‘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 sPLA 2 reactants.

and acute phase • 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… - phagocytosis.

- complement activation.

- natural killer cells.

• Aquired. Includes… - secondary antibody mediated response.

-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.

• IgG Immunoglobulins.

• Major bloodborne immunoglobulin.

• 75% total Igs.

• 150 kda mw.

• Four subtypes.

• Main antibody of the secondary immune response.

• IgA Immunoglobulins • 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 A 1 & A 2 .

• IgM.

Immunoglobulins.

• A pentameric molecule.

• Confined to the blood.

• Important in the primary immune response.

• IgD.

Immunoglobulins • A minority (1%) immunoglobulin present on B-cells.

• Short half life.

• IgE .

Immunoglobulins.

• 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 ( a,b,g,d 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.

Ab-ag, Gm neg bacteria, subcellular particles Yeasts, parasites, ab-ag.

Classical (C1,4,2 & 2) Alternate (C3) C3 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. C5

C5a Some actions of complement fragments.

C5a, C3a C5a, C5b-9 C3b C5b-9 chemotaxis, phagocyte degranulation, stimulation of O 2 .

mast cell and platelet degranulation.

enhancement of cytokine release, induction of eicosanoid synthesis.

potentiation of Ab response, opsonisation of cells and lysis.

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.

HN Histamine.

N CH 2 CH 2 NH 2 • Formed from

histidine

.

• Stored in high concentrations in mast cells and basophils together with heparin and ATP.

• Three main receptor subtypes (H 1 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

CH 2 CH 2 NH 2

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.

• Neurokins A & B.

• Mainly located in sensory neurones.

• Released on nerve stimulation.

• Act on 7TM ‘NK’ receptors (3 subtypes; NK 1 etc).

• Cause vasodilatation, vascular permeability, smooth muscle contraction, mucus secretion, pain.

• CGRP.

Tachykinins.

• A product of the calcitonin gene generated through differential splicing.

• Found in sensory neurones.

• Induces neurogenic inflammation.

• Bradykinin (9 aa) • Kallidin (10 aa).

Kinins.

• Formed from

kininogens

forms) by

kallikreins

(2 (also 2 forms).

• Inactivated by

kininases

forms).

(2 • Two receptors B 1 and B 2 (inducible) (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.

TxA2

PG G2 Arachidonic acid LTA4

PGs LT B4 E,I,F,D LTs C,D,E

• Arachidonic acid from cellular phospholipids.

• At least 2 different pathways: cycloxygenase forms

prostaglandins

and

thromboxanes

.

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.

C 12 -C 18 fatty acid.

Acetyl group Phoshatidylcholine • PAF formed from phoshatidyl choline by and acetylase.

• Key role of phospholipase A 2 (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

experimental models.

useful treatment in

Nitric oxide (NO; EDRF).

H 2 N-CH.COOH

(CH 2 ) 3 NH C H N NH 2 • Formed in many tissues from

arginine

.

• Three enzymes (NOS) described;

iNOS, ncNOS & ecNOS.

• Resonsible for NANC transmission.

• Potent

vasodilator

microbiocidal.

and • Physiological effects dependent of

guanylate cyclase

activation.

iNOS.

• Induced in cells by

cytokines

, TNF a , IL1 b • iNOS does not require Ca 2+ supply of

arginine

.

or LPS.

for activation, only a • 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 a IL1 b .

& • 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 • TNF inflammatory response.

• Two forms found, TNF a and TNF b .

• 17Kd mw.

• Produced by many cells including monocytes (TNF a ) .

• Produced by T-cells (TNF b ).

• Widespread activation of cells; apoptosis, shock, cachexia etc.

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.

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 a & b .

• Eotaxin.

• Utilise CCR 1-5 receptors.

• Main targets eosinophils and monocytes.

• C chemokines .

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.