MECHANISMS IN INFLAMMATION:

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MECHANISMS IN INFLAMMATION A common thread in
diverse diseases
David J. Grainger, Ph.D. British Heart Foundation
Senior Research Fellow Department of Medicine,
Cambridge University
What do the following diseases have in common?
Coronary heart Disease Osteoporosis
Asthma Severe non-fatal RTA trauma
Multiple Sclerosis Alzheimers Disease

Inflammation is a key pathogenic component in all
of them, and anti-inflammatory medicines have the
potential to reduce or abolish the symptoms
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What is inflammation?
Normal Lung
Bacterial sepsis
Recruitment of white blood cells
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Red blood cell
Numerous Oxygen transporters
White blood cell
Rarer Various different kinds Major
effectors of the immune system Also called
LEUKOCYTES
Picture of blood
x800
Picture of neutrophil
This leukocyte is engulfing snd killing bacteria,
a process called PHAGOCYTOSIS. This is the major
mechanism that protects you from infection.
x11,750
Images taken from Functional Histology
Churchill Livingston 1979 with permission
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The leukocyte army
LEUKOCYTES
MONONUCLEAR CELLS
GRANULOCYTES
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50
LYMPHOCYTES
MONOCYTES
NEUTROPHILS
EOSINOPHILS
BASOPHILS
B CELL
T CELL
macrophages
Mast cells
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20
10
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5
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The Mission

• Detect invasion
• Identify sites of infection
• Identify sites of tissue damage and other sites
  at high risk of becoming infected
• Differentiate enemies from friends
• Destroy invaders
• Engulf them
• Kill them
• Precipitate them

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The Mission

• Detect invasion
• Identify sites of infection
• Identify sites of tissue damage and other sites
  at high risk of becoming infected
• Differentiate enemies from friends
• Destroy invaders
• Engulf them
• Kill them
• Precipitate them
• 3. Organise and co-ordinate (logistics and HQ)

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MONOCYTES/MACROPHAGES
Patrol every tissue in the body Particularly
common in mucosal frontline Part of the
innate immune system Detect invaders
primarily using PAMPs Respond by raising the
alarm, releasing many pro-inflammatory
cytokines Direct phagocytosis function to
eliminate enemy forward guard Soon overwhelmed
by a decent attack Phagocytosis function
crucial for clearing debris following tissue
damage Clear up after the battle is won Also
function as antigen presenting cells (APC)
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BASOPHILS/MAST CELLS
Patrol mucosal tissues Part of the innate
immune system Detect invaders primarily using
PAMPs Respond by raising the alarm, primarily
by releasing histamine and IgEs Major
response to non-living invaders (bee stings,
peptides, proteins, tissue damage, heat etc).
Limited capacity to directly deal with enemy
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NEUTROPHILS/EOSINOPHILS

• Fast response foot soldiers - respond in
  hours
• Attracted to sites of activated macrophages,
  and also directly on tissue damage
• Fearsome array of chemical weapons, primarily
  free radical generators
• Generate further pro-inflammatory signals
• Huge capacity to overwhelm even large
  invasions
• BUT major capacity for collateral damage!

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B CELLS
Antibody factories Key component of the
mammalian adaptive immune system Repeated
clonal selection produces high affinity
antibodies to any given antigen - but it take
days or even weeks if naive Memory cells
persist for years or decades, and provide an
accelerated response when antigen
re-encountered Antibodies come in various
flavours pentameric IgM dominates early low
affinity response, primarily for precipitin
action, then high affinity IgG labels take
over Require T cell and APC help
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T CELLS Killer T cells (CD8)
The special forces of the immune system Key
component of the mammalian adaptive immune
system Kill cells designated as non-self
following recognition by Helper T cells
Highly specific, tightly regulated killers, with
less capacity for collateral damage Form a
Membrane Attack Complex which permeabilises the
target cell and kills it in Requires T cell help
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T CELLS Helper T cells (CD4)
Multiple sub-classes now being identified Key
component of the mammalian adaptive immune
system Important role as the army generals,
co- ordinating the immune response Clonally
selected like B cells to react with particular
foreign antigens Required to initiate the
killing by the CD8 T cells Central role in
immunity illustrated by AIDS - CD4 T cells are
the major cell type wiped out by uncontrolled
HIV replication
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• Macrophages process antigens and display them on
  cell surface
• Migrate through the lymphatic system to
  secondary lymphoid organs
• Present the antigen to naïve B and T cells
• Initiate B cell clonal selection and affinity
  maturation
• Selected B cells released into blood, and high
  levels of specific antibody are produced

