View
0
Download
0
Category
Preview:
Citation preview
SPACEFLIGHT AFFECTS IMMUNE CELLS
MARIANNE COGOLI-GREUTER, AUGUSTO COGOLI
Zero-g LifeTec GmbH
2007 ELGRA Symposium / XX National Meeting of AIMAS, Florence, September 4 - 7, 2007
STUDIES WITH IMMUNE CELLS IN CULTURE ⇒ ININ--VITRO EXPERIMENTSVITRO EXPERIMENTS- IN SPACE - ON EARTH IN MODELED µG
INVESTIGATIONS ON THE IMMUNE SYSTEM OF HUMANS IN SPACE ⇒ EXEX--VIVO / INVIVO / IN--VIVO STUDIESVIVO STUDIES
SUMMARY OF RESULTS OF SUMMARY OF RESULTS OF
25 YEARS IN25 YEARS IN--VITRO STUDIESVITRO STUDIES
REPORTS IN THE EARLY 70’S REVEALED:
LYMPHOCYTES FROM CREW MEMBERS ON US AND SOVIET SPACE MISSIONS HAD A DECREASED
RESPONSE TO MITOGEN AFTER FLIGHT
• KIMZEY ET AL. - APOLLO AND SKYLAB
• KONSTANTINOVA ET AL. - SOYUZ
EFFECTS OF WEIGHTLESSNESS ON LYMPHOCYTE EFFECTS OF WEIGHTLESSNESS ON LYMPHOCYTE PROLIFERATIONPROLIFERATION
EXPERIMENT PROPOSED BY A. COGOLI FOR SPACELAB-1
EFFECTOR CELL
RESTING CELL
MEMORY CELL
ACTIVATOR
BLAST CELL
ACTIVATION OF T-LYMPHOCYTES
ISOLATION OF LYMPHOCYTESISOLATION OF LYMPHOCYTES
Centrifugation
Blood on Ficoll Lymphocytes
RESULTS FROM SPACELAB-1 1983, Cogoli et al., Science, 225: 228- 30, 1984
ININ--VITRO EXVITRO EX--VIVO / INVIVO / IN--VIVOVIVO
IN-VITRO: ISOLATED CELLS IN CULTURE MEDIUM
EX-VIVO / IN-VIVO: IMMUNE CELLS ARE EXPOSED TO OTHER SYSTEMS, INCLUDING NEUROENDOCRINE SYSTEM
020406080
100
Act
ivat
ion
leve
l in
%
0 x gspace
1 x gspace
1 x gground
MITOGEN ACTIVATED LYMPHOCYTES MITOGEN ACTIVATED LYMPHOCYTES RESULTS FROM SPACELAB D-1, 1985
LYMPHOCYTES IN SPACELAB DLYMPHOCYTES IN SPACELAB D--1, 19851, 19853 DAYS AFTER MITOGENIC ACTIVATION
In-flight 1g control in µg samples reference centrifuge
FAST ROTATING CLINOSTAT
RANDOM POSITIONING MACHINE
APOPTOSIS IN ACTIVATED TAPOPTOSIS IN ACTIVATED T--CELLS IN MODELED CELLS IN MODELED µµGG
0
1
2
3
4
5
6
7
0 2 4 8 12 24 48 72
µg 1g
Ap
op
toti
c b
od
y fo
rmat
ion
*
**
Incubation time (hrs)
Maccarrone et al., J Leukocyte Biology, 2003
IN FLIGHT ACTIVATION OF WHOLE BLOOD IN FLIGHT ACTIVATION OF WHOLE BLOOD LYMPHOCYTES FROM 4 DONORSLYMPHOCYTES FROM 4 DONORS, SLS, SLS--1, 19911, 1991
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
donor 1 donor 2 donor 3 donor 4
0 g0.6 g1 g1.36 g1.75 g
Cogoli et al.,1996
3 SIGNALS ARE REQUIRED FOR FULL T CELL 3 SIGNALS ARE REQUIRED FOR FULL T CELL ACTIVATIONACTIVATION
Antigen/MHCanti-CD3ConA
TCR/CD3
CD28
IL-2R
B7 (Accessory cell / monocytes)
IL-2(Autocrine signal)
SIGNAL TRANSDUCTION IN T CELLS /1SIGNAL TRANSDUCTION IN T CELLS /1
TCR/CD3CD28
B7
G
Cytoskeleton
Motion
PLCPKC
c-fosc-jun AP-1
IL-2
DAG
Pi3
PIP2
IP3
ER
Ca2+
?
