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Life Sciences Research Laboratories/SD3, NASA-Johnson Space Center, Houston, Texas 77058, USA
1Correspondence: Life Sciences Research Laboratories/SD3, NASA—Johnson Space Center, Houston, TX 77058, USA. E-mail: csams{at}ems.jsc.nasa.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONAlternative modes of T...Gravity effects on T...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: microgravity culture • clinostat • DNA synthesis • CD69 • CD25
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONAlternative modes of T...Gravity effects on T...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Similarly, ground-based
studies of lymphocytes in clinorotation demonstrate a dramatic decrease
in tritiated thymidine incorporation after 72 h of activation.
These studies rely on cell culture methods using clinostats that
simulate some of the physical effects of spaceflight by providing a
vector-averaged reduction of the apparent gravity on cells in culture
without significant shear forces (3
4
5)
. Although these
investigations have clearly demonstrated inhibition of DNA synthesis in
microgravity culture and in clinorotation, little is known about the
mechanism by which this inhibition occurs. To elucidate the
mechanism(s) responsible for this inhibition, we have investigated the
progression of cellular events during the first 24 h of activation
in human peripheral T cells.
Activation of T cells plays an important role in various humoral and
immunological responses. Because of their critical role in the immune
response, circulating T cells are maintained in a resting or G0 state
of the cell cycle, and their growth and differentiation are strictly
regulated (6
7
8)
. The engagement and aggregation of the T
cell receptor complex (TCR) on the plasma membrane of the T cell is an
important signaling step that leads to cell cycle entry. Signaling via
the TCR induces phosphorylation of cellular substrates including the
TCR itself, phosphatidyl inositol hydrolysis, internalization of the
TCR from the plasma membrane (9)
, and activation of
cellular events that lead to a proliferative response within 72 h.
The entry of T cells into the cell cycle and their progression through
G1 is accompanied by the activation of numerous cellular events,
including gene transcription and the surface expression of activation
markers on the plasma membrane. The earliest surface activation marker
is the CD69 molecule, which is expressed within a few hours of
activation and does not initially require new RNA or protein synthesis
(10)
. Upon full activation of pathways necessary for gene
expression, the receptor for interleukin 2 (IL-2: CD25) is expressed on
the plasma membrane, an activation event that is crucial to regulating
the immune response. Surface expression of CD25 requires gene
transcription beginning within 2 h after TCR stimulation
(11)
, the receptor is rapidly expressed on the surface
after activation (12)
. In the current study, measurements
of the surface expression of these activation markers were used to
evaluate the entry of T cells into and their progression through the G1
phase of the cell cycle.
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Alternative modes of T cell activation |
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TOPABSTRACTINTRODUCTIONAlternative modes of T...Gravity effects on T...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
In vitro, peripheral blood mononuclear cells (PBMCs) can be
activated by anti-CD3 monoclonal antibodies (mAb) or lectins
(13
14
15)
. This mode of activation requires cellular
contact and intercellular signaling between T cells and accessory cells
(16)
. Alternatively, aggregation of the TCR and activation
of T cells can be induced by cell-sized beads that are coated with
antibodies specific for CD3. Stimulation of T cells with
bead-immobilized anti-CD3 can induce strong, sustained calcium
signaling (17
, 18)
, prolonged protein kinase C (PKC)
activation (19)
, expression of activation markers
(18)
, and proliferative response (20)
. A
major advantage of the bead activation method is its ability to
activate purified T cells in the absence of any intercellular signaling
requirements and interactions with accessory cells.
The binding of ligands to accessory molecules on the surface of T cells
has been shown to augment the biochemical signals provided by the TCR.
While full activation of T cells via the TCR alone requires
`triggering' of ~8000 TCRs, engagement of costimulatory molecules
can reduce this threshold to ~1500 TCRs (21)
. Perhaps
the most well-characterized costimulatory receptor on T cells is the
CD28 molecule (22
23
24)
. The engagement and aggregation of
CD28 regulates a signal transduction pathway that is clearly distinct
from those stimulated by the TCR complex. Costimulation of CD28 in
conjunction with immobilized anti-CD3 mAb can dramatically augment T
cell responses such as cytokine production and proliferation
(22
23
24)
. Therefore, costimulation of CD28 in conjunction
with suboptimal stimulation of CD3 by surface immobilized mAb (for
example, Bead-OKT3/CD28) can be used to elicit markedly augmented T
cell activation responses. We have used this method of activation to
determine whether costimulation with CD28 can overcome the inhibition
of T cell activation in microgravity culture.
