Transcript HU - bYTEBoss

Apoptosis and Immune Homeostasis During Hindlimb Unloading
Arthur Roberts, Erwei Sun, Lixin Wei, H.S. William Tan, Zengrong Yuan, Catherine Liu, Satish Devadas,
Duane Pierson, Satish Mehta, Kathryn Wang, David Denhardt, Yufang Shi
UMDNJ-Robert Wood Johnson Medical School & Rutgers University, Piscataway, NJ and Johnson Space Center, Houston TX
RESULTS
Spleen
30000
20000
*
10000
0
Normal
Control
Fig. 5. HU treatment inhibits ovalbuminspecific lymphocyte proliferation. Mice
were immunized with ovalbumin (OVA, 100
mg) and subjected to HU for 2 d. After 5
more days, splenocytes were harvested and
cultured in 96 well plates with or with-out
OVA stimulation (1 mg/ml), and
proliferation measured after 5 days. OVAspecific proliferation was drastically
reduced by HU stress.
4
6
8
0
10
2
4
6
8
10
Days HU treatment
Fig. 1. Hindlimb unloading (HU) causes a severe loss of splenocytes and thymocytes. HU
simulates some of the deleterious conditions of space flight, including lack of load-bearing,
fluid shift to the head, and psychological stress. In this model, mice are suspended by tail
traction so that only the forelegs contact the cage floor. Male BALB/c mice (8 wk old) were
subjected to HU for the indicated time, after which lymphoid organs were harvested, single-cell
suspensions prepared, cell subpopulations analyzed by immunofluorescence staining and flow
cytometry, and absolute numbers of each phenotype determined (mean + SD x 106 ).
6
splenocytes
thymocytes
5000
*
80
*
*
40
20
0
saline
Immun
Imm+Stress
Immun+HU: 0.13%
6
Fig. 6. Activated T cells are more sensitive to HU. Mice were immunized with OVA with or
without HU treatment, as in Fig.5, and splenocytes stained by 2-color immunofluorescence to
reveal CD8+ T cells with OVA-specific T cell receptor using fluorescent MHC class I tetramer.
Flow cytometry revealed CD8+ T cells positive for tetramer (circled red); their numbers as a
fraction of total CD8+ T cells in the spleen is given. HU treatment nearly eliminated OVAspecific T cells, indicating suppression of antigen-driven T cell expansion.
naltrexone RU486
Thymus
Thymus
Thymocytes
Spleen
Spleen
Splenocytes
HU
Unloading
4.2%
receptors and corticosteroid receptors on
HU-induced lymphocyte reduction.
Mice were treated with naltrexone (opioid
receptor blocker) or RU486 (a steroid
receptor antagonist) then subjected to HU
for 2 days, and the effect on splenic and
thymic cellularity measured by counting
total lymphocytes. HU-induced splenocyte
reduction was reversed by either naltrexone
or RU486, while thymocytes were rescued
only by RU486. *p<0.01 vs. control
4.9%
Control
1.2%
HU
Unloading
1.7%
Rad
Radiation
Rad+HU
Radiation
Rad
Radiation
+Uuloading
22.3%
100
35.6%
45.6%
19.6%
Rad+HU
Radiation
+Uuloading
+HU
53.3%
100 rads gamma rays then subjected to HU. Splenocytes and thymocytes were isolated and
apoptosis detected by staining for total DNA content with propidium iodide in a permeabilizing
buffer. The hypodiploid peak reveals apoptotic cells that have lost DNA due to cleavage.
80
80
60
*
*
40
40
20
20
0
0
control
saline
Fas-Fc
n.s IgG
HU treated
*
control
saline
*
Fas-Fc
*
n.s IgG
HU treated
Fig. 3. Blocking the Fas-FasL interaction prevents HU-induced splenocyte reduction.
Male BALB/c mice were subjected to HU with or without administration of Fas-Fc or control Ig.
Total cells in the spleen and thymus were counted. Fas-Fc treatment prevented HU-induced
splenocyte losses, but had no effect on thymocyte reductions. *p<0.001 vs. control.
Splenocyte number (x10 )
170
60
0
0
HU
control
HU
250
Fig. 12. High-dose vitamin C protects the
control
HU
200
thymus from cell depletion. Mice were fed
water supplemented with vitamin C (ascorbic
acid, 2 mg/ml) for 2 weeks before HU. The loss
of thymocytes was significantly abrogated by
vitamin C. Other antioxidants, including melatonin and N-acetylcysteine were less effective.
Vitamin C did not prevent splenocyte reduction.
150
100
50
0
control
Vitamin C
splenocytes
thymocytes
6
8
control
kelp
6
control
kelp
4
4
2
2
0
100 rads
control
100 rads
Mice were fed with a polysaccharide extract of the brown seaweed, Laminaria japonica
(part of the Eastern diet for centuries), for 3 weeks before gamma irradiation. Lymphocyte
losses in spleen and thymus were significantly improved by this dietary supplement.
CONCLUSIONS
6
6
Total cell no. (x 10 )
100
20
Fig. 13. Kelp extract protects lymphocytes from radiation-induced depletion.
Fig. 8. Radiation synergizes with HU stress to induce apoptosis. Mice were irradiated with
Thymocytes
20
control
DNA Content
Splenocytes
40
0
HU treated
120
6
0
CD8-FITC
Fig. 2. Effect of blockade of opioid
100
40
Thymus Cellularity
Control
120
60
6
2
60
transferrin increase during HU.
Sera from mice subjected to HU were
Con
analyzed by 2-D PAGE for changes in
proteins (shown). Microsequencing of
excised spots revealed high levels of Hpg
and transferrin after HU. By ELISA,
serum levels of Hpg increased 10-20 fold.