SIGNAL TRANSDUCTION IN T CELLS /2SIGNAL TRANSDUCTION IN T CELLS /2
TCR/CD3
IL-2RCD28
B7IL-2
IL-2
PROLIFERATION
JAK
STAT
STATIL-2 IL-2R
DELIVERY OF FIRST SIGNALDELIVERY OF FIRST SIGNAL
• BINDING OF MITOGEN CONCANAVALIN A NOT ALTERED IN µG
• CROSSLINKING OF THE RECEPTORS (PATCHING AND CAPPING) IS DELAYED
DELIVERY OF FIRST SIGNAL NOT DISTURBED BY GRAVITY UNLOADING
(J. Hatton, ESA)
DELIVERY OF SECOND SIGNALDELIVERY OF SECOND SIGNAL
SUSPENDED LYMPHOCYTES ARE HIGHLY MOTILE IN µG AND FORM AGGREGATES
1
55
µm
CELL A
CELL D
µm
55
1
Cell out of plane
CELL G
75 80 85 905
10
15
20
25
86 88 90 92 94 96 98 100 10228
30
32
34
36
38
40
42
µm
55
1
CELL B
125 130 135 140 145 1505
10
15
20
25
30
70 75 80 85 90 9560
65
70
75
80
85
1
55
µm
46 HOURS
78 HOURS
SINGLE CELLS (MAXUS 1)
12 HOURS 46 HOURS
CON A ACTIVATED CELLS (IML-2)
MOTILITY OF MONOCYTES IN MODELED MOTILITY OF MONOCYTES IN MODELED µµGGLOCOMOTION TRACKS OF J-111 ON GOLD-PARTICLE
COATED SLIDES
INCUBATION AT 37°C
1 HOUR
24 HOURS
CONTROL RPM
SINGEL CELL ATTACHED ON COLLOIDAL GOLD SINGEL CELL ATTACHED ON COLLOIDAL GOLD COATED COVER SLIDESCOATED COVER SLIDES
CONTROL RPM
INCUBATION TIME AT 37°C: 24 HOURS
0
20
40
60
80Fr
eque
ncy
%
1 2 3 4 5 6 7 8 9 10 11
Control 24h
FREQUENCY OF MIGRATION FREQUENCY OF MIGRATION
DISTANCE (DISTANCE (µµm) m) DURING DURING
24 HOURS (%)24 HOURS (%)
11 00--44
22 55--99
33 1010--1414
44 1515--1919
55 2020--2424
66 2525--2929
77 3030--3434
88 3535--3939
99 4040--4444
1010 4545--4949
1111 ≥≥500
20
40
60
80
Freq
uenc
y %
1 2 3 4 5 6 7 8 9 10 11
R.P.M. 24h
50
FF--ACTIN IN J111 CELLSACTIN IN J111 CELLS
INCUBATION AT 37° C
1 HOUR
24 HOURS
CONTROL RPM
STRUCTURE OF VIMENTIN IN JURKAT CELLSSTRUCTURE OF VIMENTIN IN JURKAT CELLS
MAXUS 1 (1991)
Ground control 30 sec at µg
MICROTUBULE MORPHOLOGY IN JURKAT CELLSMICROTUBULE MORPHOLOGY IN JURKAT CELLSLewis et al, 1998
Ground control fixed 4h after activation in µg
DELIVERY OF THE THIRD SIGNAL DELIVERY OF THE THIRD SIGNAL
TCR/CD3
IL-2RCD28
B7IL-2
IL-2
PROLIFERATION
JAK
STAT
STATIL-2 IL-2R
SECRETION OF ILSECRETION OF IL--1, IL1, IL--2 AND IL2 AND IL--2R OF CON A 2R OF CON A ACTIVATED LYMPHOCYTES (IMLACTIVATED LYMPHOCYTES (IML--2, 1994)2, 1994)
0
50
100
150
200
Sec
retio
n le
vel i
n %
0 x gspace
1 x gspace
1 x gground
IL-1IL-2IL-2R
IL-2Ralpha
IL-2Rbeta
IL-2Rgamma
IL-2 Receptor
IL-2
T cell membrane
α- and β-subunits: produced during activation process
χ-subunit: constitutively present on membrane
EARLY TRANSCRIPTIONAL RESPONSE OF T-LYMPHOCYTES TO MICROGRAVITY CONDITIONS
MASER 9, 2002
Gene Gene modifiedmodified** Type of Type of modulationmodulationILILββ11 inducedinduced
ILIL--4R4Rαα, IL, IL--13R13Rαα1, IL1, IL--13R13Rαα22 inducedinduced
cytokinecytokine A13A13 inducedinduced
ILIL--88 depresseddepressed
ILIL--2R2Rαα, IL, IL--7R7Rαα, IL, IL--10R10Rββ depresseddepressed
cytokinecytokine A2A2 depresseddepressed
cytokinecytokine A5 (RANTES)A5 (RANTES) depresseddepressed
* compared to inflight 1xg control Cogoli-Greuter et al.