The combination of phorbol ester and calcium ionophore bypasses surface
TCR engagement and cross-linking requirements and directly activates
intracellular signaling pathways leading to T cell activation. Phorbol
esters such as phorbol dibutyrate (PDB) are diacyl glycerol analogs
that stimulate PKC activity, while calcium ionophores such as ionomycin
(I) are membrane channels that integrate into the plasma membrane,
resulting in an increase in intracellular calcium levels due to the
higher extracellular calcium concentration. The increase in PKC
activity and cytoplasmic calcium mimics the downstream effects of PI
hydrolysis and directly activates intracellular signal transduction
pathways that lead to T cell activation (25)
.
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Gravity effects on T cell activation |
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. In addition, it has
been reported that binding of phorbol esters occurs normally during
periods of microgravity in parabolic aircraft flights
(26)
. These results indicate that diffusion and binding of
mitogens to lymphocytes are not greatly affected during microgravity
culture. Furthermore, electron microscopy observations of lymphocyte
activation indicate that surface contact between monocytes and
lymphocytes does occur in microgravity culture (27
28
29)
.
Therefore, the inhibition of T cell activation most likely occurs
downstream of cell–cell interaction and ligand binding, and prior to
DNA synthesis. In the current study, we investigate the progression of human peripheral T cell activation in clinorotation and microgravity culture in order to define the mechanism(s) responsible for the lack of T cell responsiveness in microgravity culture. These experiments used several alternative modes of T cell activation to identify which cellular responses are altered in clinorotation and microgravity culture and to determine the signaling requirements for effective cellular activation and response under these culture conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONAlternative modes of T...Gravity effects on T...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several experiments were performed to characterize the activator
concentration dependence of T cell activation. Accordingly, the optimum
concentration of activators was chosen as follows. Activation of PBMCs
was performed using a final concentration of 2 µg/ml
phytohemagglutinin (PHA-L, Sigma Chemical Co., St. Louis, Mo.) or 0.15
µg/ml FITC (fluorescein isothiocyanate) anti-Leu4 (Leu4), a
monoclonal antibody specific for the CD3-
subunit of the TCR complex
(Becton Dickinson, San Jose, Calif.) (30)
. For activation
of purified T cells with phorbol ester and calcium ionophore, 5 ng/ml
phorbol myristate acetate (PMA) or PDB and/or 0.5 µM ionomycin (I)
(Sigma) were used as indicated. For activation of purified T cells with
bead-immobilized antibodies, carboxylated beads (6.2 µm;
Polysciences, Warrington Pa.) were covalently coupled with goat
anti-mouse kappa antibody (Jackson ImmunoResearch, West Grove, Pa.), by
carbodiimide modification, followed by incubation with specific mouse
antibody, as described earlier (17
, 31)
. Anti-Leu4 and
anti-CD28 were purchased from Becton Dickinson. Orthoclone OKT3 (OKT3)
is an anti-CD3 monoclonal antibody and was generously provided by Carl
Kincaid at Ortho Biotech (Raritan, N.J.). In experiments using beads to
stimulate T cells, the final concentration of beads was 5 x
106 beads/ml whereas the final concentration of
cells was at 1 x 106 cells/ml.
Two separate donors were used for the microgravity experiments on space shuttle flights STS-81 and STS-84. The activation responses of T cells from those donors were evaluated prior to the flight experiment and exhibited positive activation responses with all of the activation modes used.
Clinostat culture (clinorotation) of T cells
The clinostat model system (clinorotation) is a ground-based
method for providing a vector-averaged reduction in the apparent
gravity on the cell culture (4
, 5)
. Clinorotation of
samples was performed at 37°C in 1.2 ml cryovials (Corning Glass
Works, Corning N.Y.) filled to capacity. These cryovials underwent
rotation at 30 rpm about the longitudinal axis (i.e., the axis of
rotation was oriented perpendicular to the gravity vector). Static
samples were maintained at 37°C in parallel for 1 g
control.
Microgravity culture of T cells
The term microgravity culture is used to refer to the culture
environment within a spacecraft in orbit around the Earth. The
microgravity experiments presented in this study were performed in the
Biorack facility of space shuttle flights STS-81 and STS-84. The
details of the experimental hardware and in-flight operations are
presented elsewhere (32)
. Briefly, cells were isolated in
the Hanger-L Facility of Kennedy Space Center and loaded in the flight
cell culture hardware (32
, 33)
. To ensure minimum elapsed
time between blood isolation and activation of T cells in microgravity,
the cell and hardware preparation procedures were initiated 26 h
prior to launch. This provided just enough time to complete the
procedures and hand over the hardware to the Biorack Team 17 h
prior to launch for transport to the space shuttle.
