Peptide mediators of inflammation

Question 1

Kallikrein - downstream pathway

Activation/ inhibition of Kallikrein?
Action of Kallikrein?

Answer 1

Activated by proteolytic enzymes released from cells by tissue damage.
A specific protease
Cleaves
- high molecular weight kininogen (blood) -> bradykinin
- low molecular weight kininogen (tissue) -> kallidin
[Also generates plasmin from plasminogen]

Natural inhibitor = C1 esterase inhibitor (C1-INH)
Artificial inhibitor = ecallantide (mimics antibody binding domain)

Bradykinin & kallidin activate constitutively expressed B2 receptors.
Removal of the C-terminal arginine (by kininase I) -> gives des-arg products

Des-arg products activate B1 receptors (induced by inflamation)

Further removal of C-terminal Phe (by kininase II aka ACE) causes complete inactivation
- thus ACE inhibitors cause BK levels to rise

Question 2

Bradykinin

Answer 2

Produced by cleavage of high molecular weight kininogen by kallikrein

Originally identified as a slow contractor of smooth muscle e.g in guinea pig ileum.

Induces vasodilatation
Activates B2 receptors

Question 3

High molecular weight kininogen

Answer 3

Found in blood
Precursor of bradykinin
Cleaved by kallikrein

Question 4

Low molecular weight kininogen

Answer 4

Found in tissues
Precursor of kallidin
Cleaved by kallikrein

Question 5

B2 receptors

Effects on endothelium?

Answer 5

Constitutively expressed
Activated by bradykinin and kallidin

Gq couples -> release IP3, increasing [Ca]i.
- in endothelium: NO released diffuses short distance to arterial smooth muscle cells- causes relaxation, increasing blood flow (rubor & calor)

Question 6

B1 receptors

Answer 6

Only expressed at low levels constitutively

• induced by inflammation
• suppressed by corticosteroids

Activated by des-arg products of kininase I cleavage of bradykinin and kallidin.

Drugs inhibiting binding of bradykinin have been developed but are not in clinical use.

Question 7

Hereditary angioedema

Answer 7

Rare acute inflammatory condition characterised by episodes of severe (often painful) swelling.

Treated by kallikrein inhibitor Kalbitor

Question 8

Tachykinins

Answer 8

Identified as fast contractors of smooth muscle

• Substance P aka NK1
• Substance K aka NK2
• Neurokinin B aka NK3

Tachykinin receptors are Gq coupled- increase [Ca]i

Question 9

Substance P

Answer 9

aka NK1
Prototypical tachykinin
Released from peripheral nerves (along with CGRP)
Causes neurogenic inflammation. May also be imp in migrane and asthma.

Question 10

Cytokines

Answer 10

= high molecular weight peptides
Unlike many other inflammatory mediators are not stored, but produced on demand by gene transcription, released by vesicle shedding
- transcription is inhibited by glucocorticoids

Produced by T cells, macrophages, monocytes

Pro-inflammatory: IL-1, IL-2, IL-6, TNF-α.
Inhibit production of other cytokines: IL-4, IL-10.

TNF-α is particularly imp in rheumatoid arthritis.

Question 11

Nerve growth factor (NGF)

Answer 11

Released from activated macrophages

Recently identified as potent agent causing pain in arthritis
- monoclonal antibody against NGF (tanezumab) has potent analgesic effects in osteoarthritis, but so effective that patients overused their damaged joint & exacerbated damage.

Question 12

Membrane phospholipid structure

Answer 12

Fatty acid at 1 location on glycerol backbone is saturated
FA at 2 location is unsaturated, often arachidonic acid.

Cleaved by phospholipases at sites depending on the phospholipase.

Question 13

Phospholipase A2 (PLA2)

Answer 13

Initiating event of production of inflammatory mediators from arachidonic acid.
Activation depends on both:
- rise in intracellular calcium
- activation of GPCRs such as B1 or B2 receptors- these activate downstream kinases which phosphorylate PLA2, switching it on.

Cleaves phospholipids at position 2- generates:

• arachidonic acid
• lyso-phosphatidylcholine (i.e the phospholipid lacking the position 2 fatty acid)

Also found in insect & snake venom- explaining the potent inflammatory action of snake bite

Question 14

Arachidonic acid

Answer 14

Generated by PLA2 cleaving phospholipids
Unsaturated fatty acid, contains 4 cis double bonds at 5, 8, 11, 14 positions.