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• Initially IgM produced pentameric, low affinity
  species with precipitin action
• After a few days, high affinit IgG produced
• Marks out cells as enemies
• Most IgGs fix complement to directly kill the
  target cell
• Also cross-link receptors on T cells stimulating
  further cytokine release, and T cell activation

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SUMMARY

• Mast cells and macrophages are early warning
  systems in the mucosa
• Early recognition through PAMPs binding to TLRs
• Cytokines are produced which recruit other
  leukocytes and establish local inflammatory
  response
• Neutrophils are usually the first responders,
  coming within hours, often in large numbers
• Neutrophils kill infectious agents primarily
  through release of free radicals
• Antigen from the invaders travels through
  lymphatics to lymph nodes
• Antigen is presented, by specialised
  macrophages, to naïve T and B cells
• Clonal selection of both T and B cells yields
  ever increasing affinity of antibodies
• Antibodies bind to infectious agents and
  preciptate them, or fix complement to directly
  kill them, or mediate recognition by T cells
• Killer T cells bind to infectious agents and
  kill them

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Peace-time duties for the leukocyte army

• Clearance of cell debris, particularly apoptotic
  cell corpses, by professional phagocytes
• Osteoclasts are specialised monocyte-derived
  macrophages responsible for bone resorption
• Macrophages clear lipids deposited into tissues

CELLS OF THE IMMUNE SYSTEM HAVE BEEN CONSCRIPTED
TO PERFORM A WIDE RANGE OF FUNCTIONS OTHER THAN
HOST DEFENCE
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When does host defense turn into pathological
inflammation?
Crohns Disease
TOO MUCH WRONG PLACE WRONG TIME
ARDS
Multiple Sclerosis
Rheumatoid arthritis
Lupus
Heart disease
Tendonitis
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ARDS

• Acute Respiratory Distress Syndrome
• An example of a whole class of acute
  inflammatory pathologies where an appropriate
  inflammatory response to trauma or infection does
  too much collateral damage (pancreatitis, sepsis,
  gun-shot wounds, head trauma etc.)
• Two major side-effects of the inflammation
• Acute edema due to vascular permeabilisation
• Fibrotic damage to sensitive structures (mainly
  lung and kidney basement membranes
• Often lethal
• No effective clinical management

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Multiple Sclerosis

• An example of a whole class of autoimmune
  disorders where the adaptive immune system fails
  to distinguish self-antigens from enemies
  (rheumatoid arthritis, lupus, Sjorgrens,
  Scleroderma, type I diabetes, Graves, B12
  resistance etc)
• Cause unknown, but cross-reaction of antibodies
  from bacterial superantigens to host proteins a
  possibility
• Two major side-effects of the autoimmune
  reaction
• Loss of specific tissue function due to
  destruction of key cell types or neutralisation
  of key proteins
• Chronic, systemic pro-inflammatory status leading
  to general tissue damage
• No effective clinical management

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Tendonitis

• An example of inflammation which was once
  appropriate but which has continued for too long
• Initiated in response to tissue damage
• Mechanisms responsible for switching off
  inflammation (such as production of
  anti-inflammatory cytokines like TGF-?) fail
• Generally respond well to clinical management
  with mild anti-inflammatory drugs - once the
  cycle of activated leukocytes producing
  pro-inflammatory cytokines has been broken, the
  inflammation resolves

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TREATING PATHOLOGICAL INFLAMMATION
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TREATING PATHOLOGICAL INFLAMMATION
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TREATING PATHOLOGICAL INFLAMMATION
THERE IS NO RIGHT LEVEL OF IMMUNE
SYSTEM ACTIVITY - IT DEPENDS ON THE CONTEXT

