PATHOPHYSIOLOGY OF THE RESPONSE AGAINST ENDOTOXIN
BIOACTIVITY H. Buchner
W. Coley
anti-tumor activity
E. Centani
O-antigen
purification Westphal
Morgan
Toxicity / pyrogenicity “PIROTOXINA”
Dermal reaction
Rietschel Takayama
Lipid A
BPI Weiss
LBP
Luderitz
Characterization
G. Shwartzman
2/ MEASUREMENTS
Lipid A antagonists
lipopolysaccharide Shear
shock / death “ENDOTOXIN”
R. Pfeiffer
1/ BIOCHEMICAL CHARACTERISTICS
BIOCHEMISTRY
fever / increased resistance to infection
Tobias & Ulevitch
Synthetic Lipid A
Boivin & Mesrobeanu
3/ TOXICITY AND PYROGENICITY
Acyloxylacyl Hydrolase Munford
Shiba
4/ COMPLEMENT AND COAGULATION ACTIVATION
1890
1900
1910
1920
1930
1940
1950
1960
1970
5/ INTERACTION WITH PROTEINS & LIPOPROTEINS
1980
Limulus
Complement Beeson
Tolerance
IMMUNO CHEMISTRY
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Sultzer
C3H/HeJ
Beutler & Cerami
-Godowski - Rothe -Beutler
TLR2/4
CD14
TNF
Endotoxic shock
IMMUNOLOGY
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
ENDOTOXIN AS AN ALARM SIGNAL OF BACTERIAL INVASION
PYROGENICITY
adapted from E.K. Legrand
INFLAMMATION COAGULATION
Many of the body’s defense system are triggered by LPS (coagulation, inflammation, immune response, acute phase response)
CAPILLARY LEAK SHWARTZMAN’S REACTION ANTI-TUMOR ACTIVITY
Pillemer
Staub Kauffmann
O-antigen
8/ MACROPHAGE ACTIVATION 9/ ENDOTOXIN TOLERANCE
Wright, Tobias & Ulevitch
B-cell act.
Besredka
Antibodies
7/ B CELL ACTIVATION
2000
Levin & Bang Oppenheim
6/ THE LPS RECEPTOR
1990
Key defense cells have receptors for LPS (macrophages, neutrophils, platelets, lymphocytes)
TISSUE TOXICITY
ADJUVANTICITY
ABORTION
RADIOPROTECTION
Some LPS-induced molecules are protective against infection (TNF, IL-1)
LETHALITY
HEMATOPOIESIS ACTIVATION
Evolution has not favor LPS-non-responding animals
COMPLEMENT ACTIVATION POLYCLONAL B CELL ACTIVATION
LPS-binding molecules are found in all species (plants, insects, crustaceans, mammals)
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
SPECIFIC O-ANTIGEN repetitive units of oligosaccharides
OUTER CORE
INNER CORE (KDO)
GlcNac Gal
Hep
KDO
LIPID A
Salmonella synthetic Lipid A 7 FA
OH
Salmonella senftenberg Lipid A 4 FA 5 FA
O =P- O O
6 FA
O
Glc -Gal-Glc- Hep-Hep-KDO GlNH2 GlNH2
O
O
Salmonella abortus equi Lipid A 5 FA
O =P- O
Salmonella minnesota R345 Lipid A
6 FA
6 FA
OH
4 FA
Enterobacteriacae Pseudomonas Vibrio
7 FA
5 FA 4 FA
Neisseria meningitidis Bordetella pertussis Acinetobacter calcoaceticus Bacteroides fragilis
Karibian et al. J. Bact. 1993, 175, 2988 4 FA
Chlamydia trachomatis Rhodopseudomonas sphaeroid 1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
5 FA
6 FA
Karibian et al. J. Bact. 1993, 175, 2988
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
1
ENDOTOXIN CONTAMINATION OF ALLERGEN EXTRACTS HUNT et al. Am J Respir Crit Care Med 1994, 149, 1471.
