation (allowing the vessels to
expand) and increased vascular permeability
- Chemotactic factors: bringing the cells in
- All combined leads to a loss of function

Tissue repair

- Inflammatory response sets in motion tissue repair processes

o Inflammation is the first step in tissue repair processes; not strictly part of
the immune response

1 2 3

o (1) Migration of neutrophils and formation of a blood clot

o (2) Fibrosis, movement in of fibroblasts that help to stabilise this and
regenerate the tissues
o (3) Then hopefully get back to normal function

Non-specific cellular responses

- 4. Natural killer (NK) cells

 o Really important cell type that are in a grey area they have some
features of innate immune cells and some features of adaptive immune
cells, however, are classified as innate immune cells
o They are large granular lymphocytes (represent 5-15% of blood
lymphocytes)
o Found in normal, unimmunised animals
o Arise from a singular precursor of T cells, independent of antigen-specific
receptors (do not have these receptors)
The hallmark of an adaptive response is an antigen-specific receptor
o Located secondary lymphoid organs as well as in the blood and they have
an immune surveillance function
o Promote cell lysis of tumour or virus infected cells
o Main function is to kill
o Mechanisms they use to kill are: direct cytotoxicity, release of granules
that can punch holes in the membrane, induction of apoptosis
(programmed cell death)
o NK cells are lymphocytes with no immunological memory part of the
innate immune system
They do not need to recognise a specific antigen before releasing
their toxins and destroying a viral infected cell or a tumour cell
In many immunodeficiency diseases (e.g. HIV), NK cell function is
abnormal, allowing the viruses to multiply inside cells
Binds to invading cells, releases its toxins which punch holes in the
membrane, inducing apoptosis

Summary

- Complement is a series of plasma proteins that act in a cascade that can lead to
formation of the MAC and other inflammatory/immune mediators
- Three main pathways that can activate the complement cascade: lectin,
classical and alternative
- Interaction between innate and adaptive immunity; complement the ability of
antibody to lyse bacteria
- Five cardinal signs of inflammation
- Vascular and cellular changes in inflammation

First and second lines of immune defence

- Defining features of innate immunity

o Equally effective against all foreign substances
o Carries no memory of an encounter with foreign substance
 ANSC3104 Lecture 6

Humoral Immunity

Review

- Barriers: e.g. epithelial cells protect from invasion by pathogens

- Innate cells/phagocytosis: oxygen radicals respiratory burst, PRRs, NETs, mast
cell degranulation (granulocytes release granules at sites of inflammation)
- Complement
o Linkage between the innate and adaptive immune responses that leads to
opsonisation and phagocytosis of targets
o Three different pathways: classical, alternative and lectin
- Inflammation:
o Five cardinal signs: swelling, heat, redness, pain, loss of function
o Fever is a corollary of inflammation but is not one of the five cardinal signs
- Natural killer (NK) cells
o Kiss of death

Review mechanisms of killing

- What are the killing mechanisms used in the innate

immune system?
o Complement
Membrane attack complex (MAC)
Opsonisation (indirect pathway including
phagocytes)
o Natural killer (NK) cells
Direct cytotoxicity
NK granules contain granzymes (proteases) and they release
perforins (which are similar to MAC from complement) which
are inserted in to the membrane of target cells to cause lysis
Induction of apoptosis (programmed cell death)
o Phagocytosis (image below)
There is a mechanism within the phagocyte to produce these very
toxic radicals or reactive oxygen species and also reactive nitrogen
species
Also a pathway where a receptor binds to the microbe, it gets
internalised into a phagosome (ingestion within the macrophage or
other phagocytic cell), this fuses with a lysosome which contains
enzymes that help digest and breakdown that microbe
There are two aspects to the mechanisms of killing used by
phagocytes
Questions

