Innate Immune System Receptors - Roger Palfree · 2012. 1. 9. · MIMM-314 -- Lecture 3 -- 2011 08/01/2012 2 January 11, 2011 MIMM-314 R.G.E.Palfree 3 Soluble receptors for common

MIMM-314 -- Lecture 3 -- 2011 08/01/2012

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January 11, 2011 MIMM-314 R.G.E.Palfree 1

IMMUNOLOGY

Year 2011 version

Dr. Roger Palfree’s Lecture 3

� Receptors

� Macrophage cytokines

� Lymphoid Tissues

� Trafficking

� From infection to Antigen Presentation

(somewhat integrated and not necessarily in that order)

Made available via www.rogerpalfree.com


Innate Immune System Receptors

Cell surface proteins, soluble proteins in body fluids, and

also cytoplasmic proteins which sense viral infection.

Commonly have several binding sites with different ligand

specificities.

Frequently polymeric, with several binding sites for the

same ligand.

Recognize (bind with functional avidity to) microbes, via

multivalent binding to so-called “pathogen-associated

molecular patterns” (PAMPs).

MIMM-314 -- Lecture 3 -- 2011 08/01/2012

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Soluble receptors for common types of foreign material

C1q(a complement

protein)

MBL(mannan-

binding lectin)

SP-A, SP-D

Ficolins

CRP(C-reactive

protein)

Noted for binding IgM, IgG in immune complexes. C1q

may also bind directly to some microbial surfaces in the

absence of antibody. (Like MBL but not a lectin).

Binds patterns of certain carbohydrates (e.g. mannose,

fucose, GlcNAc). Associates with MASPs (serine

proteases which can activate the complement cascade)

Pulmonary surfactant proteins (collectins like MBL)

opsonize microbes for alveolar macrophage.

Like collectins, but not lectins. Bind patterns of acetyl

groups (may be on carbohydrate or other molecules).

Associate with MASPs.

Pentraxin which binds PAMP via phosphocholine. Also

triggers complement by binding C1q collagen domain.


Phagocyte Membrane Receptors

Note that macrophage, in their janitorial role, have receptors for

many molecules found normally in the body.

They particularly recognize molecular surfaces associated

normally intracellular molecules (phosphatidyl-serine), or

molecules altered by abnormal conformation, degradation, or

chemical modification (e.g. oxidation).

Opsonizing protiens like Antibodies, Complement and Collectins

are similarly recognized by phagocyte receptors. Some more

specialized than others.

Phagocytes also have receptors for foreign material.

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Membrane receptors for common types of foreign material

Mannose Receptor Family

MR (CD206)

DEC205 (CD205)

Scavenger receptors

Toll-like receptors (TLR)

f-MLP receptor (7tm-GPCR)

Have multiple C-type lectin-like Carbohydrate

Binding Domains (also called CTLDs).

DEC205 is a Dendritic Cell C-type multi-lectin

receptor

Diverse ligands include negatively-charged

polymers. Includes lipoteichoic acid found in

Gram positive bacterial wall.

Whereas the above are important for uptake

of foreign material, TLR family members are

important for signalling the presence of

foreign material. More on these later.

Signal: formyl-methionine at pptd N-terminal


A pretty picture from Taylor et al., 2005, Trends in Immunology 26: 104.

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Toll-Like Receptors

Anecdote (1980):

Biologist, Christiane Nusslein-Volhard was so delighted

with a Drosophila mutant which helped in explaining

embryogenesis in the fruit fly, that she exclaimed “Toll !”,

which is German slang for “fantastic !”.

The Toll-1 Receptor and 8 others serve critical functions in

Drosophila development.

Nusslein-Volhard received a Nobel prize in 1995 for this

work.


Toll-Like Receptors and Defense

Fungal infection causes expression of antimicrobial peptides

(AMPs) in Drosophila.

These AMP genes have binding sites for a transcription

activator called “dorsal” (promotes transcription of specific

genes).

Activation of dorsal is caused by signals through the Toll

receptor.

Mutations in the Toll signalling pathway reduced the insect’s

survival after fungal infection.

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Toll-Like Receptors in Mouse and Human

Several Toll-like receptor (TLR) genes were found in mouse.

