determined by measuring the release of the radiolabel into the culture supernatant. The percentage of specific
51
Cr release was calculated as: 100 = experimental release y spontaneous release r maximum release y
Ž . Ž
spontaneous release .
.
Maximum release was determined from supernatants of cells that were lysed by adding 5 Triton X-100. Spontaneous release was determined from target cells incubated
without added effector cells.
2.3.4. Cytokine assay Ž
6
. Ž
. PBMC 1 = 10
were incubated with 20 ml of mitogen PHA; 1:10 and complete Ž
. medium total volume of 200 ml per well in a 96-well U-bottom plate for 48 h at 378C.
One hundred microliters of supernatant were then removed from each well and frozen at y708C in another 96-well U-bottom plate. At the time of assay, the plate was thawed
and assayed for cytokines IFN-g, IL-2, IL-4, and IL-10 using Cytoscreen immunoassay
Ž .
kits Biosource International , according to the manual instructions. It has been shown that the human test kits for these cytokines are acceptable for use with rhesus monkey
Ž .
cells Villinger et al., 1993 . The minimal detection limits for these assays were 4 pgrml for IFN-g; 8.7 pgrml for IL-2; 2 pgrml for IL-4; and 5 pgrml for IL-10.
2.4. Analyses Ž
. Data were analyzed using analysis of variance ANOVA techniques with housing
Ž .
Ž condition single, pair, or group as a between-subjects factor and sample timepoint 0,
. 4, 8, and 12 months as a within-subjects factor. Planned comparisons were used to
determine if singly caged subjects differed significantly from socially housed subjects Ž
. pair and group housed combined . Separate one-way ANOVAs were used to determine
if there were housing effects at baseline or at the 12-month timepoint. A few subjects were not assayed for all dependent variables at all timepoints, as reflected in the degrees
of freedom in certain analyses. Since no systematic differences in immune responses existed between pair housed
subjects that had been previously paired and those that had been group housed, or between pair housed subjects living in outdoor runs and those living in indoor double
cages, for purposes of analysis, the pair housed data were treated as one data set. Similarly, since singly housed subjects that had been previously singly housed and those
that had been group housed did not differ systematically, the single housed data were also treated as one data set in the analysis.
3. Results
3.1. T-lymphocyte subsets The ratio of CD4
q
to CD8
q
lymphocytes differed significantly across the three w Ž
. x
q
housing conditions F 2,28 s 6.14, P F 0.01 , as did the absolute number of CD4
Table 1 Ž
. Lymphocyte subsets mean cellsrml1 s.d.
Baseline 4 months
8 months 12 months
qa ,b,c,d
CD4 Single
3185899 24391276
2248608 2131701
Pair 44082768
26711417 30191314
26921373 Group
36471841 32211333
45041211 40781958
qc ,e
CD8 Single
35051021 24391276
2957829 33581165
Pair 69445533
47392443 35562138
38142585 Group
33661851 2642991
39741361 40691816
q qa,d,f
CD4 r CD8 Single
0.950.23 0.860.60
0.780.21 0.680.24
Pair 0.940.45
0.710.42 0.960.29
0.830.37 Group
1.130.29 1.230.24
1.190.28 1.150.40
a
P F 0.05 for comparison across housing conditions.
b
P F 0.05 for comparison across timepoints.
c
P F 0.05 for housing condition= timepoint interaction.
d
P - 0.05 for comparison at 12 month timepoint only.
e
P F 0.05 for comparison at baseline timepoint only.
f
Ž .
P F 0.05 for planned comparison of single housed Õs. socially housed pair and group at 8 months and 12 months.
w Ž .
x
q
lymphocytes F 2,29 s 7.42, P F 0.01; see Table 1 . The absolute number of CD8 lymphocytes did not differ. Importantly, across housing conditions, there were no
significant differences in CD4
q
rCD8
q
or in CD4
q
numbers at the baseline timepoint, but significant differences across housing conditions existed at the 12-month timepoint
q q
w Ž .
x
q
w Ž .
for both CD4 rCD8 F 2,29 s 5.38, P F 0.01 and CD4
numbers F 2,30 s 5.43, x
q
P F 0.01 . CD8 numbers differed significantly across the three housing conditions at
w Ž .
x baseline F 2,33 s 4.21, P F 0.05 , but not at 12 months. Planned comparisons re-
vealed that singly caged subjects had significantly lower CD4
q
rCD8
q
than did socially Ž
. w Ž
. x
housed in pairs and groups subjects at the 8-month F 1,30 s 9.11, P F 0.01 and the w Ž
. x
12-month F 1,29 s 6.04, P F 0.05 timepoints only.
q
w Ž .
Only CD4 numbers differed significantly across all timepoints
F 3,84 s 2.75, x
P F 0.05 . Five of six pairs of CD4
q
to CD8
q
ratios were significantly positively correlated Ž
across timepoints for all subjects combined significant Pearson’s correlation coeffi- cients ranged from 0.48 to 0.72, P ’s all F 0.009, the Bonferroni alpha level, see Table
. 2 . Only the correlation between the baseline sample and the 4-month sample was not
significant at the corrected alpha level. 3.2. Lymphocyte proliferation assays
Proliferation responses to S. flexneri differed significantly across housing conditions w Ž
. x
w Ž .