In-flight, cells were cultured in the 37°C incubator of the Biorack facility either in microgravity or on the 1 g reference centrifuge for a preincubation period (5 h:30 min for STS-81 and 6 h for STS-84). The radius of the centrifuge and its rotation rate were such that the apparent acceleration force on the cell culture was equivalent to unit gravity (1 g). The addition of activating reagents was performed in the glove box facility of Biorack, after which the cells were returned to their appropriate location in the 37°C incubator for ~24 h (22 h for STS-81 and 25 h for STS-84). Cells were then washed and fixed with paraformaldehyde (paraformaldehyde in phosphate-buffered saline (PBS) with 2 mM EGTA at pH 7.4). The fixation time for the samples was dictated by the docking timeline requirements of the space shuttle and the space station Mir. The final fixation conditions were 1 h:40 min at 1.8% fixative for STS-81 and 19 h at 1% fixative for STS-84. Cells were washed at the end of the fixation period and were stored at 4°C for return to Earth. Immunofluorescence labeling and flow cytometry of samples were performed postflight at Johnson Space Center.
Scanning electron microscopy
Cells were cultured in clinorotation or at 1 g for
17 h and fixed in 50% glutaraldehyde solution. Samples were
captured on a silver mesh, critical point dried, and gold coated for
microscopy. Images were acquired on a JEOL JSM-T330A scanning electron
microscope.
Measurement of cell proliferation and S-phase entry
The fraction of cells in S-phase was determined according
to Becton Dickinson protocol by pulsing 200 µl of cells with 20 µl
100 µM 5-bromo 2 deoxyuridine (BrdU; Sigma) in 96-well round bottom
plates for 2 h at 37°C. Cells were immediately washed in 3 ml
cold PBS, spun down, fixed in 70% cold ethanol, and stored at 4°C.
The fixed cells were stained with anti-BrdU-FITC to analyze total BrdU
incorporation, and propidium iodide was used to measure total DNA
content. Determination of percent cells in S phase was made using flow
cytometry to score the number of BrdU-positive cells (34)
.
Surface expression of activation markers and TCR internalization
Cells were harvested at the times indicated after activation and
stained for the CD3 using FITC-Leu4 and for surface expression of CD69
and CD25 (IL-2 receptor) using phycoerythrin-labeled (PE) anti-CD69 and
anti-CD25 antibodies (Becton Dickinson). Cells were stained for at
least 30 min at 4°C, washed with 3 ml PBS containing 1% serum and
0.1% sodium azide by centrifugation at 400 g for 10 min.
Cells were then lightly vortexed and fixed with 1% paraformaldehyde
(Polysciences, Inc.) in PBS. Flow cytometry gating was used (Coulter
EPICS XL cytometer, Coulter Corporation, Miami, Fla.) to analyze the T
cells (i.e., CD3-positive cells) for expression of CD69 and CD25.
Control samples were stained in an identical manner with
FITC-CD3/PE-IgG1 to determine nonspecific staining and background
levels of surface marker expression. Results are reported as percentage
of CD3-positive lymphocytes that expressed the surface marker CD69 or
CD25. For TCR internalization experiments, the level of CD3 on the
plasma membrane of the T cell population is reported as the `relative
CD3 fluorescence' and is the mean channel from the log amplifier
histogram of the fluorescence for FITC anti-Leu4.
Determination of G1 cell cycle entry
Cell cycle analysis was performed on activated T cells using
differential staining of DNA and RNA with acridine orange (AO)
(35)
. Briefly, PBMCs were isolated and activated in static
or clinostat culture with soluble Leu-4 for the amount of time
indicated. Cells were washed in cold PBS containing 0.1% fetal calf
serum/0.01% sodium azide and fixed in cold 70% ethanol. For cell
cycle determination, the cell samples were decanted to remove ethanol
and treated with 0.1% Triton X-100/0.08 N HCl/0.15 N NaCl at 4°C.
Samples were then stained with 20 µM AO/1 mM EDTA/0.15 M
NaCl/phosphate citric acid buffer, pH 6.0. Cells were analyzed on a
Coulter EPICS XL flow cytometer with 488 nm excitation, 460 nm bandpass
filter for detection of green (DNA) fluorescence and 640 nm long-pass
filter for the red (RNA) fluorescence. Histograms of total cellular DNA
and RNA fluorescence were used to resolve distinct populations in the
G0 and G1 stage of the cell cycle. Data are presented as percent of
cells in G1 = G1/(G0+1), where G0 and G1 denote the number of
cells in each population.
