Ž singly caged rhesus macaques were allowed to spend regular periods 100 minrsession,
. 3–5 sessionsrweek interacting in stable social groups, while the remaining subjects
Ž .
spent regular periods same duration and frequency as above interacting in social groups whose membership was constantly altered by the experimenters. Capitanio et al.
Ž .
1998b found that the macaques that had been in stable social groups spent significantly more time in affiliative interactions and had significantly longer survival times after
Ž .
infection with simian immunodeficiency virus SIV than macaques that had been in Ž
unstable social groups. They concluded that psychosocial factors the social opportuni- ties and relationships provided by interacting with a stable set of partners, specifically
. the amount of time spent affiliating , could influence a critical immunological factor,
Ž .
survival time after infection with a deadly pathogen SIV . This finding highlights the relevance of psychosocial factors and their effects on laboratory primates in biomedical
research. Nonhuman primate subjects for biomedical research are usually chosen in a fairly
nonsystematic fashion. Typically, available andror inexpensive animals are chosen first Ž
. Kaplan et al., 1991; Benveniste et al., 1996 , with little regard for other factors. Prior to
the beginning of an experiment, monkeys are removed from their ‘‘normal’’ housing and placed in the experimental condition. A period of acclimation to allow the subjects
to adapt to their new surroundings is usually provided. The acclimation period can vary Ž
. in duration from several weeks to several months Capitanio et al., 1996 , but is rarely
rigidly controlled or explicitly stated. There are few empirical data that demonstrate that an acclimation period of a specific length is necessary for subjects to return to baseline
Ž .
functioning. In fact, recent work by Lilly et al. 1999 shows that acclimation is unlikely to have occurred, even 28 weeks after removal from the ‘‘normal’’ housing condition.
Clearly, an enhanced understanding of the time course of acclimation processes would be germane to biomedical research with laboratory primates.
Ž . We were specifically interested in determining 1 whether there were any differences
in the cell-mediated immune responses of adult rhesus monkeys that were housed singly, Ž .
in pairs, or in small groups and 2 whether these responses changed over time. Clearly, housing adult rhesus monkeys individually is a fairly stressful psychosocial manipula-
tion; one that may be comparable to the more commonly studied mother–infant separation or reintroduction to groups of conspecifics. We measured lymphocyte sub-
sets, mitogen- and pathogen-induced lymphocyte proliferation, NK activity, and cy- tokine production on multiple occasions over a 12-month period. Measuring lymphocyte
proliferation responses to gastrointestinal pathogens is a novel approach with potential ecological relevance.
2. Animals, materials, and methods
2.1. Subjects and housing Ž
. We studied 36 adult rhesus monkeys Macaca mulatta of both sexes, that had been
Ž .
born in the U.T.M. D. Anderson Cancer Center’s specific pathogen-free SPF breeding
Ž .
colony Schapiro et al., 1994 . All subjects were at least 5.5 years of age at the time of sampling, and all had been socially housed for a minimum of 4 years of their lives. All
but two of the subjects were socially housed for the 2 years prior to the study. Ž
. The 12 monkeys in the group housed condition five males and seven females were
living in outdoor buildings in stable social groups comprised of one male, four to seven females, and their most recent offspring at the time the study began. No alterations were
made to their social or physical settings. Ž
. Eight of the twelve pair housed subjects five males and seven females were also
living in similar social groups prior to the study. On the day the study began, these animals were removed from their groups, a blood sample was obtained, and they were
reformed into pairs with a single familiar groupmate. The other four pair housed subjects were already living in pairs when the study began. No alterations were made to their
social or physical settings. Three pairs were housed in runs in outdoor buildings and three pairs were housed in double cages connected by a tunnel in indoor rooms.
Ž .
Ten of the twelve singly housed subjects five males and seven females were living in social groups prior to the study. On the day the study began, these animals were
removed from their social groups, a blood sample was obtained, and they were then Ž
2 2
placed in an appropriately sized either 0.40 m or 0.57 m , dependent on the subject’s .
Ž .
weight and enriched toys, perch, feeding devices single cage. The other two singly housed subjects were singly housed prior to the beginning of the study and no alterations
were made to their social setting, although one of these monkeys was moved from one room to another, a change in physical setting. All but one of the singly caged subjects
were housed in cages on racks in indoor rooms; the lone exception was a male housed in an outdoor run because he was too large for any of our cages.
