File Name: SLS1_09.DOC

EXPERIMENT SLS1 BSP9

EFFECT OF SPACE FLIGHT FACTORS ON THE FUNCTIONAL ACTIVITY

OF IMMUNE CELLS

[PIs:]{.underline}

[Russia:]{.underline}

I.V. Konstantinova, Institute of Biomedical Problems, Moscow

A.T. Lesnyak, Institute of Biomedical Problems, Moscow

[Co-Investigators:]{.underline}

M.P. Rykova, Institute of Biomedical Problems, Moscow

D.O. Meshkov, Institute of Biomedical Problems, Moscow

A.A. Markin, Institute of Biomedical Problems, Moscow

T.G. Orlova, Gamaleya Institute of Epidemiology and Microbiology, Moscow

[PIs:]{.underline}

[US:]{.underline}

R.D. Lange, Tennessee State University, Knoxville, Tennessee

G. Sonnenfeld, University of Louisville, Louisville, Kentucky

H. Leon, NASA Ames Research Center, Moffett Field, California

[Introduction]{.underline}

Numerous observations suggest that man and animals exposed to
microgravity develop immune changes associated, primarily, with
disorders in immunocompetent cells. However, there are many open
questions concerning the type of disorders of cell immunity, effect of
space flight factors on the kinetics of cell-mediated reactions, and
time course variations of the recovery of immunocompetent cells. SLS-1
immune experiments helped address these questions in a sophisticated
way. In addition, immune studies of rats flown on SLS-1 allowed
comparison of the effect of Space-Shuttle and Cosmos environmental
parameters. A rat tail-suspension experiment performed simultaneously
with the flight study made it possible to compare immune responses

of Sprague-Dawley and Wistar rats, the latter being a standard
experimental object in Cosmos flights.

The purpose of the study was to investigate the effect of a 9-day space
flight

on immune cells and that of a 9-day readaptation period at 1 g on their
recovery.

[Materials and Methods]{.underline}

Experimental animals were divided into the following groups, 5 rats
each:

Basal control (BC)

Flight rats sacrificed at the day of recovery (R+0)

Vivarium controls for the R+0 group (C+0)

Flight rats sacrificed 9 days after recovery (R+9)

Vivarium controls for the R+9 group (C+9)

Tail suspended rats (S+0)

Vivarium controls for S+0 group (SC+0)

Spleen and bone marrow cells were used. Cell suspensions were prepared
at the dissection site and shipped to the laboratory for further
analysis at 0 oC. Immediately after arrival cells were cultured.

The following parameters were measured:

1\. Proliferative activity of spleen T-cells stimulated by concanavallin
A (Con-A, 0.1, 1.0 and 10.0 μg/l) and interleukin-2 (IL, 2 U/ml) was
measured with respect to RNA synthesis (incorporation of 14C-uridine for
2 hours) and DNA synthesis (incorporation of 3H-thymidine for 18 hours)
48, 72 and 96 hours after cultivation at 37 oC in wet 5% CO2 containing
air. The concentration was 0.2 x 106 cells per well of the round-shaped
96-welled culture plate in each of the two parallels. After incubation
cellular residues were transferred to filters by means of a cell
harvester, and their radioactivity was measured using a beta-counter.
The results were expressed as lg DPM or as lg DPM in the mitogen
containing culture after subtraction of lg DPM in the control culture.

2\. Functional activity of cells - natural killers (NK) of the spleen
and bone marrow

was investigated in a 16 hour membrane toxic test using target cells
K562 and YAC 1 prelabeled with 3H-uridine. To prevent 3H-uridine
reutilization, effector cells were treated with actinomycin D (5 μg/ml)
for an hour at 37 oC. The reaction was performed in RNAse containing
culture media at 37 oC in wet 5% CO2 containing air. The effector:
target ratio was 100: 1, 50: 1, 25: 1, 12: 1, 6:1, 3:1 and 1.5: 1. After
incubation cellular

residues were transferred to filters by means of a cell harvester, and
their radioactivity was measured using a beta-counter. The results were
expressed as a cytotoxicity index equal to (1 - DPM in the flight cells
: DPM in the control cells) x 100%.