Intracellular levels of IL-1
The intracellular levels of the IL-1 cytokine were measured in
monocytes using the FastImmune intracellular staining system by Becton
Dickinson. Briefly, PBMCs were stimulated by Leu4 or PDB/I for 4 h
in the presence of 10 µg/ml Brefeldin A to inhibit the secretion of
IL-1. Cells were then labeled with FITC anti-CD14 for gating on the
monocyte population. Subsequently, cells were fixed, permeabilized, and
labeled with PE-anti-IL-1 (Becton Dickinson). The fraction of monocytes
that synthesized IL-1 was determined by flow cytometry and gating on
the CD14+ population to measure the fraction of IL-1+ cells.
Data analysis
Unless otherwise noted, the data presented represent at least
three experiments, and the error bars are the standard error of mean
from duplicate samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONAlternative modes of T...Gravity effects on T...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, stimulation of cells in clinorotation shows a fivefold reduction in
BrdU incorporation compared to the 1 g control. Both PHA and
Leu4 show similar reduction in BrdU incorporation during clinorotation.
These findings are consistent with earlier studies indicating a lack of
DNA synthesis in T cells activated with mitogenic lectins
(2)
, and demonstrate a clinostat-induced inhibition of T
cell activation prior to the S-phase entry of the cell cycle. To
identify the mechanism responsible for the lack of proliferative
response, it was necessary to characterize the progression of T cell
activation and cellular responses earlier in the activation pathway.
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Expression of activation markers and cell cycle entry
The progression of T cell activation during the first 24 h of
culture was examined by evaluating the surface expression of activation
markers after stimulation of PBMCs with Leu4. As shown in Fig. 2
A, the fraction of cells expressing CD25 at 1 g
began to rapidly increase 6 h after activation and reached a
maximum of 52% at ~12 h. In comparison, the expression of CD25 in
clinorotation closely followed that of the unactivated control levels
and was only slightly elevated at 24 h. Similarly, the fraction of
cells expressing CD69 at 1 g (Fig. 2B
) rapidly
increased to a maximum level of ~80% within the first 3 h of
activation. However, stimulation of cells in clinorotation resulted in
little or no expression of CD69 throughout the 24 h period
comparable to the unactivated control. The failure of these two
important receptors to be expressed even after 24 h of stimulation
indicates the existence of an early block to activation in
clinorotation.
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To determine whether stimulation of PBMCs in clinorotation induces the
transition of T cells from the G0 to G1 stage of the cell cycle, time
course analysis of total cellular RNA was performed using acridine
orange staining and flow cytometry. As shown in Fig. 2C
, the
fraction of cells in the G1 stage of the cell cycle increased steadily
starting at 6 h after activation in the 1 g control.
For cells stimulated in clinorotation, the fraction of cells in G1
remained at the level of unactivated control for the duration of the
24 h time course. Based on these findings, it would appear that
the inhibition of T cell activation occurs within the first few hours
of activation.
TCR internalization by T cells during clinorotation
One of the earliest functional responses of T cells to activation
is the internalization of the TCR from the plasma membrane. Indeed, it
has been shown that the degree of TCR internalization can be used as a
measure of T cell activation (9)
. To determine the degree
of TCR internalization during clinorotation, PBMCs were stimulated with
soluble Leu4 and cultured in clinorotation and at 1 g for up
to 24 h. As shown in Fig. 3
, measurement of the mean fluorescence of cells labeled with FITC-Leu4
indicates that the internalization of TCR occurred more rapidly during
the first 12 h of culture in both clinorotation and 1
g, followed by a small degree of internalization during the
ensuing 12 h. The degree of TCR internalization in clinorotation
reached that of the 1 g control by 24 h of culture, at
which time the majority of TCR were internalized relative to the
unstimulated control. These results indicate that the early signaling
required for TCR internalization does occur during clinorotation.
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IL-1 synthesis by monocytes
It has been postulated that the inhibition of T cell activation in
clinorotation may simply be due to lack of cell–cell contact.
Activation of PBMCs with soluble Leu4 requires the interaction of T
cells with monocytes and results in intercellular signal transduction,
which leads to IL-1 production by monocytes (36)
. To
determine whether clinorotation inhibits the intercellular interactions
and signaling between T cells and monocytes, we measured the
intracellular levels of the IL-1 cytokine in monocytes. As shown in
Fig. 4
, activation of PBMCs with Leu4 resulted in equivalent levels of IL-1
synthesis in clinorotation and in the 1 g control. This
result indicates that T cell/monocyte interactions occur during
clinorotation resulting in monocyte signaling and is consistent with
our observation that the secretion of IL-1 in clinorotation is equal to
or greater than that in the 1 g control (data not shown).
