RJ Flower the Mediators

8/3/2019 RJ Flower the Mediators

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The Holy Grail

-mediators of inflammation.

Lecture 3

Rod Flower, WHRI, London.


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The components of

inflammation. Cells..

- Fixed cells such as vascular cells.

- Migratory cells such as PMNs. Mediators..

- many chemicals released into the body.

Immune system..

-Innate.

-Acquired.


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The chemical theory.

Chemical substances,

called mediators, released

from injured or activatedcells co-ordinate the

development of the

inflammatory response.


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A chemical mediator' should. .. be found in tissues in concentrations that can explain the

observed symptoms or effects.

.. be released by the endogenous trigger which produces theresponse.

.. have the same action in all species where the phenomenonoccurs.

.. be destroyed locally or systemically to avoid undueaccumulation.

.. be blocked (directly or indirectly) by inhibitors of

inflammation.- Rocha E Silva, 1978.


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The mediators of inflammation.

Plasma proteins such as complement and antibodies.

Other proteins such as sPLA2 and acute phase

reactants. Cytokines and chemokines.

Lipids such as prostaglandins and PAF.

Amines such as histamine.

Gasses such as NO and O2-.

Kinins such as bradykinin.

Neuropeptides such as substance P.


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Mediators which suppress

inflammation. ACTH, GCs and products of the HPA axis.

Some cytokines such as IL-10.

Some induced proteins such as anti-

proteases and lipocortin 1(annexin 1).


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Two types of immunity.

Innate. Includes

- phagocytosis.

- complement activation.- natural killer cells.

Aquired. Includes

- secondary antibody

mediated response.-secondary cell

mediated response.


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Antibody mediated effects.

IgG, IgA, IgM, IgD, IgE

subtypes.

Fab region recognises

antigen.

Fc region important for

host defence functions

Responsible for antibody

mediated immunity and

some innate immunity.


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Immunoglobulins.

IgG Major bloodborne

immunoglobulin.

75% total Igs. 150 kda mw.

Four subtypes.

Main antibody of thesecondary immune

response.


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Immunoglobulins

IgA Predominant form inmucous secretions.

Occurs as a dimer

(especially in secreted

form) and also in the

plasma of some animals.

Has a secretory

component associated

with it.

Two subclasses A1& A

2.


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Immunoglobulins.

IgM. A pentameric

molecule.

Confined to the blood. Important in the

primary immune

response.


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Immunoglobulins

IgD. A minority (1%)

immunoglobulin

present on B-cells. Short half life.


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Immunoglobulins.

IgE. Pentameric heavy chain.

Low concentrations in

serum.

High concentrations on

surface of mast cells

which posses a IgE Fc

receptor.

When bound to antigen,

histamine is released

from mast cells.


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Auto-immunity.

A case of mistaken

identity.

Responsible for arange of disorders,

both trivial and

serious.


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T-cell mediated immunity.

The primary immune

response.

Immunologicalmemory.

Some effector

functions.


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T-cell mediated immunity.

T-cell receptor is aheterodimer ( , , , chains).

Recognises MHCcomplexes.

Detects antigenicfragments presented byAPC thus priming the abresponse

Unique to eachlymphocyte.


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Phagocytes.

Uptake of foreign

organisms.

Destruction of micro-organisms etc.

Many microbiocidal

weapons e.g. lytic

enzymes, active

oxygen etc.


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Natural killer (NK) cells.

A type of lymphocyte.

Cytotoxic potential.

Attacks invading, infectedor transformed cells.

Differs from T-cells in the

way in which they

recognise their targets.

Secrete toxic proteins.

Sometimes involved in

acute rejection.


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Classical

(C1,4,2 & 2)

C3

C5

Alternate

(C3)

Ab-ag, Gm neg bacteria,

subcellular particles

Yeasts, parasites,

ab-ag.

Complement.

A complex series of about20 proteolytic enzymes inthe blood.

Classical and alternatepathways act in a cascadefashion.

Accelerated in the presenceof IgGs

Lytic to many micro-organisms.

Opsonise others.


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Some actions of complement

fragments.C5a chemotaxis, phagocyte

degranulation, stimulation of O2-.

C5a, C3a mast cell and platelet

degranulation.

C5a, C5b-9 enhancement of cytokine release,

induction of eicosanoid synthesis.

C3b potentiation of Ab response,

opsonisation of cells and lysis.

C5b-9 cell lysis.


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Non-immune mediators.

Soluble chemicals released by injured,

activated or dying cells.

