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PERITOINE ET INFLAMMATION
Pierre Moine
Department of Anesthesiology
University of Colorado Health Sciences Center
Peritonitis general phases
[Bartlett JG. Arch Surg 1978]
1. Bacteriemic phase
Phase of rapid removal of contaminants from the
peritoneal cavity into the systemic circulation.
This phase predominently involves Gram-negative
aerobes
2. Aerobs and anaerobs synesgistic interaction phase
3. Infection localization
The normal peritoneal cavity
• About 300 cells/mm3
• Mainly macrophages (45%), lymphocytes (T cells
45% - NK cells 6% - B cells 2% (peritoneal CD5+ B1
cells)), dendritic cells (2%), mast cells and
desquamated PMCs
• Peritoneal-associated lymphoid tissue (omental
milky spots, lymphocytes within the peritoneal
fluid, and the draining lymph nodes)
Peritoneal mesothelial cells
PMCs have the same mesodermal origin as the
endothelial cells; both types of cell can express
specific surface markers that enable them:
1. To promote the margination and migration of
neutrophils,
2. To interact with extracellular matrix proteins,
3. To present antigens to immune cells,
4. To produce inflammatory mediators such as
proinflammatory cytokines, NO, growth factors,
tissue plasminogen activator and plasminogen
activator inhibitor.
‘‘Milky spot’’: They consist of macrophages, B and T lymphocytes and mast cells,
immersed in a network of connective tissue, vascularized by fenestrated capillaries
and lymphatic vessels. Near milky spots, the mesothelium is practically devoid of
basement membrane so that macrophages and lymphocytes do not encounter any
obstacles in the peritoneal lumen. Milky spots may also be associated with stomata.
Diagram of the proposed
sequence in the
morphogenesis of
Lymphatic stomata
[Nakatani T et al, Anat Rec 1997]
BL
E
G
L
M
P
S
Basal lamina
Lymphatic endothelial cell
Gap
Lymphatic cavity
Peritoneal mesothelial cell
Peritoneal cavity
Lymphatic stoma
The PMCs lining the muscular portion of the diaphragm are interrupted by a large
number of intermesothelial gaps (Stomata). Defects in the basement membrane
allow the stomata to communicate directly with the underlying lymphatic lacunae.
The peritoneal infection
• Leukocytes influx into the peritoneal cavity is a
characteristic and diagnostic feature of
peritoneal infection.
• Leukocyte influx is characterized by increases
in both polymorphonuclear leucocytes (PMN)
and
mononuclear
cells
(mononuclear
phagocytes and lymphocytes).
• Initially PMNs predominate and they are
subsequently replaced by mononuclear cells as
the main cell population as infection resolves.
Early phase of the peritoneal
inflammatory response
The peritoneal cavity (1)
• Under normal circumstances, 1/3 of the fluid draining from
the peritoneal cavity passes through the diaphragmatic
lymphatics, and the remainder exits through the parietal
peritoneum
• The action of the diaphragm generates a cephalad flow of
peritoneal fluid
• Relaxation of the diaphragm generates a negative pressure
that sucks fluid and particles into the stomata.
• Contraction of the diaphragm closes the stomata and
forces lymph to the mediastinum.
The peritoneal cavity (2)
• After exposure to P aeruginosa, there are larger stomata
(4-10 mm to 23 mm) and lymphatic lacunae. New stomata
are also formed. This adaptation is aimed at enhancing
peritoneal clearance.
• In experimental animals, 1/2 of the bacteria placed within
the peritoneal cavity are removed via the diaphragmatic
lymphatics, and they appear in the thoracic duct within 6
minutes.
• Depression of spontaneous respiration under general
anesthesia decreases the rate of bacterial clearance from
the peritoneal cavity
The powerful absorptive capacity of the peritoneum
Mechanical clearance of irritants from the peritoneal cavity
Diaphragm
Omentum
Mesenteric peritoneum
Pelvic peritoneum
Clinical issues
It explains
• the rapid onset of systemic signs of peritonitis
• The primacy of early antimicrobial therapy
• The need to avoid organizational delays before
surgery
Natural antibodies and complement link innate and acquired immunity
[Ochsenbein et al, Immunology Today 2000]
Mechanisms of how NAs protect against infections. Protection can occur directly or indirectly
(via the complement system), and also by enhancing the specific Ab response, leading to a
more efficient control of the infection. These links to adaptive immunity are highlighted in blue.
Abbreviations: TI, T-cell independent; TD, T-cell dependent.