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Expression of activation markers by direct stimulation of
intracellular pathways with PMA/I
To determine whether bypassing TCR mediated signaling and directly
activating intracellular signal transduction pathways can overcome
clinostat-induced arrest of G1 entry in T cells, we evaluated the
progression of T cells stimulated with a combination of phorbol ester
and calcium ionophore. As shown in Fig. 5
(lane 2), the combination of PMA/I is a potent activator of T cell
surface marker expression in both clinorotation and 1 g.
Experiments were then carried out to determine whether the
clinostat-induced inhibition of TCR-mediated activation can be rescued
by the additional stimulation of only one of these pathways. PBMCs were
stimulated with soluble Leu4 (lane 1) or PHA (inset lane 1), in the
presence of either PMA or ionomycin, and the surface expression of
activation markers was measured after 24 h of culture. The
addition of ionomycin to anti-CD3- (lane 4) or PHA- (inset, lane 3)
stimulated cells did not rescue the inhibition observed in
clinorotation. It is interesting, however, that addition of PMA to
soluble anti-CD3 (lane 3) or PHA (inset lane 2) stimulation induced the
surface expression of both CD25 and CD69 (CD69 data not shown).
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Activation of purified T cells by bead-immobilized antibodies
Direct investigation of the response of T cells in the absence of
accessory cell requirements can be performed by using bead-immobilized
ligands to activate column-purified human peripheral T cells. In
seeking to find a bead activation method that can elicit a very strong
activation response from T cells, we tested several bead preparations
with combinations of Leu4, OKT3, and CD28. Since Bead-Leu4 elicited a
good activation response, which was better than Bead-OKT3 at 1
g, it was used for characterizing T cell responses in
clinorotation and microgravity culture. For costimulation with
anti-CD28, however, Bead-OKT3/CD28 elicited an unusually strong CD25
expression and BrdU incorporation that surpassed Bead-Leu4/CD28 at 1
g.
T cells circulating in the blood are in the resting (G0) stage of the
cell cycle; scanning electron microscopy of these cells shows the
characteristic microvilli-decorated membrane morphology shown in
Fig. 6
. Stimulation of purified T cells with Bead-Leu4 induces TCR-mediated
activation of intracellular pathways leading to cellular activation and
response. These events are accompanied by alterations in the cell's
cytoskeletal systems that result in dramatic reorganization of the
plasma membrane, disappearance of the microvilli, and the appearance of
membrane ruffles in clinorotation (Fig. 6B
) and the 1
g control (Fig. 6C
).
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To determine whether the inhibition of T cell activation in
clinorotation can be overcome by stimulation with surface-attached
ligands, time course experiments were performed with purified T cells
and Bead-Leu4. As shown in Fig. 7
A, activation of T cells at 1 g resulted in
maximum expression of CD69 by 24 h of culture; the fraction of T
cells expressing CD69 remained at ~80% for the duration of the
experiment. Activation of T cells in clinorotation resulted in
expression of CD69 by greater than 50% of the cells within 24 h,
and the fraction of CD69-positive cells in clinorotation increased to
that of the 1 g control by 72 h. Analysis of total
cellular RNA (Fig. 7B
) indicates that the fraction of cells
entering the cell cycle in clinorotation increased steadily with time,
as did activation of cells at 1 g.
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Costimulation of CD28 and the TCR complex can induce strong
proliferative responses in purified T cells which can surpass even that
elicited by PDB/I. Activation of T cells with Bead-OKT3/CD28 or PDB/I
initiated BrdU incorporation by 30 h after activation, as shown in
Fig. 8
. Bead-OKT3/CD28 in clinorotation induced levels of BrdU incorporation
that were higher than those in 1 g. In addition, Bead-Leu4
(data not shown) and Bead-OKT3/CD28 (Fig. 8
, inset) elicited strong
surface expression of CD25 in 1 g and in clinorotation.
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Activation response of T cells in microgravity culture during
spaceflight
Our results from the ground-based model system of clinostats
indicate that the activation response of T cells is inhibited in
clinorotation and that this inhibition can be overcome with PDB/I and
bead-immobilized anti-CD3. To determine whether the inhibition of T
cell activation in microgravity culture can be overcome with these
modes of activation, experiments were performed in the Biorack facility
of space shuttle flights STS-81 and STS-84 to assess the activation
response of T cells relative to the on-board 1 g reference
centrifuge.
TCR internalization
The relative amount of surface TCR was measured by flow cytometry
to determine whether TCR internalization can occur effectively in
microgravity culture. As shown in Fig. 9
, stimulation of purified T cells with Bead-Leu4 induced strong
internalization of the TCR in both microgravity culture and the 1
g reference centrifuge, as indicated by the reduction in CD3
fluorescence. Similarly, Bead-OKT3/CD28 and PDB/I induced
internalization of TCR, but to a lesser degree in microgravity culture
than in the 1 g centrifuge. These findings indicate that the
early signaling required for internalization of TCR from the plasma
membrane occurs effectively in microgravity culture.