3\. Activity of immunity humoral mediators was measured in spleen and
bone marrow cell supernatants cultivated in 24-welled flat-bottom plates
at 37 oC in wet 5% CO2 containing air at a concentration of 5 x 106
cells/ 1.5 ml/ well. After incubation the supernatants were frozen and
stored at - 20 oC until analysis.

3.1. Interleukin-2 (IL2). Spleen cells were incubated for 48 hours in
the presence of Con A (1 μg/ml). Activity of the mediator was measured
in the CTLL-2 cell culture for 48 hours at 37 oC in wet 5% CO2
containing air. The supernatant was sequentially diluted (two parallels
per each dilution). The reaction was measured with respect to 3H-uridine
incorporation during the last 18 hours of cultivation. After incubation
cellular residues were transferred to filters by means of a cell
harvester, and their radioactivity was measured using a beta-counter.
The results were compared with the activity of reference interleukin-2
and expressed in activity units.

3.2. Interferon-alpha. Spleen cells were incubated for 24 hours in the
presence of poly I: C (25 μg/ml) and DEAE-dextran (50 μg/ml).

3.3. Interferon-gamma. Spleen cells were incubated for 24 hours in the
presence of Con A (1 μg/ml).

3.2 and 3.3. Interferon activity was measured in cultured L cells of
murine origin in terms of inhibition of cytopathic action of mouse
encephalomyocardite virus. Supernatants diluted with cell test-culture
were incubated for 24 hours at 37 oC in wet

5% CO2 containing air. The results were expressed as titer ln.

3.4. Tumor necrosis factor- alpha (TNF-alpha). Spleen cells were
cultivated for

24 hours in the presence of lipopolysaccharide (E. coli LPS, 10 ng/ml)
and muramyl dipeptide (MDP, 10 μg/ml).

3.5. Tumor necrosis factor- beta (TNF-beta). Spleen cell suspension was
incubated in Petri dishes twice (for 60 min and 30 min) at 37 oC in wet
5% CO2 containing air to isolate a nonadhesive fraction. Then the
nonadhesive cells were cultivated for 48 hours in the presence of Con A
(1 μg/ml).

3.4 and 3.5. Activity of TNF-alpha and TNF-beta was determined with
respect

to the cytopathic action on line L929 cells. After 24 hour incubation of
the cells with supernatants containing TNF-alpha and TNF-beta dead cells
were separated, and alive cells were stained with crystal violet.
Activity of the mediators was determined on the basis of the staining
intensity of cellular residues.

The data obtained were statistically treated to determine the mean
arithmetic (M) and mean error (m). The significant difference between
the flight and control groups was evaluated using the Student\'s t-test
and nonparametric Wilcoxon-Mann-Witney U-test.

[Results and Discussion]{.underline}

1\. Proliferative activity of spleen cells.

Figures 1 through 5 present functional activity of T-lymphocytes as
measured by the rate of RNA synthesis (14C-uridine incorporation). The
R+0 rats did not show any significant differences when compared to the
controls. In the R+9 rats the rate of RNA synthesis was increased in the
mitogen-free cultures, in the cultures stimulated by 1.0 μg/ml Con A for
72 hours, in the cultures stimulated by 10.0 μg/ml Con A for nearly all
periods of time, and in the cultures stimulated by IL2. In the
suspension rats the spontaneous incorporation of 14C-uridine was
significantly lower than in the controls. The rate of RNA synthesis was
also lowered in the cultures stimulated by 0.1 μg/ml Con A for 96 hours
and by 10.0 μg/ml Con A for 48 hours.

The R+0 rats did not show any differences when compared with the
controls in terms of the rate of DNA synthesis (incorporation of
3H-thymidine) (Figures 6-10). The R+9 rats exhibited an increased
spontaneous incorporation of the radioactive label and an enhanced rate
of DNA synthesis in the cultures stimulated by 1.0 μg/ml Con A for 72
hours, as well as in the cultures stimulated by 10.0 μg/ml Con A for 72
and 96 hours and IL2. In the suspended rats the rate of 3H-thymidine
incorporation was higher in the mitogen-free cultures than in the
controls. The low level of DNA synthesis was seen in the cultures
stimulated by 0.1. mg/ml Con A as well as in the cultures stimulated by
1.0 μg/ml Con A for 48 hours.