- Neutrophil numbers in reservoirs

o In some species there are reservoirs of neutrophils (very species specific)
o There can be a few days worth of neutrophils stored in the bone marrow
ready to be released (e.g. in dogs)
o In cattle there are very few neutrophils in reservoirs
- Neutrophil numbers in blood during inflammation
o Generally where there is an inflammatory stimuli, get an increase in
number of white blood cells because some of those mediators of
inflammation stimulate the production of granulocytes and other cells
from the bone marrow
o If there is a very overwhelming inflammatory response, those cells could
all have migrated into the tissue could potentially have neutropenia
ow number of neutrophils in the blood cell count (very uncommon), more
common that you would get an increase in the cell numbers
- Pattern recognition receptors (PRR) these are the receptors on the innate
immune cells
o PRR: e.g. TLRs are the receptors that are on the innate immune cells in
the animal
- Pathogen-associated molecular patterns (PAMPs) are the structures on the
pathogen that are recognised by PRR
o These are the things that these receptors on our innate cells are able to
recognise as foreign/non-self
- Pain in inflammation
 o Arachidonic acid pathway: can lead to eicosanoids (prostaglandins and
leukotrienes) these are mediators of inflammation
One of the enzymes involved in this process is cyclooxygenase
inhibitors of this enzyme are commonly used as anti-inflammatories
Common target for things like paracetamol (Panadol), ibuprofen
(nurofen)
COX-1 and COX-2 inhibitors target this pathway
Because PGE2 is not only involved in vascular changes but also in
the pain responses in inflammation
Dual action

- Image:
o Innate: have barriers, have enzymes and antimicrobial substances being
produced, have complement that is acting in the innate immune process,
have different cell types
o Unless an animal has a minor issue, will get induction of adaptive immune
responses; innate immune responses are really important though
 because, if there were congenital deficiencies in many of the innate
pathways any organism with those is highly susceptible to recurrent
bacterial and other types of infections (very important on a daily basis)
o However, adaptive immune responses are where evolution of the immune
response has moved on well developed

Immune system function: humoral immunity

Learning outcomes

- Describe how humoral immune responses are antigen-specific, how the

responses are generated and how the characteristics of the antigen-
specific responses differ to innate immune responses
- Outline the process of antibody production and the characteristics of a
primary and secondary antibody response
- Appreciate the ways in which antibody inactivates antigen
- Be familiar with the different antibody isotypes (IgG, IgM, IgA, IgE) and
be able to describe their functions in protecting the host from an
antigen

Antigen-specific immunity: lymphocytes

- T-lymphocytes: cell mediated immunity

o Originate from bone marrow
o Mature in thymus
- B-lymphocytes: humoral immunity
o Originate from bone marrow
o Mature in bone marrow
In chickens mature in bursa of Fabricius
In ruminants and pigs mature in Peyers patches (gut)
- Humoral immunity relates to B lymphocyte responses (B cells)
o B cells produce antibodies very important in vaccines

Properties of Innate and Adaptive Immunity

Innate Adaptive
Innate immune receptors are Adaptive immune receptors are
germ-line encoded generated by rearrangement of
gene segments
There is immediate maximal There is a lag time between
response (first line of defence) exposure and maximal response
Not antigen-specific Antigen-specific
No clonal selection Clonal selection/expansion
Exposure results in no Exposure results in immunologic
immunologic memory memory

Initiation of adaptive immune responses

 - Draining lymph nodes
o B cell zones adjacent to T cell zones
o Antigen and antigen presenting cells
travel from the site of
inflammation/infection to the lymph
node
o Activate antigen-specific lymphocytes
- If there is a distant site (e.g. leg) where
there is an inflammatory response
occurring, will get fluids and other things
draining from that site to the closest lymph
node (called the draining lymph node)
o Often will get enlargement of that draining lymph node
o Within the lymph nodes, there are cells that form the adaptive immune
response arm: B cells (follicles), T cell and APCs (T cell zone)
These zones are adjacent to each other interaction between these
cell types
Dendritic cells are present all around the body will go to the lymph
nodes to tell the immune system where there is an issue and back
up is needed
o Oedema at the site fluids going into the site increased drainage
through the lymph node can get antigen itself (APCs) coming through
the draining lymph node this is how they activate antigen specific
lymphocytes