Mice which have mutant TLR-4 genes:

are hyporesponsive (do not respond well) to LPS and are

unable to survive infection with gram-negative bacteria.

TLR-2 knockout mice:

have an impaired response to gram-positive bacterial cell

wall and to peptidoglycan from S. aureus.


Receptor

TLR-1

TLR-2

TLR-3

TLR-4

TLR-5

TLR-6

TLR-9

Known Agonist

Lipopeptide

Several, including lipoteichoic acid,

peptidoglycan, zymosan, lipoprotein, and

HSP70 (released by damaged cells)

dsRNA

LPS, Hyaluronic acid fragments, HSP60,70

Flagellin

zymosan

unmethylated CpG DNA

7th edition Immunobiology updates this with discussion of hetero and homodimers

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Some TLRs are on the cell surface.

TLR-1, TLR-2, TLR-4, TLR-5, TLR-6

Some are in the endosomal compartment and

make contact with agonist in the phagolysozome

in phagocytic cells

TLR-3, TLR-7, TLR-8, TLR-9

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TLR Signalling through TIR domains and adapters

I will not ask

questions on the

details of the

signalling pathways.

Main points are

covered in text slides.


Signalling is through the cytoplasmic TIR domains

(Toll-IL-1 Receptor Domains) which affect signalling

cascades via TIR Adapters (e.g. MyD88).

TLR-4 activates 2 pathways:

• A pathway through MyD88 which activates NFκκκκB,

which is like the Drosophila dorsal transcription

factor, and results in cytokine (esp. TNFαααα)

production.

• A so-called MyD88-independent pathway resulting

in induction of nitric oxide synthase (iNOS) and

type 1 Interferons (IFNαααα/ββββ).

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Lipopolysaccharide (LPS)

is detected via TLR-4 in a 3

step process:

1. LPS is bound by LPS-

Binding Protein (LBP) in

the fluid.

2. The LPS-LBP complex

passes LPS to CD14 on

the phagocyte.

3. The LPS-CD14

complex interacts with

TLR-4.


TLR and Antimicrobial Peptides (AMPs)

As Toll activation triggers AMP production in the fruit fly,

TLR activation is often associated with AMP production.

Bronchial epithelial cells responding to dsRNA via TLR3

produce cytokines and beta-Defensins 2 and 3.

Tracheobronchial cells responding to LPS via TLR-2 and

TLR-4 produce beta-Defensin 2.

It works the other way too:

mouse beta-defensin 2 has been found to activate

dendritic cells via TLR-4

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There are different varieties of Dendritic Cell

Their expression of TLRs differs, and they respond

differently to microbial materials.

From: Iwasaki & Medzhitov, Nature Immunology 5: 987, 2004

No question on exam for this slide – just knowledge that there is not just a single type of DC


Macrophage have many

receptors which bind common

foreign materials.

Binding through these

receptors can trigger

phagocytosis and release of

cytokines and lipid

inflammatory mediators

(including prostaglandins and

leukotrienes).

They immediately begin to clear

foreign material, and also signal

changes in neighbouring cell

behaviour, and attract other

leukocytes to the infection,

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Bactericidal agents produced by phagocytes


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To infect the body, pathogens must first cross a barrier.

Barriers separating the rest of the body from the external

environment have many defenses.

Immediately below the surface there are lymphoid

tissues which are adapted to provide protection and

efficient responses to infection.

We look at those tissues, then follow an infection through

recruitment of leukocytes to presentation of antigen.


Defense Mechanisms: Barriers

Our interface with the outside world:

Skin:

Flexible barrier.

Protects from water loss

Protects from friction and impact wounds

Produces vitamin D

Body temperature regulation - sweat

Mucosal surfaces

Transfer across the epithelial barrier

Need to be kept moist - secretions

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The Skin

Skin:

accounts for about 16% of adult total body weight

From: http://www.mederma.com/ce3.html

Besides providing a

continually renewing barrier

to microorganisms, skin

also actively challenges

microbes through sweat

components.

Antimicrobial peptides are

produced by skin cells and

secreted upon damage

along with lipid which is

transformed into a

hydrophobic barrier called

the stratum corneum.


Skin: Defence Mechanisms

Keratinocytes in the epidermis not only provide

structural and mechanical integrity, they can also secrete

cytokines which affect immune function and wound healing.