Circulating LPS in patients with severe sepsis Opal et al. J. Infect. Dis. 1999, 180, 1584
Broncho-Alveolar Lavages t = 24 h
Segmental bronchoprovocation
PMN Endotoxin-containing ALLERGEN (i.e. 1,25 ng/ml) LPS-free ALLERGEN
Eosino
68 x 10 6
0.8 x 10 6
3.1 x 10 6
8.2 x 10 6
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
A LETHAL ROLE FOR LIPID A IN SALMONELLA INFECTIONS
ENDOTOXIN TOXICITY CHICKEN EMBRYO
Khan et al. Mol. Microbiol. 1998, 29, 571
LD50
Generation of a Salmonella typhimurium mutant lacking the waaN gene which encodes the enzyme that catalyses one of the acylation reaction --> The mutant Lipid A lacks one myristic acid.
1 ng
Hexa versus penta-acyl Lipid A
RABBIT
1 - 10 µg
[minimum pyrogenic dose : 2 - 6 ng/kg]
MOUSE
270 µg
RAT
5 mg
surrenalectomized or hypophysectomized : DL50 10 000 x lower
HUMAN maximum Tolerance: 4 ng/kg
SEPTICEMIA (plasma LPS)
MORTALITY
< 1 pg/ml 1 - 4.9 pg/ml 5 - 9.9 pg/ml 10 - 14.9 pg/ml > 15 pg/ml
0% 38% 67% 80% 100%
Wild type S. typhimurium
9
Mutant
8 7 TNF
5
J774 MØ + killed bacteria (12h)
pg/ml 500
4 3
400
2
300
1
200
0
ENDOTOXIN
1
3
5
7
8
9 10 11 14 20 28 35 days
100 0
Wild type
mutant
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
ENDOTOXIN Via LIPID A moiety
Via POLYSACCHARIDE Moiety
Classical pathway C1q
CHEMOTAXIS PMN ACTIVATION MAST CELL DEGRANULATION
Log10 viable bacteria in spleen or in liver
6
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
COMPLEMENT ACTIVATION
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Alternative pathway
Endothelial cell
COAGULATION TISSUE FACTOR expression
C3
Anaphylatoxins C5a / C3a
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Monocyte / MØ
DISSEMINATED INTRAVASCULAR COAGULATION
Catalytic cofactor for Factor VIIa
PAF release
PLASMINOGEN ACTIVATOR INHIBITOR
Fibrinolysis Platelets aggregation inhibition
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
2
INDUCTION OF TISSUE FACTOR
LPS
TF expression
SYNERGIE
Human monocytes & mouse macrophages
anti-CD11/CD18
Human endothelial cell / monocyte coculture
aggregated IgG Oxidized LDL
Fan et al. J.Clin.Invest.1991,87,50
IL-1
Human monocytes Human endothelial cell and monocyte
Hypoxia -> PAF
Activated Protein C
Brand et al. Arteriosc. Thromb. 1994,14,790
Gray et al. Haemost. 1996,6,92
HUVEC
Pulmonary Surfactant protein A
LPS-Binding Protein (LBP)
Lipoproteins
histatins (saliva)
Gangliosides
COCKROACH (Periplaneta americana)
HORSESHOE CRAB HERMIT CRAB
(Tachypleus tridentatus, Limulus polyphemus)
(Diogenes affinis)
BROWN SHRIMP (Penaus californiensis)
CRAYFISH (Pacifastacus leninsculus)
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
NATURAL FACTORS WHICH INTERACT WITH LPS
+
-
ENDOTOXINS
LBP
BPI (Bactericidal/Permeabilit Increasing protein)
IFNγ
(LPS Binding Protein)
Serum Lipoproteins Neutrophil Cationic antimicrobial Proteins (CAP 18; CAP 37)
MONOCYTES / MACROPHAGES
Albumin Antibodies
BPI
Complement
IL-6
LPS-BINDING PROTEIN IS CARRIED ON LIPOPROTEINS AND ACTS AS A CO-FACTOR IN THE NEUTRALIZATION OF LPS
LBP IS REQUIRED TO COMBAT A MURINE GRAM NEGATIVE BACTERIAL INFECTION Jack et al. Nature 1997, 389, 742
( 12 h )
HOMOLOGY OF LBP with HDL-associated Protein Cholesterol Ester Transfer protein
23%
Phospholipid transfer protein
IL-8
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Wurfel et al. J. Exp. Med. 1994,180,1025
LBP
TNFα
IL-1
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LPS
Leukocytic enzyme : Acyloxyacyl hydrolase
lysozyme
CAP18 (bactericidal/ (Cationic Hemoglobin permeability Increasing antimicrobial Protein) Protein)
24%
LPS - s CD14 LPS - mbn CD14
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
100 - 300 CFU S. typhimurium i.p.