2.2. Blood collection and cell preparation All monkeys were initially sampled in February, 1996, the end of the 1995–1996
breeding season in our colony. Monkeys that had undergone a change in social andror Ž
physical settings at the initiation of the study 11 singly housed subjects and 8 pair .
housed subjects were sampled immediately prior to the change, 2 days after the change, and again 1, 4, 8, and 12 months after the change. Monkeys that had not changed social
Ž .
or physical settings 1 singly housed, 4 pair housed, and all 12 group housed subjects were sampled on four occasions; once at the beginning of the study, and again 4, 8, and
12 months into the study. Ten milliliters of peripheral blood were obtained from the femoral vein using an
EDTA-coated syringe and were then transferred to an EDTA sterile vacutainer tube Ž
. Becton-Dickinson between 08:30 and 10:45. All monkeys were restrained using light
Ž .
Ž .
ketamine anesthesia 10 mgrkg . Peripheral blood mononuclear cells PBMC were Ž
. separated using Histopaque-1077 Sigma, St. Louis, MO density gradient centrifuga-
tion. PBMC present at the plasma–Histopaque interface were harvested. These cells Ž
were washed twice and resuspended in complete medium RPMI-1640 supplemented .
with 25 mM Hepes, 5 ml glutamine, and 25 mg gentamicin; Gibco, Grand Island, NY for proliferation, natural killer cell, and cytokine assays.
2.3. Immunological assays 2.3.1. T-lymphocyte subsets
PBMC were assayed for the presence of T-cell surface antigens using a whole-blood Ž
. staining procedure and a FACScan flow cytometer Coulter . A series of flourescein-
Ž .
Ž .
FITC and phycoerythrin- PE conjugated monoclonal antibody reagents previously characterized to react with subsets of PBMC of rhesus macaques were utilized to
determine the frequency of each subset. The monoclonal reagents included PE-con-
q
Ž .
q
Ž jugated CD4 -Leu-3a Becton Dickinson and FITC-conjugated CD8 -Leu-2a Becton
. Dickinson . A total of 10,000 cells per sample were analyzed for the frequency of
FITC
q
and PE
q
cells. Calculation of the absolute numbers of cell subpopulations were based on a complete blood cell count using a Baker System 9000 hematology instrument
and a differential done from Wright–Giemsa-stained blood smears. Results are reported as absolute number of cellsrmilliliter.
2.3.2. Lymphocyte proliferation assays Ž
6
. A 0.1-ml aliquot of the cell suspension 1 = 10 cellsrml was dispensed into each
well of a U-bottom, 96-well microtiter plate and incubated in triplicate with concentra- Ž
. Ž
. tions of concanavalin A ConA; 1:100 , phytohemagglutinin PHA; 1:50 , pokeweed
Ž .
Ž .
mitogen PWM; 1:250 , lipopolysaccharide LPS; 1:100 , and staphylococcal entero- Ž
. toxin A StaphA; 1:1000 .
Ž .
In addition, we tested two concentrations each 1:5 and 1:10 of four different common gastrointestinal pathogens of rhesus monkeys: Escherichia coli, Salmonella
typhimurium, Shigella flexneri, and Campylobacter jejuni. These organisms can cause Ž
severe diarrhea in captive macaques Holmberg et al., 1982; Hird et al., 1984; Reinhardt, .
1993; Wolfensohn, 1998 . All cultures were incubated for 4 days at 378C in a humidified 5 CO atmosphere;
2
w
3
x Ž
and during the final 16–18 h, 1 mCi of H thymidine 6.7 Cirmmol; ICN Biomedicals,
. Costa Mesa, CA was added. Cells were then harvested onto filter strips for estimation
w
3
x Ž
of H thymidine incorporation using a Skatron cell harvester Skatron Instruments,
. Ž
. Sterling, VA . The specific radioactivity DCPM, counts per minute of cells treated
with various additions were calculated in each case by subtracting the values obtained with cells cultured in medium alone.
2.3.3. Natural killer cell assay NK activity against
51
Cr-labeled K562 target cells was measured. 5 = 10
6
K562 cells Ž
were incubated for 2 h at 378C, with 100 mCi of sodium chromate New England .
4
Nuclear, Boston, MA . Cells were washed twice and resuspended at 5 = 10 rml in medium containing 10 heat-inactivated fetal calf serum. 5 = 10
5
PBMC per well were Ž
. added to 96-well U-bottom microtiter plates Falcon , and cocultured with the K562
Ž .
cells in triplicate at effector:target ratios E:T of 100:1, 50:1, 25:1, and 12.5:1. The cells were then incubated for 4 h at 378C in a humid atmosphere containing 5 CO . After
2
incubation, the plate was centrifuged at 250 g for 5 min, 100 ml of supernatant were
w
Ž .
removed from each well and added to Titertube Micro Tubes Bio Rad , and the tubes
Ž .
were assayed in the 1470 Wizarde gamma counter Wallac . Cytotoxicity was
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