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CD25 and CD69 expression
To determine the functional response of T cells, the expression of
the surface activation marker CD25 (Fig. 10
A) and CD69 (Fig. 10B
) was measured for cells in
microgravity culture and in the 1 g reference centrifuge.
Stimulation of PBMCs with soluble Leu4 failed to induce expression of
CD25 or CD69 (lane 2) in microgravity culture compared to the 1
g control. Whereas Bead-OKT3/CD28 (lane 4) induced
expression of CD25 in greater than 60% of the cells in the 1
g reference centrifuge, very few cells expressed CD25 in
microgravity culture. Similarly, stimulation with Bead-Leu4 (lane 3)
failed to induce expression of CD25 in microgravity culture compared to
the 1 g control. Direct activation of intracellular pathways
with PDB/I was capable of inducing expression of CD25 in ~20% of the
cells in microgravity culture (lane 5); however, the fraction of cells
expressing CD25 was less than half of that in the 1 g
reference centrifuge.
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Although stimulation of T cells in microgravity culture elicited little or no expression of CD25, the cells were more apt at expressing CD69. Stimulation with Bead-Leu4 or Bead-OKT3/CD28 resulted in expression of CD69 by ~50% of the fraction of cells expressing CD69 in the 1 g reference centrifuge. The majority of T cells expressed CD69 upon stimulation with PDB/I in both microgravity culture and the 1 g reference centrifuge.
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DISCUSSION |
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, 26)
, indicating that a lack of
initial binding of signaling molecules by soluble ligands cannot
account for this inhibition. Here we have investigated the progression
of T cell activation downstream of ligand binding and up to the point
of DNA synthesis in order to characterize the mechanism of T cell
inhibition in clinorotation and microgravity culture.
Many of the important early signaling events such as TCR engagement and
aggregation occur at the plasma membrane of the T cell and require
cell–cell or cell–substrate contact. It is therefore important to
consider whether the more dispersed 3-dimensional environment of
clinostats and microgravity culture can interfere with the formation of
cellular contacts, thereby inhibiting the initiation of early signaling
cascades. Several lines of evidence suggest that lack of cell–cell and
cell–substrate contact cannot account for the inhibition of T cell
activation under these culture conditions. 1) Our results
with intracellular cytokine production are consistent with earlier
reports (37)
and demonstrate that activation of PBMCs with
Leu4 during clinorotation results in the synthesis of IL-1 by the
monocyte population. This synthesis of IL-1 occurs in response to
intercellular signaling between T cells and monocytes (38)
and requires cell–cell contact, suggesting that intercellular
signaling can occur effectively during clinorotation. 2) One
of the important initial signaling events in TCR-mediated activation of
T cells is the engagement and aggregation of the TCR on the plasma
membrane, which subsequently lead to its internalization
(9)
. Our data on TCR internalization in microgravity
culture indicate that the engagement and aggregation of TCR by
bead-immobilized anti-CD3 did occur, effectively resulting in the
endocytosis of the TCR despite the inability of cells to activate and
express CD25. 3) Results from monocyte/T cell recombination
experiments in clinorotation (data not shown) indicate that sizable
aggregates of T cells and monocytes form by 24 h of stimulation in
clinorotation. 4) Electron microscopy observations of PBMCs
activated with mitogenic lectins in microgravity culture indicate that
surface contact between monocytes and lymphocytes occurs in flight
samples (27
28
29)
.5) Our microscopy observations
clearly reveal intimate surface contact between T cells and Bead-Leu4
in clinorotation and in microgravity culture (data not shown), and are
consistent with earlier studies that show adherence of human embryonic
kidney cells to substrates during spaceflight (39)
. Taken
together, these results indicate that the inhibition of T cell
activation in clinorotation and microgravity culture occurs downstream
of cell–cell interaction and TCR engagement, but upstream of DNA
synthesis.
