The Inflammatory Response in the ICU:
The Good, The Bad, and The Ugly
Mark W. Hall, MD
The Ohio State University College of Medicine
Nationwide Children’s Hospital
Columbus, OH
Disclosures
• I have no conflicts of interest to disclose.
• I will be talking about off-label use of
medications during this talk.
My apologies in
advance to United
Artists. ©1966
Does this patient have an over- or under-active
innate immune response?
In other words…..does this patient need an anti-inflammatory
therapy or a pro-inflammatory therapy?
The Good and the Bad
Pro-inflammatory
PG
–
–
–
–
–
–
IL-1β
TNF
IL-6 (?)
IL-8
IL-17
Eicosanoids
Anti-inflammatory
–
–
–
–
–
–
IL-10
TGFβ
IL-11
sTNF receptor
IL-1ra
IL-4
The Good and the Bad
Pro-inflammatory
PG
–
–
–
–
–
–
IL-1β
TNF
IL-6 (?)
IL-8
IL-17
Eicosanoids
• Yeah….but therapies
targeting these guys have
failed to consistently
improve sepsis outcomes!
– Anti-LPS
– IL-1ra
– Anti-TNFα*
– Anti-bradykinin
– Anti-PAF
– Methylprednisolone/
dexamethasone
Immunologic balance
• Systemic Inflammatory
Response Syndrome (SIRS)
– TNFα, IL-1β, IL-8, IFNγ
• Compensatory Antiinflammatory Response
Syndrome (CARS)
– IL-10, TGFβ
• Homeostasis: The patient is
able to respond to a challenge
but is not actively inflamed.
Monneret, Adv in Sepsis, 2005
The Bad
The Good
The Ugly
Where can we look for markers
of immunoparalysis?
What about the monocyte?
2.
TNF
1.
3.
Activated
Monocyte
1. Phagocytosis
2. Intracellular killing
3. Antigen presentation
4. Extracellular TNF production
TNF
4.
TNF
TNF
Immunoparalysis
TNF
HLA-DR
expression
< 30%
Deactivated
Monocyte
TNF
Ex-vivo TNF
TNF
TNF
HLA-DR Quantification
simple flow cytometry
Monneret et al, Intensive Care Med, 2006
Monocyte HLA-DR expression < 30% has been associated with
adverse outcomes from sepsis-induced MODS in adults and children.
July, 2006
• 86 adults with septic shock, sampled on Days 1-2 and 3-4.
• Overall mortality 34% (61 survivors, 25 non-survivors)
Immunoparalysis
TNF
Deactivated
Monocyte
TNF
Ex-vivo TNF
TNF
TNF
Impaired ex-vivo LPS-induced TNF production
Immunoparalysis: Pittsburgh-Columbus Study
• Immunoparalysis: Ex-vivo TNFα production
< 200 pg/ml for more than 3 days
• Combined data sets from children with MODS
at Nationwide Children’s Hospital (30) and
Children’s Hospital of Pittsburgh (40).
Immune function monitored serially.
• Immunoparalysis incidence: 24/70 (34%)
Immunoparalysis in the PICU
• A state of prolonged immunoparalysis was associated
with increased relative risks for the development of
adverse outcomes in children with MODS:
Relative risks (RR) with 95%CI if immunoparalysis is
present for more than 3 days:
Outcome
RR with 95%CI
Nosocomial Sepsis
3.3 (1.8 – 6.0)
Death
5.8 (2.1 – 16)
Ex vivo TNF response
(pg/ml)
*
1800
1400
1000
600
*
200
Day 1
Day 3
Day 7
Day 14
Pediatric MODS patients who developed nosocomial sepsis (■) had lower ex vivo
TNFα production over time compared to those without nosocomial sepsis (▲).
Ex vivo TNF response
(pg/ml)
1600
*
1400
1200
1000
800
*
*
600
400
200
0
Day 1
Day 3
Day 7
Day 14
Pediatric MODS patients who died (■) had lower ex vivo TNFα production over time
compared to those who survived (▲).
Immunoparalysis in other ICU settings
Immunoparalysis doesn’t just
happen after sepsis!