F 2,30 s 3.57, P F 0.05, see Table 3 and responses to E. coli F 2,30 s 2.73,
Table 2 Pearson’s correlation matrix for CD4
q
rCD8
q
Baseline 4 months
8 months 12 months
Baseline –
4 months 0.40
–
U U
8 months 0.53
0.50 –
U U
U
12 months 0.64
0.61 0.72
–
U
P F 0.009.
x w Ž
. x
w Ž .
P F 0.08 , S. typhimurium F 2,30 s 3.01, P F 0.07 , and C. jejuni F 2,30 s 2.84, x
P F 0.08 all approached significance. There were no significant differences in prolifera- tion responses to any of the pathogens across housing conditions at the baseline
w Ž .
x w Ž
. timepoint, but responses to S. flexneri F 2,30 s 3.32, P F 0.05 , E. coli F 2,30 s
x w Ž
. x
11.59, P F 0.001 , and S. typhimurium F 2,30 s 3.97, P F 0.05 all differed signifi- cantly across housing conditions at the 12-month timepoint. For all four pathogens, pair
housed subjects had the highest proliferation responses, although planned comparisons of pair housed subjects to single and group housed subjects combined, were only
w Ž .
significant at the 12-month timepoint F 1,30 s 17.51, 5.37, 4.67, and 4.13 for E. coli, x
S. typhimurium, S. flexneri, and C. jejuni, respectively, all P ’s F 0.05 .
Table 3 Ž
. Lymphocyte proliferation responses to pathogens mean SI1 s.d.
Pathogens Baseline
4 months 8 months
12 months
a,b
S. flexneri Single
2.604.13 4.614.48
3.271.34 4.543.67
Pair 3.412.86
3.982.69 5.935.93
7.117.76 Group
2.270.98 1.400.45
3.542.34 1.831.13
b,c,d
S. typhimurium Single
6.9512.5 1.811.33
20.070.67 3.241.45
Pair 10.212.1
1.770.92 2.411.06
4.262.45 Group
2.731.11 1.070.22
2.451.47 2.051.55
b,c,d
E. coli Single
10.020.2 4.346.88
6.152.89 9.064.14
Pair 9.397.97
2.621.69 8.605.39
15.79.34 Group
6.056.00 2.200.96
7.994.80 3.323.18
c,d
C. jejuni Single
2.021.66 2.000.99
3.571.43 3.021.16
Pair 6.136.52
2.080.73 4.882.92
3.721.43 Group
5.353.68 1.610.49
5.332.99 2.891.60
a
P F 0.05 for comparison across housing conditions.
b
P F 0.05 for comparison at 12 month timepoint only.
c
P F 0.08 for comparison across housing conditions.
d
P F 0.05 for comparison across timepoints.
There were significant differences in proliferation responses to three of the four w Ž
. pathogens across all timepoints
F 3,90 s 4.40, 7.91, and 5.98 for E. coli, S. ty- x
phimurium, and C. jejuni, respectively, all P ’s F 0.01 . There were no significant housing = timepoint interactions.
w Ž .
Subjects differed by housing condition in their proliferation response to LPS F 2,30 x
w Ž .
x w Ž
. s 5.11, P F 0.05 , to PWM F 2,30 s 3.37, P F 0.05 , and to StaphA F 2,30 s 3.27,
x Ž .
P F 0.05, see Table 4 . In our immunological laboratory, a Stimulus Index SI value of Ž
at least 2.0 is required for a proliferation response to be considered positive Nehete et .
al., 1998 . All SIs less than 2.0 are considered indicative of no proliferation response, are referred to as negative, and are treated as biologically identical. Therefore, although
the difference between LPS SI values across housing conditions was statistically significant, it was not biologically meaningful, since SI values to LPS never averaged
greater than 2.0.
Planned comparisons revealed that proliferation responses of singly caged subjects Ž
did not differ systematically from socially housed subjects again, pair and group housed .
w Ž .
x subjects combined . Proliferation responses to PHA
F 2,33 s 4.85, P F 0.05 and
Table 4 Ž
. Lymphocyte proliferation responses to mitogens mean SI1 s.d.
Mitogens Baseline
4 months 8 months
12 months
a
LPS Single
1.020.40 1.100.27
1.070.15 1.290.31
Pair 1.430.77
1.360.46 1.761.16
1.600.61 Group
1.180.25 1.260.32
1.170.24 1.300.42
a,b,c
PWM Single
0.610.36 17.07.34
6.857.11 6.7917.5
Pair 0.790.30
17.813.6 8.409.41
1.460.64 Group
0.950.15 9.007.50
1.000.21 1.410.78
b
ConA Single
6.866.21 7.766.64
3.012.20 5.073.45
Pair 12.811.9
5.383.69 4.182.87
4.684.01 Group
10.19.25 3.672.09
1.360.48 2.591.32
b,c,d,e
PHA Single
6.063.47 9.328.14
1.360.68 1.520.47
Pair 13.111.7
8.468.34 2.221.57
2.381.60 Group
18.812.5 3.134.32
1.640.93 1.150.42
a,b,d,e
StaphA Single
61.349.8 51.716.5
72.715.5 110.50.0
Pair 57.447.6
43.424.2 72.326.8
120.54.0 Group
35.826.6 36.916.7
63.915.5 35.430.3
a
P F 0.05 for comparison across housing conditions.