1 / 12
Wild type
11 / 12
12 / 12
LBP -/-
2 / 12 Bacterial load on day 3 after S. typhimurium i.p.
LBP in sera (ng/ml)
[ LPS - LBP ]
( 2 weeks )
SURVIVAL
LPS (200 ng) + GalNH (50 mg) i.p.
LBP is a constituent of apoA-I containing lipoprotein
LPS - HDL
Inducible
Herbert et al. J Cell. Physiol. 1996,169,290
NATURAL LPS-BINDING COMPOUNDS
Lactoferrin
SILKWORM (Bombyx mori)
Wharram et al. J. Immunol. 1991,146,1437
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Transferrin
Constitutive
LPS-BINDING COMPOUNDS ARE FOUND NOT ONLY IN MAMMALS BUT ALSO IN INVERTEBRATES
LBP + / +
114
LBP + / -
95
LBP - / -
0
Peritoneum
liver
LBP + / -
10
14
8
LBP - / -
2 127
729
894
spleen
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
3
B.P.I. 55 kDa Glycoprotein
Granules and membranes of PMN
Chromosome : 20 q11.23 - q12 homology LBP : 44%
RELATIVE CONCENTRATIONS OF LBP AND BPI IN BODY FLUIDS
ng/ml
plasma
serum
Opal et al. Lancet 1994, 344, 429
5 - 70
0.2 - 2.0
LBP : 1 - 24 µg/ml
Abscess fluids
Kd Lipid A : rBPI23 = 2.6 nM; rLBP = 58 nM Bactericidal activity nBPI55 E. coli P. aeruginosa K. pneumoniae S. aureus
TNFα induction
rBPI23
++
nBPI55 & rBPI23
++ ++
-
-
↓ ↓ ↓
-
-
PROTECTIVE EFFECTS OF AN N-TERMINAL FRAGMENT OF BACTERICIDAL/PERMEABILITY INCREASING PROTEIN Ammons et al. J.Infect.Dis. 1994,170,1473
Species RAT
MICE
MICE
E. coli
Timing
rBPI21
3x108 CFU i.v. 07:K1
T0
none 10 mg/kg 20 mg/kg
0 / 15 5 / 15 11 / 15
107 CFU i.p. 07:K1
T0 T0 T+1h T+2h
none 200 µg “ “
0 / 15 15 / 15 9 / 15 2 / 15
2.5x109 CFU i.p. 0111:B4
T0
none 500 µg Cefamandole(100 mg/kg) BOTH
4/20 1/20 8/20 17/20
Survival (D7)
0.01
0.18 - 0.47
Su et al. Infect. Immun. 1997,65,1258
% Survival at 48 h
7%
+ Hemoglobin 60 mg i.v. simult.