In 36 children undergoing CPB, reduction in ex vivo TNFα response
was associated with the development of SIRS/sepsis.
PICUFlu Sites in the LPS-stimulation Study
10000
p = 0.002
*
1000
ve
vi
Su
r
D
d
100
ea
d
Ex vivo TNF production
(pg/ml)
The innate immune response and death
• Critically ill children who died (n = 6) had lower ex vivo LPS-induced TNFα
production capacity than did children who survived (n = 71).
• This remained true when controlling for age, PRISM, steroid use, and baseline
immune compromise (p = 0.0005, multivariable regression).
Ex vivo TNF production
(pg/ml)
Critical Trauma at NCH
p = 0.003; ANOVA
2500
2000
1500
*
control median
and
25th-75th percentile
**
1000
500
0
1-2
3-4
5-6
Post-trauma Day
n = 60, 13 developed nosocomial sepsis, 47 did not.
So what?
Evidence for role of immunomodulation
• Döcke et al (Nature Medicine, 1997)
– Survival in 6 of 9 adult patients with sepsisinduced immunoparalysis after treatment with
IFN.
• Nierhaus et al (Intensive Care Medicine, 2003)
– Normalization of monocyte function without
increased systemic inflammation in 9 adult
patients with severe sepsis and
immunoparalysis after treatment with
rhGM-CSF. Six patients survived.
GM-CSF in critically ill adults with SIRS
n = 15
30%
n = 18
30%
Rosenbloom et al, Chest, 2005
Monocyte HLA-DR expression improved in GM-CSF treated
group compared to placebo. Increased HLA-DR expression was
associated with clearance of infection (p=0.02)
• Prospective, randomized, placebo-controlled, double-blind
clinical trial
• Adults with severe sepsis/septic shock underwent immune
monitoring. Those with monocyte HLA-DR expression
< 8,000 molecules/cell for 2 days were randomized to get
SQ GM-CSF or placebo for 8 days.
• Monocyte HLA-DR expression, ex vivo LPS-induced TNFα
production, cytokines, and outcomes were measured
n = 19 subjects per group.
• There was no increase in systemic IL-6 levels in the
GM-CSF-treated group.
• There were no GM-CSF-related adverse events
reported.
• Patients in the GM-CSF arm were weaned from the
ventilator sooner. Underpowered to address
mortality.
The Good and The Bad?
Pro-inflammatory
PG
–
–
–
–
–
–
IL-1β
TNF
IL-6 (?)
IL-8
IL-17
Eicosanoids
Anti-inflammatory
–
–
–
–
–
–
IL-10
TGFβ
IL-11
sTNF receptor
IL-1ra
IL-4
What about other aspects
of immune function?
Cytopenias
• Problem: Critical
illness in the
setting of
leukopenia.
– Cancer
– Transplantation
– Drug-induced
– Sepsis-induced
Neutrophil
Lymphocyte
ANC < 500/mm3:
↑ incidence of adverse
outcomes in the
setting of malignancy
ALC < 1000/mm3
or ↓CD4 count :
↑ incidence of
adverse outcomes
in the settings of
HIV, malignancy,
transplantation
Lymphocyte apoptosis in adult sepsis
Hotchkiss, J Immunol, 2001
Lymphopenia is associated with negative outcomes in
pediatric MODS (Felmet, JI, 2005)
• Prolonged lymphopenia
– Occurred only in patients with
MODS (17/58 vs. 0/55 )
– Associated with secondary
infection with OR 5.5 (95% CI
1.7-17)
• controlling for immune
suppression and steroids
– Associated with death OR 6.8
(95% CI 1.3-34)
• controlling for immune
suppression, steroid use,
and PRISM score
B cell depletion in spleens of children
with MODS and lymphopenia
Summary: Restore Immune Balance
• Neutropenia and lymphopenia contribute to adverse outcomes in
the ICU and demand specific management:
– Neutropenia: GCSF, antimicrobial prophylaxis, removal of contributory
drugs, +/- WBC transfusion
– Lymphopenia: antimicrobial prophylaxis, removal of contributory drugs
• Innate and adaptive immune suppression is common in critically ill
children and we need to develop robust, standardized monitoring
regimens.
