Mediators of inflammation

1. Mediators of inflammation

2. 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.

3. The ‘chemical theory’.
• Chemical substances,
called mediators,
released from injured or
activated cells co-
ordinate the
development of the
inflammatory response.

4. 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.

5. 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.

6. 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).

7. Two types of ‘immunity’.
• Innate. Includes…
- phagocytosis.
- complement activation.
- natural killer cells.
• Aquired. Includes…
- secondary antibody
mediated response.
-secondary cell
mediated response.

8. 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.

9. Immunoglobulins.
• IgG • Major bloodborne
immunoglobulin.
• 75% total Igs.
• 150 kda mw.
• Four subtypes.
• Main antibody of the
secondary immune
response.

10. 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.

11. Immunoglobulins.
• IgM. • A pentameric molecule.
• Confined to the blood.
• Important in the
primary immune
response.

12. Immunoglobulins
• IgD. • A minority (1%)
immunoglobulin
present on B-cells.
• Short half life.

13. 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.

14. Auto-immunity.
• A case of ‘mistaken
identity’.
• Responsible for a
range of disorders,
both trivial and
serious.

15. T-cell mediated immunity.
• The primary immune
response.
• Immunological
‘memory’.
• Some effector
functions.

16. T-cell mediated immunity.
• T-cell receptor is a
heterodimer (a,b,g,d
chains).
• Recognises MHC
complexes.
• Detects antigenic
fragments presented by
APC thus priming the ab
response
• Unique to each
lymphocyte.

17. Phagocytes.
• Uptake of foreign
organisms.
• Destruction of micro-
organisms etc.
• Many microbiocidal
weapons e.g. lytic
enzymes, active
oxygen etc.

18. 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.

19. 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 about
20 proteolytic enzymes in
the blood.
• ‘Classical’ and ‘alternate’
pathways act in a cascade
fashion.
• Accelerated in the
presence of IgGs
• Lytic to many micro-
organisms.
• ‘Opsonise’ others.

20. 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.

21. 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.

22. 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.
Causes ‘triple response’.
N
HN
CH2CH2NH2

23. 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.

24. 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.

25. 5HT; serotonin.
• Found in platelets, neurones and in CNS. Often
stored with other transmitters.
• Inactivated by MAO.
N
H
HO CH2CH2NH2

26. 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.

27. Neuropeptides.
• Tachykinins
- substance P
- neurokinin A
- neurokinin B
- CGRP
• Kinins:
- bradykinin
- kallidin

28. Tachykinins.
• Substance P.
• Neurokins A & B.
• Mainly located in sensory
neurones.
• Released on nerve
stimulation.
• Act on 7TM ‘NK’ receptors
(3 subtypes; NK1 etc).
• Cause vasodilatation,
vascular permeability,
smooth muscle contraction,
mucus secretion, pain.

29. Tachykinins.
• CGRP. • A product of the
calcitonin gene
generated through
differential splicing.
• Found in sensory
neurones.
• Induces neurogenic
inflammation.

30. 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, smooth
muscle contraction, pain and
inflammation.
• Anti-proteases and receptor
antagonists are occasionally
useful.

31. 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.

32. Eicosanoids.
• Arachidonic acid from
cellular phospholipids.
• At least 2 different
pathways:
- 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

33. 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).

34. 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.)

35. 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.

36. Nitric oxide (NO; EDRF).
• Formed in many tissues
from arginine.
• Three enzymes (NOS)
described; iNOS, ncNOS &
ecNOS.
• Resonsible for NANC
transmission.
• Potent vasodilator and
microbiocidal.
• Physiological effects
dependent of guanylate
cyclase activation.
H2N-CH.COOH
(CH2)3
NH
C
HN NH2

37. iNOS.
• Induced in cells by cytokines, TNFa, IL1b 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.

38. 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.

39. Key cytokines which activate the
inflammatory response.
• IL1 • Two forms found IL1a & IL1
b.
• 17Kd mw.
• Soluble IL1 receptor
regulates activity.
• Produced by monocytes and
many other cells.
• Activate lymphocytes and
many inflammatory cells.

40. 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.

41. Key cytokines which activate the
inflammatory response.
• IL2 • 15Kd mw.
• Produced by T-cells.
• Activates T-cells,
monocytes and NK cells.

42. Key cytokines which regulate the
inflammatory response.
• IL10. • 17-21Kd mw.
• Produced by T-cells.
• Stimulation of mast cell
replication.
• Inhibits cellular immune
reactions.

43. 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.

44. Key cytokines which activate the
inflammatory response.
• TNF • Two forms found, TNFa and
TNFb.
• 17Kd mw.
• Produced by many cells
including monocytes (TNFa
) .
• Produced by T-cells (TNFb).
• Widespread activation of
cells; apoptosis, shock,
cachexia etc.

45. Key cytokines which activate the
inflammatory response.
• Interferons (IFNs). • 3 forms found a,b & g.
• Many different subtypes.
• Generally 19-26 Kd mw.
• Produced by monocytes (a),
fibroblasts (b) and T-cells
(g).
• Antiviral, cell activating and
tumour suppressant effects.

46. 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).

47. 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.

48. Chemokines.
• CXC chemokines. • IL8.
• Platelet factor IV.
• Granulocyte
chemotactic protein 2.
• Platelet basic protein
and related species.
• Utilise CXCR 1-5.
• Main target PMN.

49. Chemokines
• C-C chemokines.
• MCP 1,2,3,&4.
• RANTES
• MIP 1a & b.
• Eotaxin.
• Utilise CCR 1-5
receptors.
• Main targets
eosinophils and
monocytes.

50. Chemokines
• C chemokines. • Lymphotaxin.

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