From our point of view, it is not correct to compare the results in the
R+0 and R+9 rats with the BC because of significant differences between
BC and C+0 and C+9 groups, whereas there were essentially no differences
between the R+0 and R+9 rats.

2\. Functional activity of natural killers (NK)

The spleen cells cultured with target cells YAC 1 (Table 1) showed a
significant increase in the cytotoxicity index in both the R+0 rats
(except at a ratio of 100/1) and in the R+9 rats (except at a ratio of
100/1 and 50/1) as compared with the control animals. The spleen cells
cultured with target cells K562 (Table 2) also demonstrated an increased
activity of NK in the R+0 rats and in the R+9 rats at a ratio of 50/1.
The suspended rats (Table 3) also displayed an increased activity of NK
in the cultures with target cells YAC 1 and in cultures of target cells
K562 but only at one ratio of the effector/target of 50/1.

Membranotoxic activity of bone marrow NK toward YAC 1 cells (Table 4) in
the R+0 rats was significantly higher than in the controls. The R+9 rats
showed a higher cytotoxicity index only at the effector/target ratio
equal to 6/1. In the K562 cultured cells the activity of NK (Table 5)
was lowered in the R+0 rats at a ratio of 100/1 and in the R+9 rats at a
ratio of 100/1, 50/1 and 12/1. In the tail suspended rats (Table 6) the
activity of bone marrow NK was reduced with respect to K562 cells and
did not differ significantly from the controls in YAC 1 cell cultures.

3.1. IL2 production by spleen cells (Figure 11) in the R+0 and R+9 rats
did not differ significantly from the controls. The suspended rats
showed a substantial decrease of the mediator activity.

3.2. Interferon-alpha production by spleen cells (Figure 12) from R+0,
R+9 and suspension rats did not differ significantly from the controls.
This parameter tended to increase in the R+9 rats only.

3.3. Interferon-gamma production by spleen cells (Figure 13) was
increased in the R+0 rats and did not differ from the controls in all
the other groups. It should be noted that the values observed in the C+0
rats were extremely low; therefore, it appears more reliable to compare
the R+0 rats with BC and C+9 data. In this situation it can be concluded
that the production of gamma-interferon did not alter significantly.

3.4. Production of TNF-alpha by spleen cells (Figure 14) increased
significantly in the R+0 rats as compared with the C+0 rats. In spite of
the fact that this parameter in the C+0 rats was significantly lower
than in other control groups, it can be concluded that the rate of
TNF-alpha synthesis increased after flight because it remained
substantially different from the C+9 rats.

3.5. Production of TNF-beta by spleen cells (Figure 15) did not differ
significantly in the R+0 and R+9 rats while it was significantly reduced
in the suspension rats.

Thus, our investigations gave evidence that the functional activity of
spleen T-lymphocytes in the R+0 rats remained essentially unaltered. In
the R+9 rats cells showed a greater capacity for proliferation. The rate
of RNA and DNA synthesis in unstimulated cell cultures increased at both
optimal and high Con A concentrations as well as in IL2 stimulated
cultures. As a rule, Cosmos observations indicated a decline in
T-lymphocytes of the rat spleen after flight (Cosmos-1667, Cosmos-1887,
Cosmos-2044) \[2\]. The discrepancy of the results obtained seems to be
associated with differences in the flight profile, landing, exposure
time in microgravity as well as with differences in the time and
procedure of animal dissection. The changes seen in the R+9 rats are
indirectly suggestive of the fact that during readaptation T-cell
mediated immunity developed certain changes that caused enhanced
reactivity of T-cells 9 days after recovery. In the suspended rats the
proliferative response was weak in the cultures that had no or low
mitogen as well as in the cultures containing high Con A concentrations
but during short incubation periods. On the whole, these data are in
agreement with the current concepts about the effect of hypokinesia on
T-cell mediated immunity.