7%
+ Hemoglobin 60 mg i.v. T- 12 h
36%
0.001 - 0.04
BPI concentration was proportional to the quantity of white blood cells within body PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, fluids Université Pierre(r=0.8) et Marie Curie and Institut Pasteur
Evans et al. J.Infect.Dis. 1995,171,153 % ANIMALS SURVIVING
(72H)
20
40 60 80 100
J5
∗
106 CFU
Control rBPI23 (5mg/kg) T0, +20, 40 & 60 min
PLASMA TNF (ng/ml) (90 min) 10
100
∗
0111:B4
∗
2x106 CFU
07K1
2x106 CFU
0111:B4 and 07K1 lead to 1000 x higher blood bacterial count compared with J5
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
52 - 60 kDa glycoprotein enzyme 2 subunits (50 & 14 kDa) found in PMN & macrophages found in plasma, and increased 16x upon i.v. injection 0 R
0
55%
Non infected
2040 ng/ml 260 ng/ml
ACYLOXYACYL HYDROLASE
HEMOGLOBIN INCREASE MORTALITY FROM BACTERIAL ENDOTOXIN
+ Hemoglobin 60 mg i.v. T+ 10 h
0.24
BPI CAN BE PROTECTIVE ALTHOUGH ITS EFFICIENCY DEPENDS ON THE ORGANISM
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
0.5 mg LPS i.p.
2.3 - 22.1
Gram BPI Gram + 1st
fluids(pleural, joint, peritoneal)
7.6
median
BPI LBP 95% CI
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Peritoneal fluids from peritonitis
0
0
0
0 P 0 0
H O
0
HN
C 0 0
0
C 0 C 0
0
C 0 0H C 0
0
NH C 0 0H
0 P 0 0
Munford et al.
Deacylated LPS has a reduced capacity : - Dermal Shwartzman reaction - Tissue toxicity - TNF production in whole blood - Priming of PMN for superoxyde release Deacylated LPS inhibits : PMN adherence to LPSpretreated endothelial cells plasminogenactivator inhibitor-1, prostacyclin, and PGE2 production by LPS treated endothelial cells LPS induced mitogenesis
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
4
In vivo Interactions Lipoprotein - LPS
Serum Lipoproteins inhibit LPS binding to monocytes and LPS-induced cytokine production Cavaillon et al. Infect.Immun. 1990,58,2375
Harris et al. J.Clin.Invest. 1990,86,696 200 ng / GalNH2 treated mice
E.c. LPS
LPS - LP
none
4930 3375 710 1700
220 120 protection against irradiation-induced apoptosis
DAF CD55
K+ Channel
Internalisation
LPS
P2X7 IL-1β TNFα
IL-10
RP105 is largely restricted to mature peripheral B cells
IL-1ra
Chan et al. J. Exp. Med. 1998, 188
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
BACTERIA
pg/ml
pg/ml
ENDOTOXINS (LPS)
IL-1β
4000
800
3000
LYMPHOCYTE COMPLEMENT COAGULATION CELL ACTIVATION ACTIVATION ACTIVATION ACTIVATION
LYMPHOCYTE CELL ACTIVATION ACTIVATION
Th1
Th2 C3a/C5
Heat Shock IL-4 / IL-10 Proteins
macrophage
NEGATIVE CONTROL
Thrombin Xa factor
PAF
IFNγ
SYNERGY
600
2000
400
1000
200
0
0 0
GMIL-3 CSF IL-2 pg/ml 1500
IL-1α
1000
2ng
20ng 200ng N. m. LPS / ml
2µg
0
Medium + C5a U/ml
TNFα
2ng 20ng 200ng N. m. LPS /ml
2 µg
IL-6
1000 800
IL-6 GLUCOCORTICOIDS
IL-1
1000
TNF
600 400
500
200
ADRENALS
HYPOTHALAMUS PITUITARY GLAND
ACTH
0
CRF
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
BACTERIAL LIPOPROTEIN AND LPS ACT SYNERGISTICALLY TO INDUCE LETHAL SHOCK AND PRO-INFLAMMATORY CYTOKINES Zhang et al. J. Immunol. 1997, 159, 4868
MORTALITY GalNH model
LP 9 µg/kg LPS 0.6 µg/kg LP + LPS
C3H/HeN 25 % 17 % 83 %
