‘The Holy Grail’

-mediators of inflammation.
Lecture 3
Rod Flower, WHRI, London.
 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.
The ‘chemical theory’.
• Chemical substances,
called mediators, released
from injured or activated
cells co-ordinate the
development of the
inflammatory response.
 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 the response.
• .. have the same action in all species where the
phenomenon occurs.
• .. be destroyed locally or systemically to avoid
undue accumulation.
• .. be blocked (directly or indirectly) by inhibitors of
inflammation.
- Rocha E Silva, 1978.
 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.
 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).
 Two types of ‘immunity’.
• Innate. Includes… • Aquired. Includes…
- phagocytosis. - secondary antibody
- complement activation. mediated response.
- natural killer cells. -secondary cell
mediated response.
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.
 Immunoglobulins.
• IgG • Major bloodborne
immunoglobulin.
• 75% total Igs.
• 150 kda mw.
• Four subtypes.
• Main antibody of the
secondary immune
response.
 Immunoglobulins
• IgA • Predominant form in
mucous 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 & A2.
 Immunoglobulins.
• IgM. • A pentameric
molecule.
• Confined to the blood.
• Important in the
primary immune
response.
 Immunoglobulins
• IgD. • A minority (1%)
immunoglobulin
present on B-cells.
• Short half life.
 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.
Auto-immunity.
• A case of ‘mistaken
identity’.
• Responsible for a
range of disorders,
both trivial and
serious.
T-cell mediated immunity.
• The primary immune
response.
• Immunological
‘memory’.
• Some effector
functions.
T-cell mediated immunity.
• T-cell receptor is a
heterodimer (
chains).
• Recognises MHC
complexes.
• Detects antigenic
fragments presented by
APC thus priming the ab
response
• Unique to each
lymphocyte.
Phagocytes.
• Uptake of foreign
organisms.
• Destruction of micro-
organisms etc.
• Many microbiocidal
weapons e.g. lytic
enzymes, active
oxygen etc.
Natural killer (NK) cells.
• A type of lymphocyte.
• Cytotoxic potential.
• Attacks invading, infected
or transformed cells.
• Differs from T-cells in the
way in which they
‘recognise’ their targets.
• Secrete toxic proteins.
• Sometimes involved in
acute rejection.
 Complement.
Ab-ag, Gm neg bacteria, Yeasts, parasites,
subcellular particles ab-ag. • A complex series of about
20 proteolytic enzymes in
Classical Alternate the blood.
(C1,4,2 & 2) (C3)
• ‘Classical’ and ‘alternate’
pathways act in a cascade
fashion.
• Accelerated in the
presence of IgGs
C3
• Lytic to many micro-
organisms.
• ‘Opsonise’ others.
C5
 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.
 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.
 Histamine.
• Formed from histidine.
• Stored in high
CH2CH2NH2 concentrations in mast cells
and basophils together with
heparin and ATP.
• Three main receptor
HN N
subtypes (H1 etc).
• Inmportant in allergies, itch,
inflammatory response.
Causes ‘triple response’.
 Histamine.
• Synthesised as a curiosity by Windaus and Vogt,
1907.
• Extracted from putrefying mixtures by Ackerman
1910.
• Assumed to be responsible for anaphylaxis by
Dale and Laidlaw (1911, 1960) as synthetic
material had the same effects.
• Eppinger (1913) demonstrated that histamine
produced a reaction in human skin similar to that
seen with insect bites.
 Histamine.
• Lewis (1927) proposed that histamine was
released by a variety of injurious stimuli.
• Best (1927) unequivocally demonstrated the
presence off histamine in the mammalian
body.
• The development of anti-histamine in the
1940’s led to the realisation that histamine
was not the only inflammatory mediator.
 5HT; serotonin.
• Found in platelets, neurones and in CNS. Often
stored with other transmitters.
• Inactivated by MAO.

HO CH2CH2NH2

N
H
 Serotonin (5HT).
• Very potent at increasing vascular
permeability in rodents but not guinea pigs
or rabbits (various groups, 1950’s)
• A histamine releaser in man?
• Many inflammatory effects but species
specific.
• Multiple receptors.
Neuropeptides.
• Tachykinins
- substance P
- neurokinin A
- neurokinin B
- CGRP
• Kinins:
- bradykinin
- kallidin
 Tachykinins.
• Substance P. • Mainly located in sensory
neurones.
• Neurokins A & B. • Released on nerve
stimulation.
• Act on 7TM ‘NK’
receptors (3 subtypes; NK1
etc).
• Cause vasodilatation,
vascular permeability,
smooth muscle contraction,
mucus secretion, pain.
 Tachykinins.
• CGRP. • A product of the
calcitonin gene
generated through
differential splicing.
• Found in sensory
neurones.
• Induces neurogenic
inflammation.
 Kinins.
• Bradykinin (9 aa) • Formed from kininogens (2 forms)
by kallikreins (also 2 forms).
• Kallidin (10 aa). • Inactivated by kininases (2 forms).
• Two receptors B1 (inducible) and
B2 (constitutive).
• Produce; vasodilation, smooth
muscle contraction, pain and
inflammation.
• Anti-proteases and receptor
antagonists are occasionally
useful.
 The kinin system.
• Kallikrein strongly increases vascular
permeability in rabbits. Rocha E Silva 1940.
• A biologically active agent, named bradykinin was
generated by the action of trypsin on plasma.
Rocha E Silva 1949.
• BK has strong vascular permeability effects
(several groups; 1950’s).
• BK causes pain. Armstrong et al 1954.
 Eicosanoids.
• Arachidonic acid from
Arachidonic acid cellular phospholipids.
• At least 2 different
pathways:
PG G2 LTA4 - cycloxygenase forms
prostaglandins and
TxA2 PGs LT B4 LTs thromboxanes.
E,I,F,D C,D,E - lipoxygenase forms
leukotrienes.
 The prostaglandin (PG) system.
• PGs discovered in seminal vesicles and in
human plasma (1930s).
• Synthesis from essential fatty acids
demonstrated (1960s).
• Aspirin like drugs prevent PG synthesis and
this explains mechanism of action (1970s).
• Multiple forms of cyclo-oxygenase
discovered (1990s).
 Synthesis of PAF.
C12-C18 fatty acid. • PAF formed from
phoshatidyl choline by
Acetyl group and acetylase.
• Key role of
Phoshatidylcholine phospholipase A2