• Immunosuppression and anti-inflammatory
  treatment are part of an continuum
• Anti-inflammatories part of an integrated
  clinical management package, including
  anti-infectives etc.
• Continual re-assessment of the costbenefit
  balance needed for each patient
• Current anti-inflammatory medicines (e.g.
  corticosteroids) discriminate poorly between
  desirable host defence and pathological
  inflammation
• Major unmet need for improved anti-inflammatory
  therapies
• More subtle control of leukocyte behaviour
• Smart drugs distinguishing physiological
  immune function from pathological inflammation

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TREATING PATHOLOGICAL INFLAMMATION
THERE IS NO RIGHT LEVEL OF IMMUNE
SYSTEM ACTIVITY - IT DEPENDS ON THE CONTEXT

• Immunosuppression and anti-inflammatory
  treatment are part of an continuum
• Anti-inflammatories part of an integrated
  clinical management package, including
  anti-infectives etc.
• Continual re-assessment of the costbenefit
  balance needed for each patient
• Current anti-inflammatory medicines (e.g.
  corticosteroids) discriminate poorly between
  desirable host defence and pathological
  inflammation
• Major unmet need for improved anti-inflammatory
  therapies
• More subtle control of leukocyte behaviour
• Smart drugs distinguishing physiological
  immune function from pathological inflammation

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Two Broad Anti-Inflammatory Strategies
TARGET LEUKOCYTE ACTIVATION AND FUNCTION

• Stop leukocytes at the already at the site of
  inflammation from doing collateral damage
• Reduce vascular permeability and edema
• Inhibit fibrogenesis

TARGET LEUKOCYTE RECRUITMENT

• Stop being recruited to the site of inflammation
• If they are not there, they cannot do damage

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Tissue damage
Monocytes
Myeloperoxidase
Macrophages
TNF-a
Neutrophils
Chemokines M-CSF
Chemokines
Interleukins
TGF-b
Immunoglobulins
Host tissue
Chemokines Interleukins TGF-b
CD4 T-cells
TNF-a
B-cells
LPS Antigen
Interleukins
Histamines
Chemokines
CD8 T-cells
Mast cells
Pathogens
Eosinophils
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Tissue damage
Monocytes
Myeloperoxidase
Macrophages
TNF-a
Neutrophils
Chemokines M-CSF
Chemokines
Interleukins
TGF-b
Immunoglobulins
Host tissue
Chemokines Interleukins TGF-b
CD4 T-cells
TNF-a
B-cells
LPS Antigen
Interleukins
Histamines
Chemokines
CD8 T-cells
Mast cells
Pathogens
Eosinophils
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CHEMOKINES
Superfamily of structurally-related, small
pro- inflammatory cytokines that direct leukocyte
traffic
CXC family
CC family
IL-8 Gro-a, -b and -g IP-10 I-TAC SDF-1a PF-4
MCP-1, -2, -3 and -4 MIP-1a, -1b, -3a and
-3b Eotaxin 1 and 2 RANTES TARC 6Ckine / SLC
CX3C family
C family
Fractalkine
Lymphotactin
A signalling network of more than 50 ligands and
20 receptors
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CHEMOKINES DIRECT LEUKOCYTE TRAFFIC
From the bone marrow through the blood stream to
the periphery, chemokines guide various leukocyte
subsets to their targets. The redundant
chemokine signalling network provides sufficient
information density to accurately address many
leukocyte subsets simultaneously.
Chemokines have been implicated in the
inappropriate recruitment of leukocytes that
typifies diseases with an inflammatory
component CHEMOKINES ARE AN ATTRACTIVE TARGET FOR
NOVEL ANTI-INFLAMMATORY THERAPIES
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CHEMOKINE INHIBITORS

• Receptor antagonists
• Specificity problems
• Redundancy
• Broad spectrum chemokine inhibitors
• Functional inhibitors
• Toxiocology?