LP 48 µg/kg LPS 200 µg/kg LP + LPS
C3H/HeJ 20 % 0% 91 %
heat killed E. coli E. coli LP - / -
90 % 50 %
TNF in vitro (pg/ml) per. MØ
0 0
C3H/HeN C3H/HeJ
LP 250 ng/ml LPS 250 ng/ml LP + LPS
350 200 1000
65 0 155
GalNH model C3H/HeN
TNF in vivo (pg/ml) 1h LP 80 µg/kg 100 LPS 80 µg/kg 200 LP + LPS 470
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
2ng 20ng 200ng N. m. LPS /ml
2µg
0
2ng
20ng
200ng
2µg
N. m. LPS / ml
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
COMPLEMENTARY ORGAN EXPRESSION OF IL-1 & IL-6 IN LPS-INJECTED MICE Hacham et al. Cytokine 1996,8,21
10 µg LPS i.p. T+4h
0
IL-6
IL-1
BRAIN MUSCLE KIDNEY INTESTINE SPLEEN LIVER LUNGS HEART 5
10 15 20 % PRODUCTION
25
0
5
10 15 20 25 30 % PRODUCTION
BIOLOGICAL ACTIVITY IN ORGAN LYSATES 1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
7
ENDOTOXIN TOLERANCE
CLINICAL RELEVANCE
ENDOTOXIN TOLERANCE ENDOTOXIN TOLERANCE WAS FIRST DEFINED BY A REDUCED FEVER RESPONSIVENESS
SEPSIS SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS)
•deactivation
Paul B. Beeson, 1946 Proc. Soc. Exp. Biol. Med. 61, 248 1 2 3 4 5 6 7 8h
•adaptation
•Immunodepression
•desensitization
°T
Daily injection of same dose of typhoid vaccine
D1
•Immunosuppression
•anergy
1 2 3 4 5 6 7 8h
•Immunoparalysis
•refractoriness
°T
D7
•reprogrammation ENDOTOXIN TOLERANCE CAN PROTECT AGAINST A LETHAL CHALLENGE OF ENDOTOXIN 1 PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
CLINICAL RELEVANCE
ENDOTOXIN TOLERANCE IN INFECTED PATIEN
ENDOTOXIN TOLERANCE
Adaptation to bacterial LPS controls LPSinduced TNF production in rabbit macrophages
IN PATIENTS WITH PYELONEPHRITIS
Mathison et al. 1990 J. Clin. Invest. 85, 1108
Reduced fever IN PATIENTS CONVALESCENT FROM as compared TYPHOID AND PARATYPHOID FEVER to controls in Neva et al. 1950 J. Lab. Clin. Med. 35, 911 response to endotoxin or IN PATIENTS RECOVERING killed bacteria
Endotoxin Endotoxin
100
EFFECT OF ENDOTOXIN TOLERANCE OF LPS-INDUCED CIRCULATING CYTOKINES
100
1h30 3h
80
2500
60
2000
15
IL-6
Control 1X
500
2X
Control 1X
3X
**
2X
900
IL-12p70
**
*
** **
**
** **
Control 1X 2X 3X LPS pre-treatment (24h period)
0
2X
3X
1000 0
*
**
2X
600 500
300
400
500 min
Fever and lethality Hypotension and hypoglycaemia Procoagulant activity Induction of iNO synthase Synthesis of TXB2, TXA2, 6-keto-PGF1α Production of TNF, IL-8, IFN γ, IL-12
3X
IL-1β
700
** **
60
Control 1X
800
MCP-1
*
NO EFFECT OR ENHANCEMENT
400 300
40
G-CSF, GM-CSF, IL-1ra, PGE 2 production Central sensitivity to LPS : Neuroendocrine activation Corticosterone induction; Food intake Phagocytic activity Liver cell injury
200
200
20
100 0
Control 1X
80
**
400 300
100
**
120
600 500
**
0
100
700
200
**
500
3X
KC
800
ng/ml
pg/ml
pg/ml
pg/ml
0
400 300
**
20
**
pg/ml
0
**
REDUCED RESPONSIVENESS
3000 2000
1000
**
5
IL-18
4000
1500
200
Consequences of endotoxin tolerance on various LPS-induced activities
5000
40
10
6000
IFNγ
3000
ng/ml
ng/ml
0.05 < p < 0.005 p < 0.005
20
ng/ml
* **
25
3500
TNFα
30
1
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Rayhane et al. J. Endotoxin Res. 1999, 5, 319 35
Daily injection of same dose of typhoid vaccine
femoral artery
?