Serum samples from astronauts pre- and HU
post-flight are currently being analyzed.
Preliminary results indicate that Hpg is
increased immediately after space flight.
*
0
0
80
Fig. 11. Haptoglobin (Hpg) and
10000
Immun: 0.78%
80
KO mice were subjected to HU for 3 days. OPN-KO mice had fewer splenic lymphocytes,
but these were less sensitivity HU depletion. Results were similar with thymocytes. In
comparison, OPN-deficiency did not protect lymphocyte from depletion in response to
restraint stress, confinement in a 50 ml tube for alternating 12 h periods.
15000
Normal
100
Fig. 10. Osteopontin-deficiency prevents HU-induced lymphocyte loss. Osteopontin-
unstim
OVA-stim
20000
WT
KO
100
total cells (x 10 )
20
0
Total cell no. (X 10 )
25000
120
control
Total cell no. (x 10 )
CD4+ CD8+
CD8 SP
CD4 SP
40
20
control
con Ig
Normal: 0.43%
60
60
Fas-Fc
HU treated
OVA-spec tetra
40
tion depends on the Fas-FasL interaction.
Mice were subjected to HU for 2 d. with or
without administration of Fas-Fc. Splenocytes
were isolated and cultured in 96-well plates
with or without stimulation with soluble antiCD3. After 5 d, proliferation was measured by
[3H]Tdr incorporation using traditional
methods. Stress significantly reduced the
proliferative capacity of lymphocytes;
neutralization of FasL with Fas fusion protein
during HU abrogated this effect.
Thymocytes
Splenocyte
120
Thymus
100
B cells
CD4+ T cells
CD8+ T cells 80
6
Total cell number (x10 )
60
unstim
anti-CD3-stim
Proliferation (cpm)
Hindlimb unloading (HU) in rodent is a well-accepted ground-based model used to simulate some space
flight conditions and reproduce the deleterious effects on the musculoskeletal, cardiovascular and immune
systems. In this model, mice are suspended by the tail so that only their forelegs contact the ground, while
they are free to move about the cage, with food and water provided ad libitum. We have found that HU
severely deplete various cell populations in the spleen and thymus. These changes are likely to be mediated
by apoptosis, since DNA strand breaks indicative of apoptosis, were detected by TUNEL staining in splenocytes and thymocytes. Administration of opioid antagonists or interference with the Fas-FasL interaction
reduced the loss of splenocytes, but not thymocytes. On the other hand, RU486, a steroid receptor antagonist, blocked the reduction of lymphocyte numbers in both spleen and thymus. Therefore, the effects of HU
on the homeostasis of splenocytes and thymocytes must be exerted through distinct mechanisms. HU
exposure severely curtailed both non-specific TCR-induced and ovalbumin-specific splenocyte proliferation.
The delayed-type hypersensitivity response measured by footpad swelling in response to secondary exposure
to OVA was also decreased by HU. Similarly, the expansion of OVA-specific T cells was reduced. Interestingly, CD4+CD25+ regulatory T cells were found to be important in the reduction of lymphocyte number and
immune suppression. Depletion of these cells by injecting monoclonal antibody against CD25 prevented
lymphocyte reduction and improved the immune responses. Thus, lymphocytes are drastically affected by
HU in mice, and normal immune responses are severely blunted. These effects are mediated at least in part
by apoptosis, and the Fas-FasL interaction is central in splenocyte reduction, but not so in the thymus. In
addition, CD4+CD25+ T cells play a critical role in modulating immune responses during HU. Further
exploration into the mechanism of HU-induced lymphocyte apoptosis and immunosuppression will allow for
the development of more effective countermeasures for use during space flight. We have begun to explore
some of these: vitamin C to prevent HU-mediated lymphocyte loss and kelp extract as a protectant from
radiation-induced lymphocyte depletion.
Proliferation (cpm)
40000
Fig. 4. HU inhibition of T cell proliferacell number x 10
ABSTRACT
Saline
150
Anti-CD25
130
Anti-FasL
110
90
70
50
30
10
control
HU
Fig. 9. CD4+CD25+ Regulatory T cells
(Treg) are critical in HU-induced
splenocyte depletion. Treg were eliminated
in vivo by injecting anti-CD25 Ab one day
before subjecting mice to HU. Total
splenocyte counts revealed strong protection
by depletion of Treg. The effect was similar
to that found by Ab-neutralization of FasL.
1. HU in mice, which mimics some of the conditions of space flight, causes a severe loss of
lymphocytes in the spleen and thymus. This effect is mediated by apoptosis, and depends
on opioid receptors and corticosteroids receptors in the spleen, but only corticosteroid
receptors in the thymus.
2. Lymphocyte apoptosis induced by HU, as well as chronic restraint, is mediated by the
Fas-FasL interaction in the spleen, but not the thymus. Furthermore, CD4+CD25+ Treg
are required.
3. Many parameters of immune responsiveness in vitro and in vivo are inhibited by HU,
indicating a potent generalized immunosuppressive effect of this model.
4. Radiation exposure synergizes with HU to induce high levels of lymphocyte apoptosis.
5. Osteopontin, a pleiotropic cytokine, plays a critical role HU-induced lymphocyte
reduction.
6. The haptoglobin and transferrin proteins are upregulated during HU and may thus
potentially serve as practical serum biomarkers for “stress” levels in astronauts.
7. Promising candidates for dietary supplement countermeasures: Vitamin C protects thymic
lymphocytes from HU-mediated depletion; kelp extract protects from radiation-induced
lymphocyte loss.