Role of the classical pathway of complement activation
in experimentally induced polymicrobial peritonitis
[Celik I et al, Infect Immun 2001]
- Natural IgM antibodies
(peritoneal CD5+ B1 cells)
- Early adaptative immune
response
C1qa-/-: functional lectin
and alternative pathways
H2-Bf/C2 -/-: Deficient in all
3 complement activation
pathways (classical, lectin,
and alternative)
2. Bacterial activation of PMC
3. Peritoneal macrophages – PMC
interaction
1. Direct recruitment
Peritoneal cavity
IL-1
TNF-a
MCP-1
RANTES
ICAM-1
VCAM-1/2
PMC
Peritoneal
mesothelial cell
and basement
membrane
PMN
Peritoneal
macrophages
IL-8
CD11/18
Submesothelium
IL-8
Mechanisms of leukocyte recruitment
to the peritoneal cavity
[Topley N, Kydney Int 1996]
Chemokines in the septic inflammatory response
- C-X-C Chemokines [Neutrophil chemotactic and
stimulatory activity]
IL-8 (murine analog MIP-2)
ENA-78
NAP-2
IP-10
GRO-a (murine KC)
MIP-1a
MCP-1
- C-C Chemokines [Monocyte/macrophage chemotactic
and stimulatory activity]
MCP-1 (murine analog JE)
RANTES
C10
MIP-1a
MIP-1b
Peritoneal interleukin-8 in acute appendicitis
[Zeillemaker Am et al, J Surg Res 1996]
Chemokines produced by mesothelial cells
huGRO-a, IP-10, MCP-1 and RANTES
[Visser CE et al., Clin Exp Immunol 1998]
ND
TNF-a
huGRO-a
IP-10
MCP-1
RANTES
(U/ml)
(ng/ml)
(ng/ml)
(ng/ml)
(ng/ml)
0
1.3 ± 1.4
ND
4.9 ± 1.1
ND
0.6
2.3 ± 0.6
ND
1.0 ± 1.1
ND
6.25
3.7 ± 1.1
ND
1.1 ± 0.9
ND
62.5
4.3 ± 0.8
ND
2.9 ± .4
0.1 ± 0.3
625
15.7 ± 2.0
ND
9.1 ± 2.5
0.2 ± 0.3
6250
32.4 ± 4.1
ND
14.9 ± 3.2 0.5 ± 0.7
TNF-a + IFN-
6.8 ± 1.5
70.6 ± 4.5 64.3 ± 6.6 3.8 ± 1.8
TNF-a + IL-1b
2.9 ± 1.5
0.5 ± 0.3 41.8 ± 2.2 0.4 ± 0.3
Chemokines produced by mesothelial cells
huGRO-a, IP-10, MCP-1 and RANTES
[Visser CE et al., Clin Exp Immunol 1998]
mRNA expression of MCP-1
Densotometric
15
10
5
0
2 4 8 12 24
IL-1b
2 4 8 12 24
TNF-a
2 4 8 12 24
IFN-
* †
Control
* IL-1b + IFN-
† TNF-a + IFN-
Leukocyte migration across human peritoneal mesothelial
cells is dependent on directed chemokine secretion and ICAM-1
[Li FK et al, Kydney Int 1998]
Chemokine concentration pg/105HPMC
% of total chemokine secretion
20000
100
10000
50
0
0
IL-8
Control apical
IL-1bapical
MCP-1
Control basolateral
IL-1bbasolateral
IL-8
RANTES
Control apical
Control basolateral
Production of IL-1b and TNF-a by peritoneal
macrophages depends on the bacterial species
and the inoculum
[Visser CE et al, Adv Perit Dial 1997]
Peritoneal interleukin-8 in acute appendicitis
[Zeillemaker Am et al, J Surg Res 1996]
IL-8 secretion by human mesothelial cell monolayers after 24 hr incubation with
bacteria (108/ml), cultured from peritoneal fluid samples of patients with perforated
appendices.
Leukocytes (106/cavity)
Time after CLP (Days)
J Immun 1999; 163:6148
MIP-2 : human analog IL-8
Infect Immun 1997; 65:3847
Infect Immun 1997; 65:3847
MIP-2 : human analog IL-8
Infect Immun 1997; 65:3847
MIP-2 : human analog IL-8
MIP-2 : human analog IL-8
Infect Immun 1997; 65:3847
MCP-1 (ng/ml)
MCP-1 (ng/cavity)
Peritoneal fluids
Serum
Time after CLP (Days)
J Immun 1999; 163:6148
% Survival
Control
Anti-MCP-1 antiserum
injected 2 h before CLP
Time after CLP (Days)
J Immun 1999; 163:6148
Anti-MCP-1
Neutrophils
Total leukocytes
CFU/10 ml peritoneal fluids
Macrophages
Control
Time after CLP (Days)
J Immun 1999; 163:6148
Control
LTB4 receptor antagonist
MCP-1 (ng/cavity)
Leukocytes (106/cavity)
Effects of a specific LTB4 receptor antagonist on the leukocyte
infiltration and on the production of MCP-1 after CLP
Total leukocytes
Macrophages
Neutrophils
Control LTB4
antagonist
J Immun 1999; 163:6148
% Survival
Effects of a specific LTB4 receptor antagonist on the survival
of mice after LCP
Control
LTB4 receptor antagonist
Time after CLP (h)
J Immun 1999; 163:6148
Role of the resident peritoneal macrophages and mast cells
in chemokine production and neutrophil migration in acute
inflammation: Evidence for an inhibitory loop involving
endogenous IL-10 [ Ajuebor MN et al., J Immunol 1999]
Peritoneal neutrophils
x
106
mMCP-1
/ mouse
ng per cavity
10
6
5
3
0
0
Control
Resident
Mast cell
macrophages depleted
depleted
Control
Resident
Mast cell
macrophages depleted
depleted
24 hr post i.p. injection of 1 mg/kg LPS