– Monocyte HLA-DR, Ex vivo TNFα production capacity
• Immunostimulatory therapies such as GM-CSF hold promise for
the reversal of immunoparalysis and should be subject of
prospective study.
Acknowledgements
The Research Institute at Nationwide Children’s Hospital
The Section of Pediatric Critical Care Medicine
Joseph A. Carcillo, MD
Mark D. Wewers, MD
NICHD
NHLBI
CANTREAT study
Lab protocol
Mark W. Hall, MD
Director, Immune Surveillance Laboratory
The Research Institute at Nationwide Children’s Hospital
Columbus, OH
Multicenter ex vivo stimulation
TNFα quantification
LPS-stimulation protocol development
Site training and education
Stimulation solution/kit manufacture
- Quality control
Batch-ship
supernatants
Ship stimulation
reagents monthly
Stimulation
procedure
CANTREAT
sites
What you will need....
• Gloves, PPE (e.g. lab coat)
• Blood tubes
– 4 ml green top
– 6 ml dark blue top
What you do....
• Collect blood
– 4 ml green top
– 6 ml dark blue top
Ex vivo stimulation (do in duplicate)
• What you need:
– Pipettes and tips
– Stimulation tubes
– Incubator
Ex vivo stimulation (do in duplicate)
• What you need:
– Microcentrifuge
– Empty storage tubes
Ex vivo stimulation (do in duplicate)
• 1. Within 30 minutes of collecting blood in the
green top (sodium heparin) tube....
• 2. Pipette 50μl of whole blood into a labelled
stimulation tube.
Ex vivo stimulation
• 3. Incubate for exactly 4 hours at 37°C.
• 4. Spin the tube in a microcentrifuge for
5 minutes at 1000 x g.
Ex vivo stimulation
• 5. Carefully pipette the supernatant into a clean,
labelled empty storage tube.
• 6. Freeze at -80° C.
While you are waiting....
What you need....
• Centrifuge
– 1000 x g
– 4 and 6 ml tubes
What you do....
• Spin at 1000 x g
for 5 minutes
What you will need....
• Pipettes and tips
• Empty storage tubes
What you do....
• Pipette off plasma/serum
in 500μl aliquots
into labelled empty
storage tubes.
• Freeze at -80° C.
YES!
NO!
No
Each subject
will have a
sheet of preprinted labels
that looks
something like
this.....
CANTREAT
01-001-1
Green Top tube
CANTREAT
01-001-1
Dk Blue Top tube
CANTREAT
01-001-1
LPS stim tube 1
CANTREAT
01-001-1
LPS stim tube 2
CANTREAT
01-001-1
Green Top plasma 1
CANTREAT
01-001-1
Green Top plasma 2
CANTREAT
01-001-1
Green Top plasma 3
CANTREAT
01-001-1
Green Top plasma 4
CANTREAT
01-001-1
Dk Blue Top serum 1
CANTREAT
01-001-1
Dk Blue Top serum 2
CANTREAT
01-001-1
Dk Blue Top serum 3
CANTREAT
01-001-1
Dk Blue Top serum 4
CANTREAT
01-001-1
LPS stim sup 1
CANTREAT
01-001-1
LPS stim sup 2
CANTREAT
01-001-1
Case Report Form
CANTREAT
01-001-2
Green Top tube
CANTREAT
01-001-2
Dk Blue Top tube
CANTREAT
01-001-2
LPS stim tube 1
CANTREAT
01-001-2
LPS stim tube 2
CANTREAT
01-001-2
Green Top plasma 1
CANTREAT
01-001-2
Green Top plasma 2
CANTREAT
01-001-2
Green Top plasma 3
CANTREAT
01-001-2
Green Top plasma 4
CANTREAT
01-001-2
Dk Blue Top serum 1
CANTREAT
01-001-2
Dk Blue Top serum 2
CANTREAT
01-001-2
Dk Blue Top serum 3
