Inflammation &

Remodeling in Asthma
Focus on : anti-inflammatory effects
of Clarithromycin

P.G. Konthen

Division of Allergy & Clinical Immunology

Dept. of Internal Medicine
Airlangga University Faculty of Medicine
Dr. Soetomo Teaching Hospital - Surabaya
 Asthma prevalence by race Asthma statistic (2)

1982-1996
80

70
Rate per 1000 population

60

50

40

30

20

10

0
1982 1984 1986 1988 1990 1992 1994 1996

White Black

US National Center for Health Statistic (1998)

 Asthma is a syndrome
Characterized by at least three
cardinal features :

Intermittent and irreversible

airway obstruction
Airway hyper responsiveness
Airway remodelling
What we have learned
from
Postmortem biopsies
Animal model studies
Human clinical trials
of asthma
???
 Congested and dilated vessels (V), prominent smooth
muscle, inflammation, and occluded bronchial lumen (L)
Goblet cell hyperplasia, subbasement membrane thickening,
collagen deposition, and cellular infiltrate

Normal person Asthmatic person

 Asthma
is an inflammatory disease
and more than just
airway narrowing
Cells involved in asthmatic
inflammation
Mast cells & basophils
Eosinophils
Macrophages
Dendritic cells
T lymphocyte (Th-2 cytokine profile)
Structural cells
(epithelial cells, fibroblast, and
smooth muscle cells)
Inflammatory response in asthma
 Early and late phase allergic
reaction in asthma
0 1 6 8 24 48 (h)
Early phase Late phase Very late phase
APC
TNF- Epithelium
Ag IL-
MBP, ECP,
FcεRI EDN, CLC etc
IL-3 RANTES
IL-4 MCP-4 MBP, ECP,
Mast cells IL-5 Eotaxin
IL-8 EDN, CLC etc
Th2 B cells
GM-CSF
Eos
IL-4
Histamin, PGD2, IL-3
TNF-
LTs etc IL-4 IL-4
IL-5 IL-5
Th0 IL-8 IL-6
Th2
GM-CSF IL-13
MIP-1 RANTES
MCP-3
IL-4
RANTES IL-13 RANTES
Eotaxin Baso MIP-1 Eotaxin
IL-8 IL-8
GM-CSF Histamin, LTC4 GM-CSF
Endothelium PAF
VCAM- PAF
1
ICAM-1 Endothelium
VCAM-1
E-selectin
Eos Th2 Baso Eos
 Mast cell and its mediators

PLA2 AA + PAF

Mast Cell CO 5-LO

Preformed mediators
Histamine TNF-α
Heparin TGF-β
Tryptase (IL-3, 4, 5) PGD2 LTC4 LTB
(Chymase) (IL-13) LTD4
Kininogenase
LTE4

Mélanges of mast cell mediators

Nerves Glands Vessel
Itch Mucus Vasodilatation
Recruit reflexes secretions Mucosal Thickening
Sneezing Permeability
Malaise Watery rhinorrhea
 Mast-cell
Mast-cell interactions
interactions in
in asthmatic
asthmatic inflammation.
inflammation.
Mast Cell

Products
Products Target
Target

bFGF, TGF-,, Tryptase,

bFGF,TGF- TNF-
Tryptase, TNF- Fibroblast
Fibroblast
IL-5,
IL-5, GM-CSF,
GM-CSF, PAF,
PAF,Tryptase
Tryptase Eosinophil
Eosinophil
IL-4
IL-4 Th2
Th2Lymphocyte
Lymphocyte
IL-4,
IL-4, IL-13,
IL-13, IL-6
IL-6 BB Lymphocyte
Lymphocyte

LTC 4
LTC PGD 2
,, PGD ,, PAF,
PAF, Histamine
Histamine Bronchiolar
Bronchiolar smooth
smooth muscle
muscle
Chymase,
Chymase, Histamine
Histamine Airway
Airway serous
serous cells
cells

LTC 4
LTC PGD 2
,, PGD ,, PAF,
PAF,Tryptase,
Tryptase, Vascular
Vascular endothelium
endothelium
Histamine, TNF-
Histamine, TNF-
Matrix
Matrix
Tryptase,
Tryptase, Chymase
Chymase metallo-
metallo- Extracellular
Extracellular
proteinases
proteinases matrix
matrix
Chymase
Chymase
 Eosinophils and its mediators
Survival Activation Chemoattraction

IL-3 IgE (FcεRII) CD40 ligand PAF IL-5

IL-5 IgG C5a Eotaxin
GM-CSF IgA IL-16 RANTES
CD40
MCF-3

IL-10, IL-8 TNF-α, IL-1a

GM-CSF, IL-4 ICAM-1, VCAM-1
TGF-α, TGF-β1 E-Selectin
Neutrophil recruitment Hypothalamic &
Mast cell & IgE hepatic
Fibrosis responses

MBP ECP EDN EPO LTC4

Epithelial cell detachment Bronchoconstriction
Exocytosis Gland secretion
Nerve damage Vasodilatation
Free radical damage Vascular permeability
 Leukocyte, endothelium, fibroblast and mediators
interaction in inflammatory infiltrate
Rolling
Leukocyte
Extravasation Platelet