EXPERIMENT LEUKINEXPERIMENT LEUKIN
REMEMBERINGCOLUMBIA STS-107
16TH JAN - 1ST FEB 2003
SEMISEMI--QUANTITATIVE RTPCRQUANTITATIVE RTPCR
T cells activated with Con A / anti CD28 / Experiment in modeled µG
IL-2RαIL-2IFNγ
IL-2RγIL-2Rβ
Blue dots: 217 genes aresignificantly upregulated
T cells activated for 4 h with Con A / anti-CD28
1g 4h
RPM 0h
RPM 4h
GRAVITY-DEPENDENT DISTRIBUTIONOF GENE EXPRESSION IN T CELLS ACTIVATED WITH CON A / ANTI CD28
99 genes upregulated above 2-fold
Drastic drop in gene expression
Little change in gene expression
Key genes and transcription factorsKey genes and transcription factors
IMPORTANT CONTRIBUTIONS TO THE UNDERSTANDING OF THE BEHAVIOUR OF IMMUNE CELLS IN SPACE AND SIMULATED MICROGRAVITY HAVE BEEN MADE BY THE TEAMS OF:
DIDIER SCHMITT
BEN HASHEMI
NEAL PELLIS
CLARENCE SAMS
MARIAN LEWIS
LAURENCE SCHAFFAR
LUDMILA BURAVKOVA
THE IMMUNE SYSTEM OF HUMANS IN SPACE
⇒ EXEX--VIVO / INVIVO / IN--VIVO STUDIESVIVO STUDIES
IMMUNE RESPONSES SHOWN TO BE ALTERED IMMUNE RESPONSES SHOWN TO BE ALTERED DURING OR AFTER SPACEFLIGHTDURING OR AFTER SPACEFLIGHT
• LEUKOCYTE BLASTOGENESIS – SEVERELY INHIBITED
• CYTOKINE PRODUCTION – INTERFERON-α/β INHIBITED
• LEUKOCYTE SUBSET DISTRIBUTION – REDUCTION IN T-CELLS, NK-CELLS, INCREASE IN NEUTROPHILS
• NATURAL KILLER CELL ACTIVITY - REDUCED
• DELAYED-TYPE HYPERSENSITIVE REACTIONS TO COMMON RECALL ANTIGENS - REDUCED
NOT ALTERED: CONTENT OF IMMUNOGLOBULINS
IMMUNOKITIMMUNOKIT flown on MIR(Mission 7, 8 and 9)
DELAYED-TYPE HYPERSENSITIVITY REACTION
Commercially available applicator for “Skin test” (Merieux)
adapted to space
"SKIN TEST" on 5 MIR COSMONAUTS"SKIN TEST" on 5 MIR COSMONAUTS
Score (mm)
05
1015202530
Pre-flight In-flight Post-flight
A
BC
D
E
EFFECT OF SPACEFLIGHT CONDITIONS EFFECT OF SPACEFLIGHT CONDITIONS ON LYMPHOCYTES OF DON LYMPHOCYTES OF D--1 ASTRONAUTS1 ASTRONAUTS
0
20
40
60
80
100
120
140
160
A B C
L-9 dL-2 dL+3 d at 1 gR+0 dR+7 dR+13 d
Crew member
Mitotic indexin cpm
Baseline
MONOCYTE DIFFERENTIATION IN SPACEFLIGHTMONOCYTE DIFFERENTIATION IN SPACEFLIGHT
•• BLOOD SPECIMENS FROM 25 ASTRONAUTS BLOOD SPECIMENS FROM 25 ASTRONAUTS COLLECTEDCOLLECTED POST FLIGHT: POST FLIGHT: MONOCYTES EXHIBITED REDUCTIONS IN ABILITY TO MONOCYTES EXHIBITED REDUCTIONS IN ABILITY TO ENGULF E. COLIENGULF E. COLIELICIT AN OXIDATIVE BURST AND DEGRANULATE ELICIT AN OXIDATIVE BURST AND DEGRANULATE PHAGOCYTIC INDEX WAS SIGNIFICANTLY REDUCEDPHAGOCYTIC INDEX WAS SIGNIFICANTLY REDUCED
•• BLOOD SPECIMENS FROMBLOOD SPECIMENS FROM 27 RETURNING 27 RETURNING ASTRONAUTS: ASTRONAUTS: SIGNIFICANT DECREASE IN PERCENTAGE OF SIGNIFICANT DECREASE IN PERCENTAGE OF CD14+CD16+ MONOCYTE DIFFERENTIATION CD14+CD16+ MONOCYTE DIFFERENTIATION MARKERS IN ALL SUBJECTS TESTEDMARKERS IN ALL SUBJECTS TESTED
Slide provided by M. Hughes-Fulford
CHANGES IN MONOCYTE POPULATION AND CHANGES IN MONOCYTE POPULATION AND PHAGOCYTIC POTENTIAL IN ASTRONAUTSPHAGOCYTIC POTENTIAL IN ASTRONAUTS
16 Day Missions, 12 AstronautsSmall but significantchange in monocyte
5-11 Day Missions, 25 AstronautsLarge and significant change in
Phagocytic potential
Graphed from data in Stowe et al., Aviation, Space and Envir. Med. 2003 Dec;74(19):1281-1284.