Langerhans cells are antigen-presenting cells (APC)

Mast cells within the dermis are close to blood vessels.

They can be stimulated to release a multitude of powerful

regulatory molecules (e.g histamine). Some stimulate vascular

endothelial cells to express adhesion molecules which

encourage leukocytes to pass between the cells from blood to

tissue.

Lymphocytes which traffic through the skin, and

Langerhans cells, are components of

the SkinSkin--Associated Lymphoid Tissue Associated Lymphoid Tissue -- SALTSALT

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Mucosal Surfaces

Mucosal surfaces - a vast surface area

Fluid flow carries microbes away from vulnerable surfaces

Non-specific surfactants and specific innate (SP-A, SP-D)

and adaptive (IgA) receptors in the mucus reduce microbe adhesion

to the epithelial cells, opsonize for phagocytosis (e.g. by alveolar

macrophage in lung), and neutralize toxins (esp. IgA).

Enzymes and antimicrobial peptides attack microbes and

toxins. Many microbes will not survive the low pH in the stomach

The tissue just beneath the epithelial layer contains

structures which promote efficient antigen presentation to

lymphocytes. Broadly called

the MucosaMucosa--Associated Lymphoid Tissue Associated Lymphoid Tissue -- MALTMALT


MALT

Cellular mass exceeds total lymphoid cells in

bone marrow, thymus, spleen, and lymph nodes

MALT includes:

Nasal-associated lymphoid tissue (NALT).

tonsils, adenoids.

Gut-associated lymphoid tissue (GALT).

Peyer’s patches.

Bronchus-associated lymphoid tissue (BALT)

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Cells such as keratinocytes, mast cells and macrophage

Soluble factors like the anaphylotoxins from complement

Signal neighbouring cells to change behaviour

Some act long-range and involve the endocrine system

When microbes penetrate the barrier

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Activated Cells and Complement produce Inflammatory Mediators

Inflammatory mediators = regulatory molecules which cause:

� Vasodilation

� Increased local blood flow (redness, heat)

� Vascular Permeability

� Fluid and proteins enter tissue (edema)

� Expression of adhesion molecules; chemotaxis

� Recruits neutrophils, monocytes, other leukocytes

� Clot formation

� Helps prevent spread of infection through blood

vessels


� Lipid mediators

� Prostaglandins, leukotrienes, Platelet Activating Factor

� Tumor Necrosis Factor alpha (TNF- αααα)

� Histamine

� Complement derived polypeptides – esp. C5a

� Bradykinin

What are the Inflammatory Mediators?

Please read and learn about them.What cells or cascades produce them ?

What are their targets and effects ?

You will encounter them again later

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Infection

triggers

inflammatory

mediators

Blood vessel

endothelial cells

and leukoctes

express CAMs

1

2

3

Blood vessels

become

permeable

4

Extravasation

Blood Clotting


Systemic infection

causing systemic

TNF-alpha

Septic Shock

May be fatal

Local TNF-alpha

release at site of

infection provides

important functions

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CXCL8

(IL-8)


MCP-1


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Chemokine Receptors are G-protein coupled receptors.

Thus, they are in the same family as the receptors for the

anaphylatoxins C5a, C3a.

Neutrophils are attracted and activated by a small peptide

of bacterial origin called f-MLP (formyl-Met-Leu-Phe). This

also acts through a G-protein coupled receptor.

(TNFα plus f-MLP activates the oxidative burst in

neutrophils)

Note: You may be aware that the single letter nomenclature for

amino acids uses F for Phenylalanine and P for Proline, so you

might have thought f-MLP stood for f-met-leu-pro

Adhesion Molecules


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L-selectin on naive lymphocytes is an important cell

adhesion molecule in their trafficking through high

endothelial venules into lymph nodes (see later).

Two other selectins are involved in leukocyte

extravasation into infected tissue. They are induced on

blood vessel endothelial cells by various combinations

of inflammatory mediators and exposure to microbial

components

P-selectin is preformed and stored in vesicles (Palade

bodies), and is very quickly expressed.

E-selectin gene expression is induced, so E-selectin

must be synthesized first and is expressed later.