Endothelium
Glycosaminoglycan

Fibroblast

RANTES
Cellular infiltrate
Inflammation
 Microenvironmental control of inflammation
Tissue Cells T Cells
fibroblasts
smooth muscle cells IL-3
IL-4
epithelial cells IL-5
endothelial cells GM-CSF

GM-CSF
G-CSF Inflammatory Cells Blood Stream
M-CSF
IL-1 proliferation mature and
IL-6 survival immature
IL-8 activation inflammatory cells,
differentiation progenitors
NGF
TNF
Asthma is a disease
of inflammation and
remodeling :
new insights …
 Th-2 Cytokines

Dendritic cell

Airway microenvironment
Th-2 cell
Th2-inflammation

IL-3
IL-9 IL-5
TGF-β
IL-4 GM-CSF
(myo) fibroblast

.. .. .. IL-3 ET-1
.... . VEGF
Eos
Mast cell

Smooth muscle
Basophil

Blood vessels
Chemoattractants,
Proinflammatory mediators

Davies DE. J Allergy Clin Immunol 2003,111,215-225

 ENVIRONMENT

GENES Atopy

Initiation Epithelial
susceptibility T-lymphocytes

Prolonged IL-4, IL-13

EGFR
growth low epithelial TGF-β high
repair
Propagation

TGF-β +

IL-4, IL-13
Myofibroblast activation

Growth Cytokines and

factors chemokines
Amplification

Airway wall remodeling Inflammation

Steroid refractory Steroid sensitive

CHRONIC ASTHMA

Davies DE. J Allergy Clin Immunol 2003,111,215-225

 Airway remodeling

Epithelial fragility and disruption

Subbasement membrane thickening
Deposition of collagen I, III, and fibronectin
Smooth muscle hypertrophy and hyperplasia
Enlarged mucous gland & increased goblet cells
Increased vascularity
Elastic fiber fragmentation
Altered neural morphology and function
 Consequences of airway
remodeling
Smooth Inflamma- Growth
Mucous Elasto-
muscle tory cell factor
gland persistence release lysis
mass

Severe Increased Ongoing Collagen Reduced

broncho- mucous inflamma- deposition elasticity
contriction secretion tion on RBM of airway
during during and ECM wall
exacer- exacer-
bation bation
 Consequences of airway
remodeling
Recruitment and activation of inflamma-
tory cells and perpetuation of airway
inflammation
Persistent irreversible airflow
obstruction
Accelerated decline in lung function
Enhanced bronchial hyperresponsiveness
 Various trigger of asthma
Chlamydial
Bacterial
infection

Respiratory
viral
infection
Specific Asthma
allergen
exposure attacks
Air
pollutants

Physical
exercise
Irritant
Cold air vapor
Respiratory infection & asthma

Virus or bacteria may increase airway

hyperresponsiveness
Bacterial antigen may elicit an IgE
mediated response
Virus or bacteria may enhance airway
inflammation by inducing NF-κβ
activity through Toll like receptor 4
Pathways of airway inflammation
INNATE ACQUIRED
LPS ALLERGEN

CD23
CD14 TLR4 OMD2 Macrophage
Macrophage

AP-1 NF-κB
B7 MHC II
IL-10 CD28
IL-12 IL-1 (CD3) T cell
Helper Receptor
IL-8 TNF T Cell
AP-1 NFκB

Eos IL-4
IL-5
IL-1
Elastase Eotaxin
MBP
IL-8

ICAM-1 VCAM-1

Reed CE, et al. J Allergy Clin Immunol 2001;108:157

Pathway of microbes triggered
inflammation
microbes

antigen specific B cell trigger tissue injury

IgE
IgG + IgM sensitization mast cell

alternative classical
pathway pathway
complement C3a/C5a inflammatory
mediators

complement antibody polymorph kinins fibrinogen

Endothelial cells Blood vessel

 Chlamydia associated
airway inflammation
Chlamydial heat shock protein 60 and
Chlamydial lipopolysaccharide may induce
NF-κβ activity through activation of Toll
like receptor 4
Production of nitric oxide
Secretion of TNF-α , IL-1, IL-6,
IL-8, IL-12
Expression of adhesion molecules
E-selectin, ICAM-1, VCAM-1
Proliferation of smooth muscle cells
Is there a role for
macrolide antibiotics
in asthmatic
inflammation ?
Three independent action
of macrolides
3 14 membered

Anti-inflammatory &
immunomodulatory
action
Chronic inflammatory airway
2 14 membered disease i.e. Diffuse Pan bronchitis
Chronic lung injury of
Motilin action prematurity etc..
Motilin like action as
a GI tract motility hormone

1 14,15,16 membered

Four decades as
antimicrobial agents

Kudoh S. Iyaku Journal 1999,21:14 (partial modification)