Our results with BrdU incorporation indicate a dramatic inhibition of
DNA synthesis in clinorotation within 48 h of activation. These
findings are consistent with earlier reports indicating lack of
3H-thymidine incorporation 72 h after
activation (1
, 2
, 28
, 37)
, and demonstrate that this
inhibition of T cell activation is a result of a block in activation at
a time earlier in the cell cycle than S-phase entry. To better
understand the mechanism(s) underlying this inhibition, we have
characterized the progression of activation using several modes of T
cell activation in clinorotation and microgravity culture. Stimulation
of PBMCs in clinorotation with soluble Leu4 shows inhibition of surface
expression of CD25, CD69, and CD71 (CD71 data not shown), as well as
proliferative response relative to the 1 g control. The
absence of significant bulk RNA synthesis in the first few hours of
activation indicates that stimulation with soluble Leu4 does not induce
transition of T cells from G0 to the G1 stage of the cell cycle during
clinorotation. PBMC activation experiments with soluble anti-CD3 in
microgravity culture exhibit a pattern of inhibited surface marker
expression very similar to that observed in clinorotation. The absence
of CD25 expression in microgravity culture would certainly impair the
responsiveness of T cells to the cytokine IL-2, an event that is
required for proliferative response.
Complementation of TCR-mediated signaling by phorbol ester restores the ability of T cells to express CD69 and CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. The results indicate that bypassing TCR-mediated signaling and directly activating the intracellular signaling pathways in clinorotation can induce normal progression of the cells into the G1 phase of the cell cycle leading to surface expression of activation markers. This rescue of T cell activation indicates that either PKC activity or earlier steps that serve to activate PKC are compromised in clinorotation and that direct activation of PKC with phorbol ester can compensate for the alterations of cellular events that occur during clinorotation. Since costimulation with ionomycin was insufficient to overcome the inhibition, it is unlikely that altered intracellular calcium fluxes are responsible for the inhibition of T cell activation in clinorotation.
To determine the efficacy of cell–substrate interaction and activation response in clinorotation and microgravity culture, we have examined the response of purified T cells to surface immobilized anti-CD3 using covalently modified 6 µm beads. One advantage of this mode of activation is that the concentration of the beads can be increased dramatically compared to the concentration of accessory cells in PBMC, thereby increasing T cell contact and TCR stimulation. Titration experiments showed that a bead:T cell ratio of 5:1 induced similar levels of CD69 and CD25 expression in clinorotation as compared to the 1 g control, indicating that the inhibition of T cell activation in clinorotation can be overcome by cell–substrate interactions that occur with this mode of activation. Surprisingly, however, Bead-Leu4 was not capable of inducing surface expression of CD25 (IL-2R) in microgravity culture.
It has been shown that the absolute number of TCRs on the T cell and a
minimum threshold of activated TCRs are key factors in determining T
cell responsiveness (21)
. Our flow cytometry data indicate
that the absolute numbers of TCRs on the surface of T cells before
activation were equivalent in microgravity culture and the
1-g centrifuge. However, the dramatic reduction in CD25
expression with Bead-Leu4 suggests that that the lack of T cell
responsiveness in microgravity culture may involve an increase in the
TCR triggering threshold for activation.
Reports of costimulation through CD28 indicate a decrease in TCR
triggering threshold by as much as a factor of five (21)
,
resulting in dramatically augmented T cell activation responses.
Costimulation with anti-CD28 can 1) enhance the metabolic
activity of T cells, 2) decrease the number of TCR
`triggerings' required for activation, 3) increase the
levels of cytokine production and secretion by 5- to 50-fold, and
4) substantially increase the proliferative response of T
cells (21
22
23
24)
. Therefore, it was important to determine
whether costimulation through CD28 can overcome the inhibition of CD25
expression in clinorotation and microgravity culture. Activation of
purified T cells with Bead-OKT3/CD28 in clinorotation elicited strong
CD25 expression and a proliferative response similar to PDB/I
activation. We then examined whether coimmobilization with anti-CD3 and
anti-CD28 could overcome the inhibition of T cell activation in
microgravity culture. Whereas stimulation of T cells with
Bead-OKT3/CD28 in the 1 g reference centrifuge elicited
substantially higher levels of CD25 expression compared to Bead-Leu4, T
cells were still unable to exhibit normal levels of CD25 expression in
microgravity culture.
Stimulation of purified T cells by the direct activation of intracellular signal transduction pathways with PDB/I also elicited dramatically lower levels of CD25 expression in microgravity culture compared to the 1 g control. This observation was consistent among multiple sample replicates and among the two sets of flight experiments when using two different T cell donors. Taken together, these results demonstrate that the inhibition of proliferative response in microgravity culture involves alterations in cellular events that lead to the surface expression of important regulatory molecules such as CD25.
CD69 is a calcium-dependent, type II lectin receptor that appears on
the surface of T cells beginning 2–4 h after stimulation, and its
initial surface expression requires no new transcription or protein
synthesis (10)
. Cells in microgravity culture were
partially capable of expressing CD69 when stimulated with either
Bead-Leu4 or Bead-OKT3/CD28, and the fraction of CD69-positive cells
was approximately half of that in the 1 g centrifuge
control. In addition, PDB/I elicited similar levels of CD69 expression
in microgravity culture compared to the reference centrifuge.
