Post on 06-Apr-2018
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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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Key cytokines which activate the
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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Key cytokines which activate the
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.
Produced by T-cells.
Stimulation of mastcell replication.
Inhibits cellular
immune reactions.
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Key cytokines which activate the
inflammatory response. IL5 45-60Kd mw.
Produced by T-cells.
Increases B-cellproliferation.
Promotes eosinophil
maturation and
inhibits macrophage
activation.
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Key cytokines which activate the
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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Key cytokines which activate the
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.