 Molecular Structure of
Clarithromycin

H CH3 H3C H3C OCH

3 O
H
H
CH3
HO O H
HO H CH3 H O
O
CH3 N(CH3)2
H3C H O H
CH2CH3 O
HO O OH
CH3
OCH3

CH3
 The potential role of
Clarithromycin as an anti-
inflammatory drug

Effect on Chlamydia associated airway

inflammation
Effect on the production of inflammatory
cytokine
Effect on phagocytosis of apoptotic cells
by alveolar macrophages
Clarithromycin active against
a wide range of microbes
Gram (+) : Staphylococcus aureus
Streptococcus pneumonia
Streptococcus pyogenes
Gram (─) : Haemophilus influenzae
Moraxella catarrhalis
Atypical : Chlamydia pneumoniae
Mycoplasma pneumoniae
Legionella pneumophila

Eradicating respiratory tract infection could prevent the

activation of Toll like receptor 4 and the subsequent
inflammatory signal transductions
Clarithromycin
Chlamydia pneumoniae
 Clarithromycin showed
a concentration-dependent
suppressive effect on IL-18
release by human eosinophils
from atopic donors

Kohyama T, et al. Antimicrob Agent Chemother 1999;43:907-911

 Effects of macrolides and other antibiotics
on IL-8 release by human eosinophils

ERYTHROMYCIN
CLARITHROMYCIN
JOSAMYCIN
TETRACYCLINE
CEFAZOLIN

120 120

*
IL-8 release (%)
(control=100%)

100 100
*
80 * 80 *
60 * * 60
*
*
*
40 40
*
20 20

0 0
0 1 5 25 0 1 5 25

Concentration (μg/ml)
Ca ionophore (─) Ca ionophore (+)
 Clarithromycin inhibit IL-8
production in human bronchial
epithelial cells by repressing
IL-8 gene transcription via
AP-1 binding site

Abe S, et al. Am J Respir Cell Mol Biol 2000;22:51-60

Dose-dependent inhibition of IL-8 gene expression
by Clarithromycin in BET-1A cells
TNF
+
Rest alone CAM
0.01 0.1 1 10 (g/ml)

IL-8 1.8 kb

GAPDH 1.4 kb
IL-8/GAPDH (densitometry unit)

0
1 2 3 4 5 6
Effects of incubation time with Clarithromycin on
IL-8 gene expression in BET-1A cells
TNF
+
CAM
Rest alone
24 48 72 96 (hours)

IL-8 1.8 kb

GAPDH 1.4 kb
IL-8/GAPDH (densitometry unit)

0
1 2 3 4 5 6
Effects of Clarithromycin on IL-8 protein release from
BET-1A cells with mediation by TNF-
3000

2500
Il-8 in Culture Supernatant (pg/mg)

2000
*
1500

1000

500

0
Resting TNF CAM + TNF
 Clarithromycin are
capable of inhibiting
pro-inflammatory cytokine
production in vitro and
as potent as prednisolone

Wallwork B, et al. Laryngoscope 2002;112:1827-1830

Interleukion-5 production in the presence of clarithromycin
and prednisolone compared with control specimens
350

300
Il-5 (pg/mg total protein)

250

200

150

100

50

0
10-5 10-6 10-7 Control 10-4 10-5 10-6 10-7 Control
Clarithromycin (M) Prednisolone (M)
Interleukion-8 production in the presence of clarithromycin
and prednisolone compared with control specimens

1500

1250
Il-8 (pg/mg total protein)

1000

750

500

250

0
10-5 10-6 10-7 Control 10-4 10-5 10-6 10-7 Control
Clarithromycin (M) Prednisolone (M)
 GM-CSF production in the presence of clarithromycin
and prednisolone compared with control specimens
150

125
GM-CSF (pg/mg total protein)

100

75

50

25

0
10-5 10-6 10-7 Control 10-4 10-5 10-6 10-7 Control
Clarithromycin (M) Prednisolone (M)
 Clarithromycin significantly
reduce all markers of mucosal
inflammation in adult with
chronic sinusitis
including CD68, EG2, elastase,
IL-6, IL-8, and TNF-α

MacLeod CM, et al. Advances in Therapy 2001;18:75-82

Markers of inflammatory cell function before and after
Clarithromycin treatment

Pre Treatment Post Treatment

16
* p < 0,05
14
# p < 0,01
12 *
10

8 #
*
6

0
CD68* Eosinophils EG2* Elastase#
Clarithromycin at clinically achievable
levels significantly promote the
phosphatidylserine receptor-
dependent phagocytosis of apoptotic
neutrophils by alveolar macrophage

Yamaryo T, et al. Antimicrob Agent Chemother 2003;47:48-53

Effect of various antibiotics on the phagocytosis of
apoptotic neutrophils by alveolar macrophage

* p < 0,01 versus control

70

* * * *
*
Percent phagocytosis

60

50

40

30

20

10

0
Ctrl 0,1 1,0 10 0,1 1,0 10 Amp Cef Dexa

Erythro Clarithro
 Summary
Asthma is a disease of inflammation and
remodeling
Asthma exacerbation could be triggered
by specific allergen exposure and various
nonimmunologic stimuli including
respiratory tract infection
Clarithromycin, a 14-membered macrolide,
could be used as an anti inflammatory
drug in patient with asthma
 Sunset in Makassar 2003

Thank you for your kind attention …