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 antiproteases 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 microorganisms 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, subcellular particles Yeasts, parasites, ab-ag.

Classical (C1,4,2 & 2)

Alternate (C3)

C3

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 microorganisms. Opsonise others.

C5

Some actions of complement fragments.

C5a C5a, C3a C5a, C5b-9 chemotaxis, phagocyte degranulation, stimulation of O2-. mast cell and platelet degranulation. enhancement of cytokine release, induction of eicosanoid synthesis. C3b C5b-9 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.

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.

CH2CH2NH2

HN

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 1940s 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, 1950s) 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; 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) 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.

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; 1950s). BK causes pain. Armstrong et al 1954.

Eicosanoids.
Arachidonic acid

PG G2

LTA4

TxA2

PGs E,I,F,D

LT B4 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.
C12 -C18 fatty acid. Acetyl group Phoshatidylcholine

PAF formed from phoshatidyl choline by and acetylase. Key role of 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).

H2N-CH.COOH (CH2)3 NH C HN NH2
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.

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.