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
40
2
5h
FROM MALARIA Heyman et al. 1949 J.Lab.Clin.Med. 34, 1400
Serum TNF U/ml x 10-3 Endotoxin
McCabe 1963 J. Clin. Invest. 42, 618
ENDOTOXIN TOLERANCE
CYTOKINES
Endotoxin
FEVER
st
Control 1X 2X 3X LPS pre-treatment (24h period)
0
100
Control 1X 2X 3X LPS pre-treatment (24h period)
0
Control 1X 2X 3X LPS pre-treatment (24h period)
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
CONTROVERSIALS DATA
IL-1, IL-6, IL-10 & NO productions
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
8
ENDOTOXIN TOLERANCE
ENDOTOXIN TOLERANCE
Glucocorticoid-dependent and -independent mechanism involved in LPS tolerance
Role of macrophages in LPS tolerance Freudenberg & Galanos Infect. Immun. 1988, 56, 1352
Evans & Zuckerman Eur. J. Immunol. 1991, 21, 1973 Adrenalectomized mice
LPS
LPS
TNF
20
0 0 0 +
hours
&
C3H/HeN
2 x 107 i.v. 20 µg i.v.
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
ENDOTOXIN TOLERANCE
MØ
LPS
C3H/HeN
Adrenalectomized mice
control mice 10
0
24 h
&
MØ
& GalNH2 LPS
2 x 107 i.v.
18 mg
20 µg
0 + + +
+ + + +
+ + + +
0 0 + +
C3H/HeJ
LETHALITY
0% 100 % 100 % 0%
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
IN VITRO TOLERIZATION OF MACROPHAGES
Human neutrophils cannot be “tolerized” in vitro
ENDOTOXIN TOLERANCE
TNF alpha (pg/ml) 15000 60000
10000
0
0 2
0 - 24 h
20
LPS (ng/ml)
200 2000
20
2
200
Activation
ENDOTOXIN TOLERANCE
1 0.1
0
Pre-treatement 0 - 24 h
1
0.01 0.1
0.01 0 LPS E.c (µg/ml)
LPS + Anti TGFß LPS + anti-IL-10 + anti-TGFß 0 25
Randow et al. J. exp. Med. 1995, 181, 1887
LPS T0 0 25 µg 0 1 µg
50 75 100 TNF (% control)
LPS T24h 200 µg 200 µg 10 µg 10 µg
SURVIVAL 0% 72 % 0% 87 %
Berg et 1al.PSU-ITP J. Clin. 2339 Workshop Invest on Cytokine,1995, September 96, 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
Activation 24 - 48 h
LPS E.c (µg/ml)
0 Marie et al. Blood 1998, 91, 3439
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ENDOTOXIN TOLERANCE
in vitro 0 pre-treatment LPS human PBMC LPS + anti-IL-10
st
20000
24 - 48 h
Cavaillon et al. J. End. Res. 1994, 1, 21
IL-10 - / -
30000
10000
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
in vivo IL-10 KO mice Wild Type
40000
2000 LPS (ng/ml)
Pretreatment
IL-8 pg/ml
5000
50000
REVERSAL OF ENDOTOXIN TOLERANCE BY IFNγ & GM-CSF
0 - 24h
24-48h
48-72h
0 LPS 0 LPS 0 LPS 0 LPS
0 0 IFNγ IFNγ GM-CSF GM-CSF IL-12 IL-12
LPS LPS LPS LPS LPS LPS LPS LPS
human PBMC
0
5000
10000
Randow et al. 1997 J. Immunol. 158, 2911
15000
TNF (pg/ml)
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
9
ENDOTOXIN TOLERANCE IS NOT A LPS-SPECIFIC PHENOMENON
ENDOTOXIN TOLERANCE IS NOT A LPS-SPEC PHENOMENON AND CAN BE INDUCED BY
Greisman et al. J. Clin. Invest. 1969, 48, 613
TNF IL-1 Alcohol Complet Freund adjuvant Sodium arsenite, PMA Heat stress Exercice ...