Antigens and epitopes

- Antigenic specific receptors

o Recognise specific antigen
- An antigen is substance that binds to a
lymphocyte receptor
o Foreign antigens, autoantigens,
alloantigens
- Epitope
o Part of an antigen
o Most potent immunodominant
The one looked for when
determining whether there
is an immune response occurring or not
- Antibodies recognise different epitopes
- A T cell would also only recognise a particular epitope

Bacterial genome

- This bacteria has a very large

number of proteins
- All these proteins are
potential antigens of this
bacteria
- A bacteria doesnt have just
one thing that can be
recognised as foreign
 - Adaptive system is geared more towards recognising multiple potential things
that are foreign about a target
- The innate system can recognise key structures (PRRs)

Epitopes

- Each pathogen may have hundreds of

epitopes
- Some epitopes may be more important
than others for protective immunity
- Lymphocyte receptor is specific for the
epitope, not the whole antigenic molecule

Antigen recognition for lymphocyte activation

- The way lymphocytes recognise antigens is

different between B cells and T cells
- T-cells require processing and presentation of
antigen via special antigen-presenting cells
(APC)
o Dendritic cells will go to the lymph node
to show the T cells the antigen (use MHC
molecules)
- B-cells can recognise native antigen
o Can see a foreign agent directly, does
not need to be broken down
o Also present antigen to T cells, get T cell
help
Once the antibody binds to a
foreign agent, it very rapidly gets
internalised into the B cell, broken
down, and the B cell can then act
to show the immune system the
epitopes associated with the
foreign agent
B cells need T cell help in order to respond in a particular challenge
(antigen)
Usually will not get an adequate B cell response without T cell help

Overview of acquired immunity

- From the development of those mature B and T cells, get activation of particular
cell types that are the responder cells of the adaptive immune response
o For B cells, these are the plasma cells: produce lots of antibodies
releasing it into the blood stream or to the site
 o Can also form memory: memory cell populations come after there is
activation of a particular immune response long-lived cell population
that is produced in order for this to occur
o There are also different antibody isotypes: not just one type of antibody
different as they have different function (e.g. might need to be in blood,
tissues, lumen of the gut structures need to be different)

Antibodies (immunoglobulins, Ig)

- Antibodies are also called immunoglobulins (Ig)

- Antibodies are synthesised only by B cells
- Can be either membrane-bound (on the surface of B lymphocytes) or secreted
- Membrane-bound antibodies function as antigen receptors to activate the B cell
- Secreted antibodies are present in the plasma and other tissue locations
(multiple locations)
o Neutralise toxins
o Prevent the entry and spread of pathogen s
o Role in eliminating microbes

Basic structure of antibodies

- Like a Y
shape
because the
majority of
antibody
isotypes are
quite flexible
- These arms
(Fab
fragment)
can move to
adjust for a
different type
of antigen
- The arms have variable regions that are the parts of the antibody that can
recognise an antigen
- Constant regions: are referred to as the FC region, they do not vary this is
important because it is the FC portion of the antibody that can be recognised by
other cells of our immune system (e.g. phagocytes have receptors for that FC
portion)
- Have two chains: heavy and light chains called this because they have
different molecular weights
o In the antibody pictured above, it is symmetrical, so the heavy chains are
the same in any particular antibody and the light chains are the same
So an antibody, either arm, will recognise exactly the same epitope
of an antigen
- Top image:
o When you have the secreted
form of an antibody, it loses
a chunk which is this
transmembrane region and
cytoplasmic tail
o The membrane bound
versions are slightly larger,
they are inserted into the
membrane of the B cells and
include the transmembrane
region and cytoplasmic tail
o The secreted versions are
truncated
o Can make exactly the same
molecule but for different
purposes
- Bottom image:
o Can see there are some
that look very much like
the basic structure of
an antibody and others
look like joined
together versions of
that basic structure
o All mammalian species
have these represented
classes of antibody, but
there are species
specific variations
Some species
only have certain
subclasses,
others have a
slightly different
structure
IgM