(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine.)
 PAF (platelet activating factor).
• Modified phospholipid.
• 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 signs
and symptoms of inflammation.
• PAF receptor antagonists useful treatment in
experimental models.
 Nitric oxide (NO; EDRF).
• Formed in many tissues
H2N-CH.COOH from arginine.
• Three enzymes (NOS)
(CH2)3 described; iNOS, ncNOS &
ecNOS.
NH • Resonsible for NANC
transmission.
C • Potent vasodilator and
microbiocidal.
HN NH2 • Physiological effects
dependent of guanylate
cyclase activation.
 iNOS.
• Induced in cells by cytokines, TNF, IL1 or LPS.
• iNOS does not require Ca2+ for activation, only a supply
of arginine.
• GCs, IL10 and some other factors can inhibit iNOS or its
induction.
• With active oxygen, NO can form peroxynitrite which is
a potent cytotoxic agent.
• Can be blocked in (e.g.septic shock) by arginine
analogues such as L-NMMA.
• NO is scavenged by haemoglobin and reacts with thiols.
 Cytokines.
• All are proteins.
• Mainly synthesised by immune cells.
• Regulate differentiation and activation of
immune cells.
• Partly responsible for coordination of the
inflammatory response.
• Act through high affinity receptors on target
cells.
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.
Key cytokines which activate the
inflammatory response.
• IL6 • 26Kd mw.
• Produced by T-cells
but also by many other
cells too.
• Activates B & T-cells
and other cell types.
Key cytokines which activate the
inflammatory response.
• IL2 • 15Kd mw.
• Produced by T-cells.
• Activates T-cells,
monocytes and NK
cells.
Key cytokines which regulate the
inflammatory response.
• IL10. • 17-21Kd mw.
• Produced by T-cells.
• Stimulation of mast
cell replication.
• Inhibits cellular
immune reactions.
Key cytokines which activate the
inflammatory response.
• IL5 • 45-60Kd mw.
• Produced by T-cells.
• Increases B-cell
proliferation.
• Promotes eosinophil
maturation and
inhibits macrophage
activation.
Key cytokines which activate the
inflammatory response.
• TNF • Two forms found, TNF
and TNF.
• 17Kd mw.
• Produced by many cells
including monocytes
(TNF ) .
• Produced by T-cells
(TNF).
• Widespread activation of
cells; apoptosis, shock,
cachexia etc.
Key cytokines which activate the
inflammatory response.
• Interferons (IFNs). • 3 forms found , & .
• Many different subtypes.
• Generally 19-26 Kd mw.
• Produced by monocytes
(), fibroblasts () and T-
cells ().
• Antiviral, cell activating
and tumour suppressant
effects.
 Strategies for inhibiting cytokines.
• Reduce cytokine producing cells (e.g. with
cytostatics).
• 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).
 Chemokines.
• At least 3 families of small proteins mw
usually 7-15Kd.
• Relative position of Cys residue determines
nomenclature e.g. CXC, CC or C.
• Act through 7TM receptors which also
function as co-receptors for HIV entry into
immune cells.
 Chemokines.
• CXC chemokines. • IL8.
• Platelet factor IV.
• Granulocyte
chemotactic protein 2.
• Platelet basic protein
and related species.
• Utilise CXCR 1-5.
• Main target PMN.
 Chemokines
• C-C chemokines. • MCP 1,2,3,&4.
• RANTES
• MIP 1 & .
• Eotaxin.
• Utilise CCR 1-5
receptors.
• Main targets eosinophils
and monocytes.
 Chemokines
• C chemokines. • Lymphotaxin.
 The plurality of mediators.
• “..it would be very unfortunate if any of the
above mentioned mediators might constitute
the final answer to the problem, because
that would mean to shut our laboratories.,
or do something else!”.

Rocha E Silva, 1973.

 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 to
produce the full response.
 The ‘Holy Grail’ hypothesis.
• “One mediator, or family of mediators, is
responsible for the majority of inflammatory
signs and symptoms. By inhibiting the
formation or antagonising the action of this
mediator(s), a resolution of most types of
inflammatory disease would be possible”.

- Flower, 1986.
 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’.
 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.