Graphed from data in Kaur et al., Brain Behav. Immun. 2005 Nov;19(6):547-54.
Slide provided by M. Hughes-Fulford
INFECTION IN SPACEFLIGHTINFECTION IN SPACEFLIGHT
•• 15 OF THE 29 APOLLO ASTRONAUTS HAD A 15 OF THE 29 APOLLO ASTRONAUTS HAD A BACTERIAL OR VIRAL INFECTION EITHER BACTERIAL OR VIRAL INFECTION EITHER DURING FLIGHT OR WITHIN ONE WEEK AFTER DURING FLIGHT OR WITHIN ONE WEEK AFTER FLIGHTFLIGHT
•• APOLLO 13, ONE ASTRONAUT DEVELOPED AN APOLLO 13, ONE ASTRONAUT DEVELOPED AN URINARY TRACT INFECTION DURING THE FLIGHTURINARY TRACT INFECTION DURING THE FLIGHT
⇒⇒ COUNTERMEASURER: CREW ISOLATION COUNTERMEASURER: CREW ISOLATION BEFORE SPABEFORE SPACEFLIGHTCEFLIGHT
Slide provided by M. Hughes-Fulford
LATENT VIRUS REACTIVATIONLATENT VIRUS REACTIVATION
• VARICELLA ZOSTER VIRUS PRESENT IN 30% OF 200 SALIVA SAMPLES FROM 8 ASTRONAUTS COLLECTED IN AND POSTFLIGHT
• EPSTEIN-BARR VIRUS DNA IN 16% OF INFLIGHT SAMPLES FROM 25 ASTRONAUTS (SIGNIFICANTLY GREATER THAN PRE/POSTFLIGHT)
• 11 OF 28 ASTRONAUTS SHOWED POSTFLIGHT EPSTEIN-BARR VIRUS ACTIVATION
SPACE FLIGHT SPACE FLIGHT ““STRESSORSSTRESSORS”” POSSIBLY ASSOCIATED POSSIBLY ASSOCIATED WITH ALTERED IMMUNE RESPONSEWITH ALTERED IMMUNE RESPONSE
• INTENSE PRE-LAUNCH TRAINING• LAUNCH• MICROGRAVITY• RADIATION• MICROBIAL CONTAMINATION• SLEEP DISRUPTION / CHANGES IN CIRCADIAN
RHYTHM• PSYCHOLOGICAL DYNAMICS OF CONFINED
ENVIRONMENT• PRESSURE OF HIGH PERFORMANCE
EXPLORATION BEYOND LOW EARTH ORBITEXPLORATION BEYOND LOW EARTH ORBIT
POTENTIAL NEW RISKS FOR EXPLORATION POTENTIAL NEW RISKS FOR EXPLORATION MISSIONSMISSIONS
• NEW FORMS OF RADIATION
• CREW ISOLATION WITH NO CHANGE OF RAPID RESCUE OR TREATMENT
• ALTERED IMMUNE SYSTEM WITH POTENTIAL ALTERED VIIRULENCE OF PATHOGENES
• ALTERED NUTRITION
ACKNOWLEDGEMENT
Millie Hughes-Fulford and her team, Univ. California, S. Francisco
Proto Pippia, M.A. Meloni, G. Galleri, G. Pani, A. Saba, L. Sciola, A. Spano, Univ. Sassari
Mauro Macarrone and his team, Univ. Teramo
Otfried Müller, Univ. Bern
Rodolfo Negri and his team, Univ. Rome
Alex Tschopp, Pia Fuchs-Bislin, Birgitt Bechler, Thomas Schopper, ETHZ
Isabelle Walther, Zero-g LifeTec GmbH
The teams of ESA, NASA, IBMP MoscowThe astronauts of SL-1, D-1, SLS-1, IML-1, IML-2The cosmonauts of MIR and Soyuz/ISS
The financial support: ETHZ, ESA/PRODEX, ASI, NASA, SNSF
I THANK YOU FOR YOUR ATTENTION
SPACE AGGREGATE 78 Hours after addition of Con ANIZEMI / Spacelab IML-2, 1994
Cogoli-Greuter et al., 1996
SINGLE CELL MOVING OUT
OF AGGREGATE