- Largest, pentamer (because it needs to be in the

blood)
- Primary response
- First antibody, made quickly (2-3 days) to
fight infection
- Main job is to trigger complement responses
and to form agglutination (to bind up
antigens)
- Effective defence in blood stream

IgG

- Protects extravascular spaces

- Neutralises toxins
- Readily binds to foreign substances i.e.
opsonisation
- Main Ig in secondary immune response
- Passed into colostrum (maternal
antibodies)
o Important in terms of
vaccination: initially, colostrum
is provided to newborn infant
(not born with fully mature
immune system), so for
protection, the other provides
them with a form of humoral
protection in the form of
maternal antibodies (highest
straight after birth and then
decrease in the plasma of neonates over time)
o If you were to vaccinate a very young animal that had just been exposed
to the maternal antibodies, those antibodies would bind the components
of the vaccine meaning the animal would not develop the appropriate
immune response no protection
o Balancing act: right time to vaccinate is when there is a decrease in the
maternal antibodies to a sufficient degree that the vaccine will be
effective but also before the animal is expose to a potential infection
- Isotypes: can have slightly different functions

IgA

- Mucosal immunity: released into

mucosal sites
- Isotypes
- In image: IgA is binding to
cholera directly as well as the cholera toxin
o Two ways this is being protective against the infection with cholera at the
lumen of the gut
IgE

- Very important for intestinal

parasites: it associated with the cell
types in the innate immune
response that respond to parasites
(eosinophils, basophils and mast
cells)
- Allergic reactions
o Image: have initial
contact with an
allergen, B cells
recognise this and
go on to have
production of
antibodies that are
released (plasma
cell formation and
production of
soluble antibodies),
these can associate
with mast cells;
mast cells have FC
receptors that binds
to the end portion of
the antibody for an IgE type of antibody
o IgE particularly associates with those cells so when an individual is re-
exposed to an allergen, they are right there on the site ready to trigger
that mast cell to release histamine and other mediators (an acute reaction
to the allergen)

IgD
 - Expressed only on the surface of mature,
nave (havent seen anything before, not
activated) B cells

B cell activation and T help

- Signal 1: the direct binding of the B cell receptor (an antibody that is bound to
the surface of the B cell) and the signal (whatever the antigen is)
- B cells need two more signals to become fully activated; need to bind to T cells
that are specific for that same antigen (via MHC II and costimulatory molecules
e.g. CD4), and
o CD40 and CD40L (ligand): CD40 is on the surface of the B cell and CD40L
is on the surface of the T cell. Once there is binding of both and the MHCII
to the T cell receptor, can then get activation of the B cell
o T cell is also providing cytokines, mediators that are helping the B cell to
respond and these are IL-4 etc.

Clonal expansion

- Other key difference between innate and

adaptive immunity
o If can recognise an antigen, can then go on
and make many clones of itself to
effectively respond to the antigen
- Effector cells
o Plasma cells
o Ab production
- Memory cells
o Long lifespan
o Fewer cycles to be effector cells
- In image: exposure of a B cell to an antigen,
needs to make lots of clones of itself, with T
cell help, it will go on to be activated, start dividing and making clones which
ends in the formation of effector cells (plasma cells) quite large and will
produce lots of antibodies to be released and be effectors of this immune
response
o Also get some memory cells produced: long lifespan = faster immune
response next time this antigen is encountered
Sir Frank Macfarlane Burnet

- Australian scientist and Nobel prize winner

- Published his Clonal Selection theory in the Australian Journal of Science (1957)
- Burnets paper was the first to theorise that continued somatic mutations after
antigenic selection would result in affinity maturation. In addition, he proposed
that repertoire purging could explain the phenomenon of tolerance. Finally, he
proposed that aberrant cell division of clones bearing self-specific antibodies
could lead to autoimmunity. Significantly, Burnets theory set the scene for
modern cellular immunology research, and his paper should be considered a
seminal landmark in the history of immunology
- Repertoire: removal of self-reactive cells before they can get into circulation