Many important inflammatory mediators are produced from arachadonic acid by COX enzymes or by 5, 12- or 15-lipoxygenase.
Products are collectively called eicosanoids. Include:
- 12-HETE (produced by 12-lipoxygenase)
- cyclic endoperoxides -> prostanoids (prostaglandins & thromboxanes)
- 5 HPETE -> leukotrienes

Question 15

12-HETE

Answer 15

aka Hydroxyeicosatetraenoic acid
Produced from arachidonic acid by 12-lipoxygenase

Chemotactic messenger employed in the migration of leukocytes to site of injury

Question 16

Leukotrienes

Answer 16

Produced by 5-lipoxygenase from arachadonic acid.
NB zileutin used clinically as a 5-lipoxygenase inhibitor

Produced predominantly by inflammatory cells e.g mast cells, macrophages.
LTB4, LTC4, LTD4, LTE4

Question 17

LTB4

Answer 17

Leukotriene

Stimulates

• neutrophil and macrophage adherence, chemotaxis & proliferation
• cytokine production by macrophages & lymphocytes

Question 18

Cysteinyl leukotrienes

Answer 18

LTC4, LTD4, LTE4
Constrict coronary vessels but increase blood flow & vascular permeability elsewhere
Powerful spasmogens: constrict bronchilar smooth muscle

Imp in asthma & other inflammatory disorders.

Used in maintenance of asthma:

• zileuton = 5-lipoxygenase inhibitor
• zafirlukast is a specific cysteinyl leukotriene receptor antagonist

Question 19

Platelet activating factor (PAF)

[a lipid compound]

Answer 19

Carboxylic acid linker at position one of phospholipid can be replaced by an ester bond:

• PLA2 removes 2-position FA from this precursor molecule
• another enzyme subsequently acetylates the 2 position
• result is PAF.

Extremely potent platelet aggregator
Vasodilator
Chemotactic agent for leukocytes

Rapidly degraded in vivo by an acetyl hydroxylase which removes the 2-position acetyl group.

Question 20

Prostanoids

Answer 20

= prostaglandins & thromboxanes
Derived from arachidonic acid by COX enzymes.
Short lived mediators (~1 min) involved in many normal cellular functions as well as inflammatory reactions.

Inflammatory effects: swelling, fever, pain

Question 21

COX enxymes

Mechanism?
COX1 vs COX2?

Answer 21

Bi-functional, catalyse 2 distinct reactions

1) cyclo-oxygenase reaction leading to PG-G [inhibited by NSAIDS]
2) peroxidase reaction resulting in PGH2 (then processed by various downstream enzymes to produce different end-products)

COX1: constitutively expressed, involved in secretion of stomach acids & production of prostanoids in tissue
COX2: induced by inflammation

Question 22

Inflammatory effects of prostanoids: Swelling

Answer 22

PGI2 (prostacyclin) acting on IP receptors

PGD2 acting on DP1 receptors -> increase in cAMP -> vascular smooth muscle relaxation -> increased blood flow

Question 23

Inflammatory effects of prostanoids: Fever

Answer 23

PGE2 locally produced in the hypothalamus (probably in response to IL-2 secretion from leukocytes)
Acts locally on neurones via EP3 receptor -> decrease in cAMP

Question 24

Inflammatory effects of prostanoids: Pain

Answer 24

PGE2 effects on EP2/4 receptors -> increase in cAMP -> enhances sensory fibre response to other painful signals (hyperalgesia)

Question 25

Actions of prostanoids on blood vessels and in platelet aggregation

Answer 25

PGI2 produced by endothelial cells has vasodilatory & anti-aggregatory actions
TXA2 produced by platelets has vasoconstrictor & pro-aggregatory actions

In undamaged blood vessels, these actions are balanced & platelets do not aggregate. Endothelial damage alters balance in favour of platelet aggregation.

TXA3 derived from eicosapentaenoic acid (from fish oil) has lower pro-aggregatory actions- lower probability of thrombus.

Question 26

Physiological actions of prostaglandins

• smooth muscle
• kidney
• stomach

Answer 26

Smooth muscle: contract or relax uterine, bronchial and GI smooth muscle

Kidney: regulate renin release & modify tubular reabsorption of sodium

Stomach: stimulate mucus & bicarbonate secretion that provides protection from acid attack, & inhibit acid secretion from parietal cells

Believed to enhance tumour growth in some cases, by inhibiting apoptosis, promoting invasion & stimulating angiogenesis (finding that COX inhibitors reduce growth rate of colon carcinomas)