E. coli LPS i.v.
T+5 h FEVER INDEX : 71% versus CONTROL 1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
NONE
6000
1 µg LPS N.m.
Zymosan
5000
200 µg/ml
4000
0 2500 2000
NONE
PMA
1 µg LPS N.m. p = 0.02
10000
NONE
1 µg LPS N.m.
6000
70 4000
0
60 50 40 0
Pre-treatment 0-24 h
20 ng 200 ng 2 µg
20 µg/ml
LPS Pre-treatment 0-24 h
1000
0
E. coli Re LPS N. meningitidis LPS
1 µg LPS N.m.
Pre-treatment 0-24 h
Cavaillon et al. J. End. Res. 1994, 1, 21
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
12
105 10 4
6
20
2 0
D8
10 2
D13
**
D1
D3
D8
10 8 *
10 7
*
106
8 4
D3
107
10
**
10 3
108
0 LPS SAC
14
30
10 NONE
16
**
106
10 5 D1
TNF (ng/ml)
10 4 BLOOD
**
CFU / ml
% surviving mice 100 90
500
1000 0
2 µg/ml
2000
60 ng/ml
1500
p = 0.01
Staph. aureus Cowan I
80
3000 2000
TNFα (pg/ml)
8000
5000
SPLEEN
CFU / g
p = 0.002
10000 p = 0.003
2000
10 7 CFU / g
15000
4000
0
TNF alpha (pg/ml)
2x105 bact/ml
20000
CFU / g
TNF alpha (pg/ml)
2 µg/ml
6000
Endotoxin tolerance protects against fungal infection
KILLED S. pyogenes
25000
*
LPS
ENDOTOXIN TOLERANCE
*
D13 **
10 6 10 5 *
LUNGS D1
**
8000
ENDOTOXIN-INDUCED TOLERANCE ON HUMAN MONOCYTES IS NOT A LPS SPECIFIC PHENOMENON
*
ENDOTOXIN TOLERANCE
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
**
24 h
*
TURPENTINE (4 sites i.m.)
D3
D8
D13
10 4
BRAIN D1
D3
D8 D13 * p < 0.05 ** p Š 0.01
Saline / Cn LPS / Whole Cn blood
0 5
Whole blood HEALTHY CONTROLS CARDIAC ARREST PATIENTS + + Cardiac arrest Healthy35 controls40 10 15 20 25 30 plasmawith Cryptococcus neoformans plasma Days post-infection
Rayhane et al. 2000 Infect. Immun. 68, 3748
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
ENDOTOXIN TOLERANCE Heterodimer : activator
p65
p65 p50
p50
NF- κ B
SIGNALING
X
Homodimer : inhibitor
p50
p50 SIGNALING
Endotoxin tolerance : A. Cross et al. J. End. Res. 2002, 8, 83 M. West et al. Crit. Care Med. 2002, 30(S1), S64 1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
p50
Modification of NF-κB expression
p50
Global decrease of NF-κB (both hetero- & homodimers) Blackwell et col. Decrease of the ratio p65p50 / p50p50 Ziegler-Heitbrock et col.
1st PSU-ITP Workshop on Cytokine, September 9-14, 2002. Prince of Songkla University, Université Pierre et Marie Curie and Institut Pasteur
10