Regulate, activate and terminate the

inflammatory response.

Some are fairly insult specific, others

more generally found in lesions.


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Histamine.

Formed from histidine.

Stored in high concentrations in

mast cells and basophils

together with heparin and ATP.

Three main receptor subtypes

(H1 etc).

Inmportant in allergies, itch,

inflammatory response. Causestriple response.

NHN

CH2CH2NH2


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Histamine.

Synthesised as a curiosity by Windaus and Vogt,1907.

Extracted from putrefying mixtures by Ackerman1910.

Assumed to be responsible for anaphylaxis by Daleand Laidlaw (1911, 1960) as synthetic material hadthe same effects.

Eppinger(1913) demonstrated that histamineproduced a reaction in human skin similar to thatseen with insect bites.


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Histamine.

Lewis (1927) proposed that histamine wasreleased by a variety of injurious stimuli.

Best (1927) unequivocally demonstrated thepresence off histamine in the mammalianbody.

The development of anti-histamine in the1940s led to the realisation that histaminewas not the only inflammatory mediator.


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5HT; serotonin.

Found in platelets, neurones and in CNS. Often

stored with other transmitters.

Inactivated by MAO.

N

H

HO CH2CH2NH2


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Serotonin (5HT).

Very potent at increasing vascular permeability

in rodents but not guinea pigs or rabbits

(various groups, 1950s) A histamine releaser in man?

Many inflammatory effects but species specific.

Multiple receptors.


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Neuropeptides.

Tachykinins

- substance P

- neurokinin A- neurokinin B

- CGRP

Kinins:

- bradykinin- kallidin


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Tachykinins.

Substance P.

Neurokins A & B.

Mainly located in sensoryneurones.

Released on nerve

stimulation. Act on 7TM NK receptors

(3 subtypes; NK1 etc).

Cause vasodilatation,vascular permeability,

smooth muscle contraction,mucus secretion, pain.


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Tachykinins.

CGRP. A product of the

calcitonin gene

generated throughdifferential splicing.

Found in sensory

neurones.

Induces neurogenic

inflammation.


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Kinins.

Bradykinin (9 aa)

Kallidin (10 aa).

Formed from kininogens (2 forms)by kallikreins (also 2 forms).

Inactivated by kininases (2 forms).

Two receptors B1 (inducible) and B2(constitutive).

Produce; vasodilation, smoothmuscle contraction, pain andinflammation.

Anti-proteases and receptorantagonists are occasionally useful.


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The kinin system.

Kallikrein strongly increases vascular permeability in

rabbits. Rocha E Silva 1940.

A biologically active agent, named bradykinin wasgenerated by the action of trypsin on plasma. Rocha

E Silva 1949.

BK has strong vascular permeability effects (several

groups; 1950s). BK causes pain. Armstrong et al1954.


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Eicosanoids.

Arachidonic acid from

cellular phospholipids.

At least 2 differentpathways:

- cycloxygenase forms

prostaglandins and

thromboxanes.- lipoxygenase forms

leukotrienes.

PG G2 LTA4

TxA2 PGs LT B4 LTs

E,I,F,D C,D,E

Arachidonic acid


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The prostaglandin (PG) system.

PGs discovered in seminal vesicles and inhuman plasma (1930s).

Synthesis from essential fatty acidsdemonstrated (1960s).

Aspirin like drugs prevent PG synthesis andthis explains mechanism of action (1970s).

Multiple forms of cyclo-oxygenasediscovered (1990s).


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Synthesis of PAF.

PAF formed from

phoshatidyl choline by

and acetylase. Key role of

phospholipase A2

C12 -C18 fatty acid.

Acetyl group

Phoshatidylcholine

(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine.)


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PAF (platelet activating factor).

Modifiedphospholipid.

Synthesised by many cells including PMN,monocytes, mast cells and eosinophils.

Acts through specific G-protein linked receptors.

Sometimes acts intracellularly.

Causes increased vascular permeability, PMN

migration, brochoconstriction and many other signsand symptoms of inflammation.

PAF receptor antagonists useful treatment inexperimental models.


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Nitric oxide (NO; EDRF).

Formed in many tissuesfrom arginine.

Three enzymes (NOS)

described; iNOS, ncNOS &ecNOS.

Resonsible for NANCtransmission.

Potent vasodilatorand

microbiocidal. Physiological effects

dependent ofguanylatecyclase activation.

H2N-CH.COOH

(CH2)3

NH

C

HN NH2


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iNOS.