Study of the functional activity of NK of spleen and bone marrow
demonstrated an increase in the cytotoxicity index toward target-cells
YAC 1 in the R+0, R+9 and suspension rats (in the last case only spleen
cells). Membranotoxic activity of spleen cells toward target cells K562
was also increased (in the S+0 rats the increase was insignificant) and
that of bone marrow cells was diminished (in the R+0 the decrease was
insignificant while in the R+9 and S+0 rats it was more noticeable).

Conversely, the rats flown on Cosmos-1887 and Cosmos-2044 displayed a
reduced capacity of spleen and bone marrow cells to lyze YAC 1 cells.
The response of spleen cells to K562 cells did not change significantly
and that of bone marrow cells either diminished (Cosmos-1887) or
enhanced (Cosmos-2044) \[4\]. Comparison of the suspension data
(Cosmos-2044) with the S+0 data also revealed different changes. In this
experiment the activity of spleen NK toward target cells YAC 1 remained
unchanged and toward K562 cells diminished. In bone marrow cell cultures
the cytotoxic index was increased toward both YAC 1 and K562 cells.

The different responses of NK of Cosmos and SLS-1 rats can be attributed
to the above-mentioned causes as well as to the differences between
Wistar and Sprague-Dawley strains.

The spleen cell capability of the flight and suspension rats to produce
interferon-alpha remained essentially unaltered, which is in agreement
with Cosmos-1887 data \[3\].

The fact that the production of interferon-gamma and IL2 by spleen cells
of SLS-1 rats did not decline, in contrast to what was observed in
earlier Cosmos-1887 \[3\] and Space-Shuttle rat experiments \[5\], finds
an indirect support in a high level of spleen T-

cell and NK activity which is closely associated with these lymphokins.
The S+0 rats showed a significant decrease in the production of IL2,
which is consistent with the state of spleen T-cells of these rats.

Activity of TNF-alpha in spleen cell supernatants in the SLS-1 R+0 rats
was increased while in the Cosmos-1887 rats it was essentially unaltered
\[1\]. Activity of TNF-beta in the R+0 and R+9 rats was identical to
that in the controls. The S+0 rats showed a lower production of TNF-beta
which was similar to a decrease of IL2.

[Conclusions]{.underline}

1\. When compared to the corresponding controls, functional activity of
spleen T-lymphocytes remained unchanged in the R+0 rats and increased in
the R+9 rats (in cultures containing no mitogen, in cultures containing
the mitogen in optimal and high concentrations, in cultures with low Con
A concentrations and short incubation times).

2\. Membranotoxic activity of spleen and bone marrow cells toward target
cells YAC 1 was increased in the R+0, R+9 and S+0 rats (in the latter
case only spleen cells). Activity of spleen NK toward target cells K562
was also increased (in the S+0 rats insignificantly), while that of bone
marrow NK was decreased (in the R+0 rats insignificantly and in the R+9
and S+0 rats to a greater extent).

3\. Production of interferon-alpha by spleen cells did not change
substantially.

4\. Production of interferon-gamma by spleen cells did not decline
either after flight or after suspension.

5\. Production of IL2 remained unchanged in the R+0 and R+9 rats and
diminished in the S+0 rats.

6\. Immediately after recovery activity of TNF-alpha in supernatants of
rat spleen cells increased while that of TNF-beta did not change. In the
S+0 rats the production of TNF-beta diminished.

[References]{.underline}

1\. Konstantinova, I.V. Insufficiency of the Immune System in Response
to Space Flight Stressor Factors. Abstr. First Natl. Immunology Congr.,
Sochi, 15-17 Nov., 1989. Moscow, 1989, vol. 2, p. 225 (in Russian).

2\. Lesnyak, A.T., Vorotnikova, I.E., Bozhikov, N.V. et al. Rat
T-Lymphocytes in Cosmos Experiments. Abstr. Intern. Symp. Cosmos
Biosatellites. Leningrad, 12-15 Aug., 1991. Moscow, 1991, pp. 73-74 (in
Russian).

3\. Lesnyak, A.T., Vorotnikova, I.E., Serov, I.V. et al. Production of
Humoral Mediators by Immunocompetent Cells of Rats in Cosmos
Experiments. Abstr. Intern. Symp. Cosmos Biosatellites. Leningrad, 12-15
Aug., 1991. Moscow, 1991, pp. 71-72 (in Russian).