Primary and secondary responses

- Vaccinations: so there is a secondary immune response

- Primary immune response has a delay time of up to seven days; long delay
before you get an effective response the first time a pathogen is seen
- If a challenge of a bad pathogen is encountered, need to respond much faster
than that in order to control, contain and subsequently recover from that
pathogen
- Top image
o If there is a primary exposure to an
antigenic challenge, this graph shows
the serum antibody titre
o Initially a lot of IgM is made and there
will be an antibody type switch to IgG
o This will diminish over time there will
be some that remains in circulation
over time but this level is low
o When you encounter this antigenic
challenge for a second time, there is a
massive increase in serum antibody
titre and it is very rapid
o Happening almost immediately as
there is now a memory cell population,
they are now kicking in; they are
mainly IgG
- Bottom image:
o Shows affinity maturation occurring in the secondary response

B cell maturation and receptors

- Only mature cells can go on to produce these antibody-secreting plasma cells

- Mature cells have IgD and IgM on their surface (membrane bound forms)
- When they are activated, then get isotype switching and can then start
producing other types of antibodies
Characteristics of antibody responses

- What are the effector mechanisms of the antibody? What do they actually do?
o Can bind directly to viruses and bacteria, and neutralise them
o Neutralisation of toxins: binding to a toxic production produced by a
bacteria for example, can then remove it and stop it from having that
toxic effect
o Agglutination: so can get removal by phagocytes
o Precipitation of soluble antigens: so can get removal by phagocytes
Phagocytes: have an FC receptor, they can see/recognise that tail
portion of the antibody and realise this needs to be internalised
(form of opsonisation)
o Interaction with the complement system: getting opsonisation or
triggering directly of cell lysis by the MAC
- There are multiple ways antibodies can have an effect

Monoclonal antibodies
 - Before monoclonal
antibodies, could
generate a type of
immune response
that was non-specific
(very difficult to see
differences between
cell populations to
see these
differences, need to
be able to see what
their surface
receptor population
looks like
- To see this, need a
monoclonal antibody,
something that recognises a particular receptor on a cell type
- Produced by the fusion of splenocytes from a mouse that has been immunised
against whatever you want to look for
- Wil have a B cell response, these B cells will be present in the spleen of the
mouse some time later with a kind of cancer cell (myeloma cell line)
- If these are fused together, can create a very long lived cell lined called a
hybridoma
- Through a process of clonal selection of the individual cells and growing those
up, can get a clone that produces just the type of antibody that you want to
produce
- These clones can be grow in in the lab, can be frozen and then brought back up
- Allows production of many of a particular antibody to anything you want it to
recognise
- Some are even being used as biological therapeutics

Serological testing

- Serological testing is the other main use for antibodies

- Serology: looking for in vivo antibody responses in an animal
o Immunofluorescent testing
In this example, have serum from an animal and then antiserum
that is binding to a flurochrome in order to visualise the pathogen

ELISA (enzyme-linked immunosorbent assay)

 - For this process, the
bottom of a 96 well
plate is coated with a
particular antigen
- The antigen coming
from the particular
pathogen you want to
see if the animal has
been exposed to
- Then add serum from
the animal which
may or may not
contain antibodies
that recognise that
antigen
- If it contains antibodies, they bind to the antigen
- Can look for those antibodies using a secondary antibody that only recognises
the FC portion of that particular type
- This has a way to amplify and then produce a colorimetric response (as shown in
image below) from that; it is a way to see if there are antibodies circulating in
the system of an animal that are particularly responsive to the antigen of
interest
- Used as a diagnostic because it is an indirect way to say an animal is infected
with a certain pathogen or has been exposed to it in the past

Summary

- Humoral immune responses are mediated by B cells and are antigen-specific

- The B cell receptor consists of membrane-bound antibody (Immunoglobulin)
- Antibody classes include IgM, IgG, IgA, IgE and IgE
- Different antibody classes have different functions and tissue distributions
- B cell activation and clonal expansion leads to plasma cell development and
antibody production, with different kinetics and characteristics of primary and
secondary responses
- B cell activation requires T cell help