Induced in cells by cytokines, TNF , IL1 or LPS.

iNOS does not require Ca2+ for activation, only a supply ofarginine.

GCs, IL10 and some other factors can inhibit iNOS or its induction.

With active oxygen, NO can formperoxynitrite which is a potentcytotoxic agent.

Can be blocked in (e.g.septic shock) by arginine analogues such asL-NMMA.

NO is scavenged by haemoglobin and reacts with thiols.


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Cytokines.

All are proteins.

Mainly synthesised by immune cells.

Regulate differentiation and activation ofimmune cells.

Partly responsible for coordination of the

inflammatory response. Act through high affinity receptors on target

cells.


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Key cytokines which activate the

inflammatory response. IL1 Two forms found IL1 &

IL1 .

17Kd mw.

Soluble IL1 receptor

regulates activity.

Produced by monocytes

and many other cells.

Activate lymphocytes and

many inflammatory cells.


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inflammatory response. IL6 26Kd mw.

Produced by T-cells

but also by many othercells too.

Activates B & T-cells

and other cell types.


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inflammatory response. IL2 15Kd mw.

Produced by T-cells.

Activates T-cells,monocytes and NK

cells.


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Key cytokines which regulate the

inflammatory response. IL10. 17-21Kd mw.


Stimulation of mastcell replication.

Inhibits cellular

immune reactions.


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inflammatory response. IL5 45-60Kd mw.


Increases B-cellproliferation.

Promotes eosinophil

maturation and

inhibits macrophage

activation.


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inflammatory response. TNF Two forms found, TNF

and TNF .

17Kd mw.

Produced by many cellsincluding monocytes(TNF ) .

Produced by T-cells(TNF ).

Widespread activation ofcells; apoptosis, shock,cachexia etc.


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inflammatory response. Interferons (IFNs). 3 forms found , & .

Many different subtypes.

Generally 19-26 Kd mw.

Produced by monocytes

( ), fibroblasts ( ) andT-cells ( ).

Antiviral, cell activating

and tumour suppressant

effects.


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Strategies for inhibiting cytokines.

Reduce cytokine producing cells (e.g. withcytostatics).

Inhibitory cytokines (e.g. IL 10).

Inhibitors of signal transduction (e.g.cyclosporin).

Regulation of gene expression (e.g. glucocorticoids)

Inhibitors of release (e.g. ICE inhibitors)

Reduction in circulating cytokines(e.g. monoclonals,soluble receptors)

Receptor blockade (e.g. antagonists or monoclonals).


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Chemokines.

At least 3 families of small proteins mw

usually 7-15Kd.

Relative position ofCys residue determinesnomenclature e.g. CXC, CC or C.

Act through 7TM receptors which also

function as co-receptors for HIV entry intoimmune cells.


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Chemokines.

CXC chemokines. IL8.

Platelet factor IV.

Granulocytechemotactic protein 2.

Platelet basic protein

and related species.

Utilise CXCR 1-5.

Main target PMN.


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Chemokines

C-C chemokines. MCP 1,2,3,&4.

RANTES

MIP 1 & . Eotaxin.

Utilise CCR 1-5

receptors. Main targets eosinophils

and monocytes.


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Chemokines

C chemokines. Lymphotaxin.


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The plurality of mediators.

..it would be very unfortunate if any of the

above mentioned mediators might constitute

the final answer to the problem, becausethat would mean to shut our laboratories.,

or do something else!.

Rocha E Silva, 1973.


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How can we understand the

plurality of mediators? Different mediators are required to produce

different aspects of the inflammatory

response. Sequential release is necessary throughout

to co-ordinate the process.

Synergism between mediators is required toproduce the full response.


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The Holy Grail hypothesis.

One mediator, or family of mediators, is

responsible for the majority of inflammatory

signs and symptoms. By inhibiting the formationor antagonising the action of this mediator(s), a

resolution of most types of inflammatory disease

would be possible.

- Flower, 1986.


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Summary of lecture 3.

Inflammation is regulated by a great many

factors including immune and non-immune

chemical mediators. There is considerable redundancy.

There is a degree of insult specificity.


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Picture credits.

Life Art.

Austrian Rheumatology Teaching slides.

Mediators of Inflammation, GP Lewis .

Cellular and Molecular Immunology,Abbas et al.

N Goulding.

St Barts Hospital Medical Illustration service.

A du Vivier. Leo & Astra.

Atlas of Clinical Endocrinology,Besser et al.

RJ Flower the Mediators

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