4\. Rykova, M.P., Meshkov, D.O., Lesnyak, A.T. et al. Activity of
Natural Killers of Rats in Cosmos Experiments. Abstr. Intern. Symp.
Cosmos Biosatellites. Leningrad, 12-15 Aug., 1991. Moscow, 1991, pp.
106-107 (in Russian).

5\. Gould, C.L., Lyte, M., Williams, J. et al. Inhibited
Interferon-gamma but Normal Interleukin-3 Production from Rats Flown on
the Space Shuttle. Aviat. Space Environ. Med., 1987, vol. 58, No. 40,
pp. 983-986.

[Table 1]{.underline}

Cytotoxic Activity of Spleen Natural Killers (YAC 1 Target Cells)

  ----------- -------- -------- -------- -------- -------- -------- --------
  Effector/   Basal    C + 0    R + 0             C + 9    R + 9    
  target                                                            

  ----------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  ratio    M ± m    M ± m    M ± m    P        M ± m    M ± m    P

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  100/1    79.9     65.7     76.5     \-       63.7     61.4     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.71    ±9.91    ±2.01             ±2.18    ±2.02    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  50/1     79.0     56.4     71.5     \<0.01   66.4     66.5     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.52    ±2.10    ±1.93             ±1.81    ±0.93    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  25/1     75.3     54.4     62.6     \<0.01   62.3     69.8     \<0.05

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.62    ±1.48    ±1.11             ±2.09    ±0.91    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- --------- -------- -------- ---------
  12/1     77.8     54.5     83.9     \<0.001   54.6     74.8     \<0.001

  -------- -------- -------- -------- --------- -------- -------- ---------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.89    ±0.71    ±0.72             ±3.27    ±0.89    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  6/1      75.1     45.1     60.5     \<0.01   57.1     68.8     \<0.01

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.50    ±3.82    ±1.48             ±1.48    ±2.21    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  3/1      73.4     46.7     65.8     \<0.01   52.4     66.2     \<0.01

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.58    ±3.18    ±2.21             ±2.01    ±2.38    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- --------- -------- -------- --------
  1.5/1    69.5     44.2     63.7     \<0.001   46.5     65.9     \<0.01

  -------- -------- -------- -------- --------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.72    ±1.32    ±1.63             ±1.62    ±5.31    

  -------- -------- -------- -------- -------- -------- -------- --------

[Table 2]{.underline}

Cytotoxic Activity of Spleen Natural Killers (K562 Target Cells)

  ----------- -------- -------- -------- -------- -------- -------- --------
  Effector/   Basal    C + 0    R + 0             C + 9    R + 9    
  target                                                            

  ----------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  ratio    M ± m    M ± m    M ± m    P        M ± m    M ± m    P

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  100/1    72.6     81.2     88.3     \<0.05   50.6     56.2     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.84    ±2.06    ±1.19             ±2.9     ±2.61    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  50/1     66.6     76.1     82.5     \-       42.2     56.0     \<0.05

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.55    ±5.07    ±1.61             ±4.45    ±2.09    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  25/1     61.3     71.1     78.3     \<0.05   34.8     42.3     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.81    ±1.93    ±1.05             ±1.24    ±5.02    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  12/1     52.8     62.8     72.6     \<0.05   28.2     35.9     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±4.61    ±3.54    ±1.52             ±4.33    ±2.01    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  6/1      48.2     58.7     68.2     \<0.05   29.7     32.4     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.57    ±3.02    ±0.62             ±4.47    ±4.28    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  3/1      35.9     48.3     64.3     \-       29.3     29.1     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±4.33    ±6.80    ±0.93             ±2.69    ±1.37    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- --------- -------- -------- --------
  1.5/1    26.4     30.9     60.3     \<0.001   28.5     25.7     \-

  -------- -------- -------- -------- --------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±2.51    ±2.80    ±1.90             ±1.31    ±4.90    