b
P F 0.05 for comparison across timepoints.
c
P F 0.05 for comparison at baseline timepoint only.
d
P F 0.05 for housing condition=timepoint interaction.
e
P F 0.05 for comparison at 12-month timepoint only.
w Ž .
x PWM F 2,33 s 4.35, P F 0.05 differed statistically across housing conditions at the
baseline timepoint, but mean SI values for PWM for all housing conditions were less than 1.0 at baseline and were therefore also considered negative. At the 12-month
w Ž .
timepoint, PHA responses still differed across housing conditions F 2,30 s 4.71,
x P F 0.05
and StaphA responses, which did not differ at baseline, now differed Ž
. x
F 2,30 s 11.74, P F 0.001 . There were significant differences in proliferation responses to four of the five
w Ž .
mitogens across the four timepoints F 3,90 s 23.76, 13.14, 24.29, and 11.80 for PHA, x
w Ž .
ConA, PWM, and StaphA, respectively, all P ’s F 0.001 . Both PHA F 6,90 s 4.80, x
w Ž .
x P F 0.001 and StaphA F 6,90 s 4.07, P F 0.001 also evidenced significant housing
= timepoint interactions.
3.2.1. Natural killer cell assays Ž
. There was an important trend in NK activity at an effector to target E:T ratio of
w Ž .
x 100:1 across the three housing conditions
F 2,30 s 2.86, P F 0.07, see Table 5 . w Ž
. x
Timepoint also affected NK activity F 3,90 s 9.07, P F 0.001 , but there was no
significant housing by timepoint interaction effect. At the baseline timepoint, NK w Ž
. x
activity differed significantly across housing conditions F 2,33 s 6.62, P F 0.01 ,
with group housed animals showing the lowest NK activity. 3.2.2. Cytokine assays
Cytokine samples were only assayed from the baseline and 12-month timepoints. There were significant differences across housing conditions in production of IFN-g
w Ž .
x w Ž
. x
w Ž .
F 2,30 s 64.49, P F 0.001 , IL-2 F 2,30 s 3.72, P F 0.05 , and IL-10 F 2,30 s x
26.57, P F 0.001 . Significant differences in production of IFN-g and IL-10 across w Ž
. housing conditions existed at the baseline timepoint
F 2,33 s 69.22 and 49.87, x
respectively, both P ’s F 0.001 . Planned comparisons of singly caged subjects to socially housed subjects revealed that singly caged subjects produced significantly lower
w Ž .
Ž .
levels of IFN-g at both timepoints F 1,30 s 32.37, P F 0.001 and F 1,30 s 9.47, x
P F 0.01 for baseline and 12-months, respectively and of IL-10 at the baseline w Ž
. x
timepoint only F 1,30 s 18.89, P F 0.001 . There were significant differences across timepoints for these same three cytokines
w Ž
. Ž
. Ž
. IFN-g: F 1,30 s 17.58; IL-2: F 1,30 s 131.77; and IL-10: F 1,30 s 105.46, all
Table 5 Ž
. Natural killer cell responses mean lysis1 s.d.
a,b,c
E:T s100:1 Baseline
4 months 8 months
12 months Single
20.111.1 22.89.09
22.55.99 15.38.76
Pair 19.07.65
24.69.54 18.69.96
17.29.20 Group
8.885.24 22.29.67
13.59.08 13.38.29
a
P F 0.07 for comparison across housing conditions.
b
P F 0.05 for comparison across timepoints.
c
P F 0.05 for comparison at baseline timepoint only.
Table 6 Ž
. Cytokine production mean pgrml1 s.d.
Baseline 12 months
a,b,c,d,e
IFN-g Single
18.012.9 28.216.7
Pair 25.213.4
32.519.0 Group
190.67.5 75.329.0
a,b
IL-2 Single
20.08.67 4.746.86
Pair 22.45.78
2.694.82 Group
25.23.86 8.755.37
f
IL-4 Single
0.781.47 1.402.37
Pair 0.420.97
1.592.11 Group
0.230.79 2.242.13
a,b,c,d
IL-10 Single
20.55.68 10.76.02
Pair 19.65.25
12.16.43 Group
40.56.40 10.03.72
a
P F 0.05 for comparison across housing conditions.
b
P F 0.05 for comparison across timepoints.
c
P F 0.05 for housing condition=timepoint interaction.
d
P F 0.05 for planned comparison at baseline timepoint only.
e
P F 0.05 for planned comparison at 12 month timepoint only.
f
Results are reported, but the values are below the accurate detection limits of the test kit.
x w Ž
. x
P ’s F 0.001 and significant interactions for IFN-g F 2,30 s 31.89, P F 0.001 and w Ž
. x Ž
. IL-10 F 2,30 s 22.11, P F 0.001
Table 6 .
4. Discussion