Differing responses of mast cells to allergen and to bacteria
Piecemeal
Selective release
of
Histamine
Leukotrienes
TNF, IL-6
Th2-associated
cytokines
(IL-10, IL-5, IL-13)
Neutrophil
recruitment
Bacterial
clearance
Caveolar chamber
with viable bacteria
IgG opsonized
bacteria
Phagolysosome
with degraded bacteria
Malaviya R & Abraham SN, Immunological reviews 2001
Histamine
Serotonine
LTB4
LTC4
IL-10
IL-5
IL-13
TNF-a
LTB-4
Mast cell
MN
TNF-a
MCP-1
IL-1
RANTES
IL-17
LTB-4
VCAM-1
HLA-DR
CystLT
PGF1a
PGE2
PAF
IL-8
LTB-4
LTB-4
IL-8
Bacteria
IFN-
IL-10
MCP-1
PMC
GRO-a
IP-10
CD40
IL-15
PMN
NO
CD154
IL-6 sIL-6R
MCP-1
RANTES
CD4 T cell
ORS
Late phase of the peritoneal
inflammatory response
Chemokine C10 promotes disease resolution
and survival in an experimental model of
bacterial sepsis [Steinhauser ML et al., Infect Immun 2000]
C10 is involved in the late stages of
the inflammatory process
Kaplanski G et al., Trends in Immunology 2003
Modulation of the PMN recruitment
IFN- deficiency
IL-6 deficiency
1. Delay in the initial rate of PMN
accumulation
2. Delay in neutrophil clearance
3. Reduction in overall PMN
numbers
4. Increased C-X-C KC/MIP-2
expressions
5. Defective PMN apoptosis
1. Greater PMN infiltration
(but the initial rate of PMN
infiltration is unaffected)
2. Increased KC/MIP-2 expressions
3. Delay in neutrophil clearance
4. Defective PMN apoptosis
Innate immunity
CXC chemokines
IFN-
IL-1b - TNF-a dependant MIP-2 (IL-8) - KC
CD40-CD154 dependant
CD40-CD154
IL-6-sIL-6R
+
IL-1b - TNF-a dependant +
IL-17
-
TNF-a
IL-1b
+
IFN-
+
PMC
-
IL-6
IFN-
CD40-CD154
+
IFN- - IL-1b - TNF-a dependant
IFN-
Neutrophils
Macrophages
Natural killer cells
+
“Switching”
between
infiltrate
phenotypes
+ IL-6-sIL-6R
+
CC chemokines
MCP-1 - RANTES – C10
Lymphocytes
Acquired immunity
Current paradigm of contemporary
sepsis research
Uncontrolled inflammatory response
vs
Inadequate immune response
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
IL-10-/- mice have an enhanced bacterial clearance
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
IL-10 mice have elevated TNF and chemokine concentrations in peritoneal fluid and plasma
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
IL-10-/- mice have more severe multiple organ damage
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
IL-10-deficient mice demonstrate multiple organ failure and increased
mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance [Sewnath ME et al., J Immunol 2001]
Control
Anti-IL-4
Anti-IL-13
J Immunol 2000
Signal transducer and activator of transcription (STAT) STAT 4 and STAT6 are transcription
factors that are essential in mediating responses to IL-12 and IL-13 respectively
WT
J Exp Med 2001
J Exp Med 2001
Peritoneal levels
IFN-y level was not
augmented in STAT6-/Mice
J Exp Med 2001
STAT6-/- mice showed an
altered cytokine profile in
the peritoneum in favor
of bacterial clearance.
J Exp Med 2001
These data suggest that
more effective bacterial
clearance in the
peritoneum may result in
a reduction in organ
inflammation and damage
J Exp Med 2001
J Exp Med 2001
J Exp Med 2001
Systemic organ damage
initiated by CLP was
ameliorated in STAT4-/mice without affecting
bacterial load in the
peritoneum and peripheral
blood
J Exp Med 2001
Aberrant inflammation and lethality to septic peritonitis
In mice lacking STAT3 in macrophages and neutrophils
[Matsukawa A et al., J immunol 2003]
- Increased lethality to CLP in mice lacking STAT3
- Exacerbated systemic inflammation and organ damage
- Failure to facilitate effective bacterial clearance despite
increased numbers of infiltrating leukocytes and
enhanced cytokine production
-Impaired innate immune response in leukocytes
* These data suggest that impaired bacterial clearance is not
likely the cause of increase lethality
* The present data highlight evidence that the innate immune
cells appear to be essential not only for the initiation and
development of inflammatory response but also the
resolution of the response, playing multifunctional roles
in innate immunity during sepsis.
Conclusions
1. Cellular interactions: PMCs / macrophagesmonocytes / Mast cells / Lymphocytes /
2. Neutrophil recruitment
3. Uncontrolled inflammatory response