  -------- -------- -------- -------- -------- -------- -------- --------

[Table 3]{.underline}

Cytotoxic Activity of Spleen Natural Killers in Suspended Rats

(K562 and YAC 1 Target Cells)

  -------- ----------- ----------- -------- ----------- ----------- --------
                       K 562                            YAC 1       

  -------- ----------- ----------- -------- ----------- ----------- --------

  ----------- ----------- ----------- -------- ----------- ----------- --------
  Effector/   C + 0       R + 0                C + 9       R + 9       
  target                                                               

  ----------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  ratio    M ± m       M ± m       P        M ± m       M ± m       P

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  100/1    62.6±0.46   65.7±2.05   \-       57.0±2.33   68.3±1.63   \<0.01

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  50/1     57.5±3.47   60.0±2.88   \<0.05   46.4±3.32   60.3±1.09   \<0.01

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  25/1     51.4±2.13   50.9±2.39   \-       35.1±3.13   53.1±1.86   \<0.01

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  12/1     46.5±1.65   45.8±1.70   \-       36.7±5.32   52.0±1.24   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  6/1      40.7±0.24   48.6±3.15   \-       36.8±1.78   52.2±3.88   \<0.01

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- ---------
  3/1      39.8±1.39   40.9±2.29   \-       43.6±1.38   56.6±0.99   \<0.001

  -------- ----------- ----------- -------- ----------- ----------- ---------

  -------- ----------- ----------- -------- ----------- ----------- ---------
  1.5/1    38.7±4.51   38.4±2.70   \-       43.0±1.94   54.7±1.65   \<0.001

  -------- ----------- ----------- -------- ----------- ----------- ---------

[Table 4]{.underline}

Cytotoxic Activity of Bone Marrow Natural Killers (YAC 1 Target Cells)

  ----------- -------- -------- -------- -------- -------- -------- --------
  Effector/   Basal    C + 0    R + 0             C + 9    R + 9    
  target                                                            

  ----------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  ratio    M ± m    M ± m    M ± m    P        M ± m    M ± m    P

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  100/1    54.8     60.2     73.4     \<0.01   54.5     55.5     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.48    ±3.63    ±1.07             ±4.01    ±5.38    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  50/1     56.2     59.5     67.3     \<0.05   62.0     57.8     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.78    ±2.46    ±1.37             ±1.47    ±1.58    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  25/1     54.9     56.8     65.7     \<0.01   54.5     62.7     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.90    ±2.34    ±0.78             ±5.34    ±3.14    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  12/1     50.9     57.0     66.5     \<0.01   45.7     57.2     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.40    ±1.80    ±1.08             ±3.8     ±3.51    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- --------- -------- -------- --------
  6/1      56.6     52.7     65.9     \<0.001   38.4     54.3     \<0.05

  -------- -------- -------- -------- --------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.58    ±2.11    ±1.05             ±1.62    ±3.06    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  3/1      58.6     61.0     65.2     \-       42.8     41.7     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.91    ±3.36    ±1.42             ±1.26    ±1.65    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  1.5/1    56.1     57.9     68.3     \<0.05   36.3     35.0     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.65    ±3.74    ±1.54             ±2.77    ±3.31    

  -------- -------- -------- -------- -------- -------- -------- --------

[Table 5]{.underline}

Cytotoxic Activity of Bone Marrow Natural Killers (K 562 Target Cells)

  ----------- -------- -------- -------- -------- -------- -------- --------
  Effector/   Basal    C + 0    R + 0             C + 9    R + 9    
  target                                                            

  ----------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  ratio    M ± m    M ± m    M ± m    P        M ± m    M ± m    P

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  100/1    60.0     85.1     76.6     \<0.05   58.8     41.2     \<0.05

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±0.62    ±1.86    ±0.87             ±2.1     ±6.92    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  50/1     56.8     80.7     77.5     \-       53.3     38.7     \<0.01

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.52    ±1.40    ±2.36             ±1.97    ±3.51    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  25/1     51.1     76.6     72.5     \-       48.5     34.2     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±1.59    ±1.63    ±1.93             ±2.94    ±6.56    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  12/1     49.8     74.6     72.7     \-       38.8     26.1     \<0.05