MET 3-18:44:01 3-18:48:00 3-18:52:00
3-18:56:00 3-19:00:35
Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change
0.1 1 5.0
Chemokine Receptors
IL2CD28
IL2Rα
CD45
PLCPLC
DAGDAG
PKCPKC
JNKJNK
VAVRAC
ELKELKNFKB1
JUN
RAFRAF
MEKMEK
ERK 1/2ERK 1/2
RASRAS
[Ca++]
IPIP33
NFATNFAT
P38P38MAPKMAPK
con A α-CD28 Ab
CD4
JAK1/3JAK1/3
STAT5STAT5
MYCMYCPI3K
AKT
E2F6
Responsive Genes:BCL2CCL20IER3
A1
JAK
STAT1
IFNγ
IFNγR
TCR/CD3
CCL20CSF2CXCL10WARS
AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2
BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12
6
A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2
We performed a series of experiments using:
Forskolin as PKA activatorH89 as PKA inhibitorGF109203X as PKC inhibitorU0126 as MEK1 inhibitorWestern blot with anti-p-CREBEMSA assay for activated transcription factor AP1
AP-1
Gq
cAMPcAMP
CREB1CREB1
Gαs
PKAPKA
Gαi
TRAF1
BIRC3
1214
69
TNFRSF
TNFSF
Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change
0.1 1 5.0
Chemokine Receptors
IL2CD28
IL2Rα
CD45
PLCPLC
DAGDAG
PKCPKC
JNKJNK
VAVRAC
ELKELKNFKB1
JUN
RAFRAF
MEKMEK
ERK 1/2ERK 1/2
RASRAS
[Ca++]
IPIP33
NFATNFAT
P38P38MAPKMAPK
con A α-CD28 Ab
CD4
JAK1/3JAK1/3
STAT5STAT5
MYCMYCPI3K
AKT
E2F6
Responsive Genes:BCL2CCL20IER3
A1
JAK
STAT1
IFNγ
IFNγR
TCR/CD3
CCL20CSF2CXCL10WARS
AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2
BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12
6
A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2
Conclusions:
1. PKA in addition to PKC and MAPK pathways plays a role in Early T cell activationand induction of IL-2, IL-2Rα and IFNγ Gene expression
2. ERK2 is phosphorylated within minutes of activationwhile P38 and JNK remain unchanged
3. Probable cross-talk between the MEK and PKA pathways and between the PKC and PKA pathways
AP-1
Gq
cAMPcAMP
CREB1CREB1
Gαs
PKAPKA
Gαi
TRAF1
BIRC3
1214
69
TNFRSF
TNFSF
Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change
0.1 1 5.0
Chemokine Receptors
IL2CD28
IL2Rα
CD45
PLCPLC
DAGDAG
PKCPKC
JNKJNK
VAVRAC
ELKELKNFKB1
JUN
RAFRAF
MEKMEK
ERK 1/2ERK 1/2
RASRAS
[Ca++]
IPIP33
NFATNFAT
P38P38MAPKMAPK
con A α-CD28 Ab
CD4
JAK1/3JAK1/3
STAT5STAT5
MYCMYCPI3K
AKT
E2F6
Responsive Genes:BCL2CCL20IER3
A1
JAK
STAT1
IFNγ
IFNγR
TCR/CD3
Gq
cAMPcAMP
CREB1CREB1
Gαs
PKAPKA
Gαi
CCL20CSF2CXCL10WARS
AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2
BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12
6
A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2
AP-1
TRAF1
BIRC3
1214
69
TNFRSF
TNFSF
Recommended