CANTREAT
01-001-2
Dk Blue Top serum 4
CANTREAT
01-001-2
LPS stim sup 1
CANTREAT
01-001-2
LPS stim sup 2
CANTREAT
01-001-2
Case Report Form
CANTREAT
01-001-3
Green Top tube
CANTREAT
01-001-3
Dk Blue Top tube
CANTREAT
01-001-3
LPS stim tube 1
CANTREAT
01-001-3
LPS stim tube 2
CANTREAT
01-001-3
Green Top plasma 1
CANTREAT
01-001-3
Green Top plasma 2
CANTREAT
01-001-3
Green Top plasma 3
CANTREAT
01-001-3
Green Top plasma 4
CANTREAT
01-001-3
Dk Blue Top serum 1
CANTREAT
01-001-3
Dk Blue Top serum 2
CANTREAT
01-001-3
Dk Blue Top serum 3
CANTREAT
01-001-3
Dk Blue Top serum 4
CANTREAT
01-001-3
LPS stim sup 1
CANTREAT
01-001-3
LPS stim sup 2
CANTREAT
01-001-3
Case Report Form
CANTREAT
01-001-4
Green Top tube
CANTREAT
01-001-4
Dk Blue Top tube
CANTREAT
01-001-4
LPS stim tube 1
CANTREAT
01-001-4
LPS stim tube 2
CANTREAT
01-001-4
Green Top plasma 1
CANTREAT
01-001-4
Green Top plasma 2
CANTREAT
01-001-4
Green Top plasma 3
CANTREAT
01-001-4
Green Top plasma 4
CANTREAT
01-001-4
Dk Blue Top serum 1
CANTREAT
01-001-4
Dk Blue Top serum 2
CANTREAT
01-001-4
Dk Blue Top serum 3
CANTREAT
01-001-4
Dk Blue Top serum 4
CANTREAT
01-001-4
LPS stim sup 1
CANTREAT
01-001-4
LPS stim sup 2
CANTREAT
01-001-4
Case Report Form
For one sampling time....
CANTREAT
01-001-1
Green Top tube
CANTREAT
01-001-1
Dk Blue Top tube
CANTREAT
01-001-1
LPS stim tube 1
CANTREAT
01-001-1
LPS stim tube 2
CANTREAT
01-001-1
Green Top plasma 1
CANTREAT
01-001-1
Green Top plasma 2
CANTREAT
01-001-1
Green Top plasma 3
CANTREAT
01-001-1
Green Top plasma 4
CANTREAT
01-001-1
Dk Blue Top serum 1
CANTREAT
01-001-1
Dk Blue Top serum 2
CANTREAT
01-001-1
Dk Blue Top serum 3
CANTREAT
01-001-1
Dk Blue Top serum 4
CANTREAT
01-001-1
LPS stim sup 1
CANTREAT
01-001-1
LPS stim sup 2
CANTREAT
01-001-1
Case Report Form
CANTREAT
01-001-2
Green Top tube
CANTREAT
01-001-2
Dk Blue Top tube
CANTREAT
01-001-2
Subject
number
LPS stim tube 1
CANTREAT
CANTREAT
01-001-2
01-001-2
Green Top plasma 1 Site number
Green Top plasma 2
CANTREAT
01-001-2
Dk Blue Top serum 1
CANTREAT
01-001-2
Dk Blue Top serum 2
CANTREAT
CANTREAT
01-001-2
01-001-1
Green
Top plasma 3
Green Top tube
CANTREAT
01-001-2
Dk Blue Top serum 3
CANTREAT
01-001-2
LPS stim tube 2
CANTREAT
01-001-2
Sample
number
Green
Top plasma
4
CANTREAT
01-001-2
Dk Blue Top serum 4
What you’ll get...
• First shipment:
–
–
–
–
–
–
–
24 stimulation tubes
Green top tubes
Dark Blue top tubes
Empty storage tubes
Labels
SOP
Incubation log
Save the boxes!
Stimulation tubes should be cool, not frozen.
Let me know if otherwise!
What you’ll get...
• Monthly shipments:
– 24 stimulation tubes
– Incubation log
– Green/blue tubes,
storage tubes
periodically
– If you need more....
just let me know!
Save the boxes!
Each quarter...
• Ship your samples back to me:
– Group samples from each subject into individual
plastic bags.
– Ship back in one of the monthly shipping
containers on dry ice. (2.5 kg should do)
– Overnight FedEx per Janet.....
Questions????