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±2.32    ±1.88    ±1.39             ±3.71    ±1.19    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  6/1      43.7     67.5     65.3     \-       22.9     19.6     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±2.74    ±4.39    ±1.95             ±1.05    ±3.61    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  3/1      37.9     56.6     63.9     \-       18.4     20.5     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±2.64    ±1.54    ±4.24             ±2.74    ±2.09    

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
  1.5/1    29.9     55.4     47.2     \-       23.1     21.5     \-

  -------- -------- -------- -------- -------- -------- -------- --------

  -------- -------- -------- -------- -------- -------- -------- --------
           ±3.18    ±3.76    ±5.29             ±4.34    ±2.10    

  -------- -------- -------- -------- -------- -------- -------- --------

[Table 6]{.underline}

Cytotoxic Activity of Bone Marrow

(K562 and YAC 1 Target Cells)

  -------- ----------- ----------- -------- ----------- ----------- --------
                       K 562                            YAC 1       

  -------- ----------- ----------- -------- ----------- ----------- --------

  ----------- ----------- ----------- -------- ----------- ----------- --------
  Effector/   R + 0       C + 0                R + 9       C + 9       
  target                                                               

  ----------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  ratio    M ± m       M ± m       P        M ± m       M ± m       P

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  100/1    56.4±2.90   40.9±3.82   \<0.05   58.2±0.89   58.6±1.93   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  50/1     47.8±1.78   34.9±5.13   \<0.05   50.6±1.78   50.4±2.80   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  25/1     43.3±2.65   32.7±4.99   \-       42.3±2.00   44.2±1.53   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  12/1     42.1±0.97   29.2±4.33   \<0.05   37.8±5.92   44.5±0.46   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  6/1      36.1±1.41   27.6±4.92   \-       43.4±5.32   42.9±6.75   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  3/1      39.1±0.87   27.5±2.30   \<0.01   51.3±3.62   58.9±5.39   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

  -------- ----------- ----------- -------- ----------- ----------- --------
  1.5/1    39.2±2.53   26.2±2.87   \<0.01   63.0±0.49   61.9±2.08   \-

  -------- ----------- ----------- -------- ----------- ----------- --------

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[Summary]{.underline}

The goal of the experiment was to study the effect of a 9-day SLS-1
space flight

on the function of immunocompetent cells of rats (basal control, R+0 and
C+0 rats) and to follow the dynamics of its recovery after return to the
Earth\'s gravity (R+9 and C+9 rats). Also, an additional tail suspension
experiment was performed (S+0 and CS+0 rats).

Proliferative activity of spleen T-lymphocytes was measured in 48-, 72-
and 96-hour tests with concanavallin A (0.1, 1.0 and 10.0 μg/ml) and
interleukin-2 (2 U/ml) with respect to the incorporation rate of
14C-uridine and 3H-thymidine. When compared to the corresponding
controls, T-cell activity remained unchanged in the R+0 rats, increased
in the R+9 rats (in the cultures containing no mitogen, containing the
mitogen in optimal and high concentrations, or stimulated by IL2) and
decreased in the S+0 rats (in the cultures containing no mitogen,
containing low concentrations of concanavallin A or incubated for short
times).

Natural cytotoxicity of spleen and bone marrow cells in the
membranotoxic test with target cells YAC 1 was increased in the R+0, R+9
and S+0 rats (in the latter case only in spleen cells). Activity of
spleen natural killers toward target cells K562 was also increased (the
increase was insignificant in the S+0 rats) and that of bone marrow was
decreased (in the R+0 rats the decrease was insignificant and in the R+9
and S+0 rats it was more noticeable).

The capability of spleen cell cultures to synthesize humoral mediators
of immunity was investigated. The production of interferon-alpha
remained essentially unaltered. The rate of interferon-gamma synthesis
did not go down either after flight or after suspension. Activity of IL2
in cellular supernatants did not change in the R+0 and R+9 rats and
decreased in the S+0 rats. Activity of TNF-alpha in the supernatants of
rat spleen cells was increased after flight (R+0 rats) while that of
TNF-beta did not change. The production of TNF-beta declined in the S+0
rats.