Therefore, the transport and expression of CD69 on the plasma membrane
can occur under these conditions in microgravity culture.
The surface expression of CD25 requires induction of gene transcription
and new protein synthesis within hours of activation (11)
,
and its expression is required for the progression of S-phase entry and
proliferative response in T cells (40)
. Whereas we were
not able to directly investigate the transcription and translation of
molecules in these experiments, the absence of CD25 expression with
bead and PDB/I stimulation are consistent with a model in which the
transcription/translation of CD25 is inhibited in human peripheral T
cells during microgravity culture. In addition, the surface expression
of receptors that are presynthesized and stored in lymphocytes, such as
CD69, can occur to a great extent in microgravity culture. This
differential regulation of CD69 and CD25 expression indicates that
microgravity culture has a complex pattern of inhibition that is
dependent on the specific requirements for surface expression of the
protein in question. Our results are consistent with reports of other
cellular systems indicating that the transcription of EGF- or phorbol
ester-induced fos and jun oncogenes in A431 fibroblasts is inhibited in
microgravity culture and in clinorotation (41
, 42)
. It has
been suggested that this inhibition is specific to PKC-mediated
pathways, since other fos inducers such as A23187 (a calcium ionophore)
and forskolin (a PKA inducer) were not inhibited by microgravity
culture. Collectively, these results indicate that microgravity effects
on transcription and translation of proteins are complex and depend on
the specific signaling mechanisms involved and their integration at the
nuclear level.
The experiments outlined in this report clearly indicate that the activation response of human peripheral T cells via accessory cells, by bead-immobilized mAb, or by phorbol ester and calcium ionophore is dramatically inhibited in microgravity culture as determined by surface expression of CD25. Although these findings are in part consistent with our results from the ground-based model system of clinostats, the full expression of CD25 upon activation with bead-immobilized mAb or PMA/I during clinorotation demonstrates a clear distinction between clinorotation and microgravity culture. These results indicate that whereas clinostats are a very good model system for mimicking some of the effects of microgravity culture, they are only an approximation, and experimental results must be interpreted accordingly. However, in view of the complexity of spaceflight experiments and the fact that clinorotation is the best available terrestrial model system for studying the effects of reduced gravity on cells, clinostat studies continue to play an important role in developing experimental systems and hypotheses concerning gravitational cell biology.
The data presented here clearly indicate a dramatic alteration in the
surface expression of important regulatory molecules that can account
for the inhibition of DNA synthesis during microgravity culture.
However, the mechanism by which these cellular processes are altered in
microgravity culture is still unknown. To fully understand gravity
sensitivity of cellular processes, it will be necessary to also
consider the interactive coupling of the cytoskeleton and signal
transduction systems that play an important role in cellular responses.
Our results with the rescue of PBMC inhibition with phorbol esters
during clinorotation implicate PKC in gravity sensitivity of cellular
response and are consistent with earlier reports indicating sensitivity
of PKC to gravity changes (41
42
43
44)
. Of the six PKC
isoforms identified in T cells, PKC-
exhibits a unique polarization
toward the region of T cell contact site upon activation
(45)
. This redistribution of PKC- follows the same
pattern as the cytoskeletal proteins talin (46)
and actin
(47
, 48)
. Furthermore, increasing evidence suggests a
functional association between the cytoskeletal protein spectrin and
PKCß in the lymphocyte cytoplasm (49
, 50)
. Although
speculative at this stage, such a cytoskeletally linked process could
provide a mechanism by which microgravity culture could affect T cell
activation. Such processes are intricately linked to the integrity and
function of the cytoskeleton, especially the microtubule and
microfilament cytomatrix. Perturbation of cytoskeletal dynamics can
affect early signaling and the subsequent responses of T cells such as
protein synthesis, exocytosis, and initiation of DNA synthesis
(51)
. Investigations are under way to characterize PKC
translocation and microtubule rearrangement in T cells activated in
microgravity culture to determine the role of cytoskeletal systems and
their interactive coupling to signal transduction elements in gravity
sensitivity of T cell activation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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during T-cell activation. Nature (London) 385,83-86[Medline]
)
or at 1 g (•), and the surface expression of CD25
(A) and CD69 (B) was determined at the
indicated period of time after activation. Unstimulated cells were
cultured in parallel as a control (
). The fraction of cells in the
G1 stage of the cell cycle was determined using acridine orange
staining and flow cytometry (C).
) or at 1 g (•). The surface
expression of CD69 (A) and the fraction of cells in the
G1 stage of the cycle (B) were determined by flow
cytometry. Unstimulated cells were cultured in parallel as a control
(
).