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LTB4, C5a, Histamine

Endothelial cell expression

of P-selectin within minutes

(from Weibel-Palade bodies –

granules)

LPS, TNF-alpha

Endothelial cell expression

of E-selectin and ICAM-1

within a few hours

IL8 (and other chemokines)

act on leukocytes to increase

integrin affinity, promote

diapedesis and chemotaxis.


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See movie I_2_6_Leukocyte_Rolling-H264.mov on CD (7th edition)


This Movie is on the 7th Ed Immunobiology CDI_2_6_Leukocyte_Rolling-H264.mov


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Local dendritic cells are activated


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Light micrograph of

section through lymph

node.

Follicles prominent

containing germinal

centers where

activated B cells

proliferate and

differentiate.


Lymph Node

Adapted From Bloom & Fawcett - Histology

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Sinus of a Lymph Node - Scanning Electron Micrograph

From Bloom & Fawcett - Histology. Original: Fujita T. et al., Zellforsch, 133:147, 1972


Lymphocyte Migration

Lymphocytes migrate from blood into lymph node through the

High Endothelial Venules( Postcapillary Venules )

1. Adhere 2. Squeeze between endothelial cells

? Why do they adhere to these capillary

endothelial cells?

What molecules are involved

on the lymphocyte on the endothelial cell

From Bloom & Fawcett - Histology

MigratingLymphocytes

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Naïve T cells have surface adhesion molecules (homing

receptors) which bind structures (vascular addressins) on

vascular endothelial cells in the lymphoid tissues

L Selectin - on the T cell

sulfated sialyl Lewisx (SLeX) molecules:

CD34 and GlyCAM-1 on LN HEV

MAdCAM-1 on endothelium in the mucosa.

bind to

This is just the first step …


Secondary Lymphoid Tissue Chemokine (SLC),

produced by • the high vascular endothelium• Lymphoid tissue stromal cells• dendritic cells

increases the affinity of T cell integrin LFA-1 (lymphocyte function-associated antigen-1)

for its ligands on the endothelial cells,

and promotes migration into the lymphoid organ.

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Lymphocyte entering LN through HEV

A similar process happens in leukocyte extravasation into

infected tissues during inflammatory responses

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Please read about Spleen and Peyer’s Patches

in the text book.

I will only cover Lymph Node in the lecture, but

you are expected to see the similarities and

differences between these lymphoid tissues.


Spleen

Note similarities

to lymph node,

but catches

antigen from

blood, and the

lymphocytes

move from blood

back to blood.

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Transverse section

through white pulp

showing the central

arteriole, PALS, B

cell corona and

germinal center.


GALT – gut associated lymphoid tissue

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Multi-fenestrated cells facilitate antigen uptake


Antigens are taken up by M cellsand carried across the epithelial

barrier by a process called

transcytosis

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Lymphocyte Recirculation

Lymphocytes circulate through lymphoid tissues.

Lymphocytes enter lymph nodes from blood through the

walls of high endothelial venules. If they do not

encounter antigen there, they leave via the efferent

lymphatic vessel, and eventually reenter the blood via

the thoracic duct.

Naïve lymphocytes (not of the differentiated form arising

from an activation event) gain necessary survival

signals while passing through the lymphoid tissues.

After activation, lymphocytes also recirculate, but not

randomly: E.g. Lymphocytes activated within a mucosal

tissue migrate back into MALT.

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Within the lymphoid tissues

T cells and B cells may become activated


Effector T cells then

perform several important

functions illustrated here.

Each interacts specifically

with cells presenting the

same peptide-MHC

combination which

activated the naïve T cell.

Most B cells require specific

help from CD4 T cells to

become activated.

They present a set of

peptides from any protein

within the material which

bound to their surface Ig.

62

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Th1 effector helps

macrophage clear

mycobacterium

Whether or not

there is a strong

Th1 response to

Mycobacterium

leprae has striking

consequences

with respect to the

form of the disease

and survival of the

infected individual.

When the macrophage needs help

Documents

Innate Immune System Receptors - Roger Palfree · 2012. 1. 9. · MIMM-314 -- Lecture 3 -- 2011 08/01/2012 2 January 11, 2011 MIMM-314 R.G.E.Palfree 3 Soluble receptors for common