BX471 AMD3100
IL-10 agonists Peptide 3
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ALIGNMENT STRATEGY TO IDENTIFY CHEMOKINE
INHIBITORS
AQPDAINAPV TCCYNFTNRK ISVQRLASYR
RITSSKCPKE .. . .
. AQPDAVNAPL TCCYSFTSKM IPMSRLESYK
RITSSRCPKE AVIFKTIVAK EICADPKQKW VQDSMDHLDK
QTQTPKT . . . .
AVVFVTKLKR EVCADPKKEW VQTYIKNLDR NQMR...
PEPTIDE 1
PEPTIDE 2
human
mouse
PEPTIDE 3
Alignment of different chemokines from more than
10 species identified regions of homology across
the whole superfamily
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Our primary functional screen for in vitro BSCI
activity

TRANSWELL FILTER MIGRATION ASSAY
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PROPERTIES OF PEPTIDE 3
The first broad-spectrum chemokine inhibitor
(BSCI)

• Sequence specific
• Effective against all chemokines tested, with
  similar potency
• Completely inhibits migration
• Not very potent

Reckless Grainger (1999) Biochem. J. 340803-10
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MEDICINAL CHEMISTRY PROGRAM IDENTIFIED FOUR
STRUCTURALLY DISTINCT FAMILIES OF BSCIs
PEPTIDES
AMINO-GLUTARIMIDES

• NR58-3.14.3
• BIM 58171
• BIM 58189

• NR58,4
• NR58,33

YOHIMBAMIDES
AMINO-CAPROLACTAMS

• Yohimban-
• 16-amide

• Olfoxamide
• BN 83250
• BN 83470

BN83470 most promising candidate for further
development
Grainger et al. (2005) Mini Rev. Med. Chem.
5825-32
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Proof of concept Inject bacterial endotoxin
into rat skin to elicit massive skin
inflammation. Does systemic pre-treatment with
BSCI reduce leukocyte recruitment?
WHITE SPOTS ARE LEUKOCYTES
Endotoxin - - BSCI
- -
Endotoxin plus BSCI
Endotoxin only

• Suppresses macrophages plus B- and T- cell
  recruitment to MCP-1
• Additionally blocks neutrophil recruitment to
  LPS
• Markedly reduces pro-inflammatory cytokine
  production

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Animal model
Rats are sensitised with an injection of
an
ovalbumin

i
.p. 21 days later,
animals are treated with
BSCI via the subcutaneous route, 24 hours prior
to further
intranasal challenge with ovalbumin.
Mac-1
CD4



OVA
Treat
active
inactive
active
inactive
MACROPHAGES
LYMPHOCYTES
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Animal model
Rats are sensitised with an injection of
an
ovalbumin

i
.p. 21 days later,
animals are treated with
BSCI via the subcutaneous route, 24 hours prior
to further
intranasal challenge with ovalbumin.
UNTREATED
BSCI
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CHEMOKINE INHIBITORS

• BN83470 is an orally acting anti-inflammatory
  agent
• Efficacy data in 10 animal models of
  inflammation
• Active at 1µg/kg doses - equivalent to
  corticosteroids
• Excellent side effect profile
• Beginning Phase I trials in 2006
• Development programme in place for allergic
  rhinitis, asthma, COPD
• Other aminocaprolactam BSCIs being developed for
  surgical adhesions, stroke and heart disease

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BSCIs are SMART anti-inflammatory agents
BSCIs do not affect resting macrophage numbers in
bone liver, skin and brain even after 6 months
continuous treatment
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SUMMARY

• Inflammation is normal, essential physiological
  process
• Pathological inflammation is too much of a good
  thing
• Improved anti-inflammatory strategies need to
  discriminate between physiological host defence
  and pathological inflammation (I.e. be SMART!)
• Two major strategies are to block leukocyte
  activation (e.g. COX-2 inhibitors) and to block
  leukocyte recruitment (e.g. chemokine inhibitors)
• Most new programmes target the soluble mediators
  that communicate between leukocytes and
  co-ordinate the immune system
• Immune regulation is very complex, and at present
  pragmatism beats theory
• Improved anti-inflammatory treatments will likely
  find utility in a very broad spectrum of diseases

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Digital copies of these slide presentations, as
well as supplementary material on many of the
topics covered, can be found at
www.graingerlab.org