Characterization of Sociability
Rhesus macaques were classified as High Sociable (HS: falling in the upper 1/3 of the Sociability distribution) or Low Sociable (LS: falling in the lower 1/3 of the Sociability distribution) based on trained observer ratings of affiliative/companionable behavior, warm/affectionate behavior, and (reverse-scored) solitary behavior in their field cages. HS and LS animals differed substantially in rated levels of ‘affiliation’ (HS: range 4.5 – 6.0 vs. LS: 3.0 – 3.5), ‘warm’ (HS: 4.5 – 5.5 vs. LS: 1.0 – 3.0) and ‘solitary’ (HS: 1.5 – 3.0 vs. LS: 3.5 – 5.5) (). Analysis of specific behaviors confirmed the trait rating-based distinction between HS and LS animals, with HS animals showing significantly greater frequencies of initiating and receiving approaches to within arm’s reach (HS: 98.3 ± 4.9 (mean ± SEM) vs. LS: 67.5 ± 6.8; p =.02), and marginally greater frequencies of physical contact (HS: 204.3 ± 10.1 vs. LS: 131.0 ± 23.6; p =.053) and grooming (HS: 125.7 ± 7.88 vs. LS: 90.5 ± 11.20; p =.063).
Observer trait ratings of High vs. Low Sociable rhesus macaques. Mean score ± standard error.
Lymph node sympathetic innervation
To determine whether lymph node innervation patterns differed in HS vs. LS animals, we used glyoxylic acid chemofluorescence to map the distribution of parenchymal and perivascular catecholaminergic neural fibers in randomly selected 16 μm sections from 13 axillary lymph nodes (). LS animals showed significantly greater density of lymph node sympathetic innervation than did HS animals (). Across 4,574 individual 250 μm2 lymph node tissue units analyzed in a hierarchical linear model (tissue units nested within lymph node, lymph node nested within animal, and animal nested within LS vs HS group), the absolute density of parenchymal catecholaminergic varicosities averaged 1.08 per unit (± 0.11) for HS animals vs. 3.05 (± 0.32) for LS animals (p < 0.0001). Individual differences across animals accounted for 42.5% of the total systematic variance in innervation density across lymph nodes (p <.0001), with localized effects specific to each lymph node (i.e., nested within individual) accounting for 57.5% (p <.0001). Of the 42.5% of systematic variance attributable to total individual differences, variations in Sociability accounted for 38.6% (i.e., 16.8% of total variability; p <.0001), with 61.4% attributable to other individual-specific factors (i.e., 26.7% of total variability; p <.0001). Expressed in terms of effect size correlations, replicate observations from the same animal showed a correlation of r = +.132, localized effects specific to each individual lymph node contributed a correlation of r = +.194, and effects of Sociability contributed r = +.105 (all correlations adjusted for confounding with other effects). Similar results emerged in analyses treating Sociability z-scores as a continuous measure of individual differences, with each 1-SD increase in Sociability associated with 0.79 (± 0.12) fewer parenchymal catecholaminergic varicosities per 250 μm2 tissue unit (p <.0001; ). Sociability-related differences occurred specifically within the parenchyma of the lymph node and did not affect the density of perivascular catecholamineric varicosities (absolute densities averaged 0.18 ± 0.06 for HS animals vs. 0.23 ± 0.08 for LS animals, difference p =.641) ().
Sociability and lymph node innervation
Analyses carried out within functionally distinct anatomic subregions of the lymph node found elevated parenchymal innervation in LS animals within all major anatomic subcompartments (). These effects were most pronounced in the paracortex, where innervation density was elevated by 4.12-fold in LS animals relative to their HS counterparts (p <.0001). Innervation density was increased by 3.26-fold in the medulla (p =.0070), and by 3.10-fold in the cortex (p =.0011). Across these anatomic subcompartments, the magnitude of innervation difference associated with Sociability increased in direct proportion to the basal density of sympathetic innervation (i.e., the largest differential density was observed in the paracortex, which also showed the highest basal innervation density). Similar results emerged in analyses of continuous individual differences, with each 1-SD increase in Sociability associated with an average 4.47 ± 0.92 fewer catecholaminergic varicosities per tissue unit in the paracortex (p <.0001), an average −0.91 ± 0.25 fewer in the medulla (p =.0003), and an average −0.24 ± 0.09 fewer in the cortex (p =.0082; data not shown).
Sociability and innervation of lymph node anatomic subcompartments
NGF gene expression
To determine whether observed differences in the catecholaminergic innervation of lymphoid tissue might stem from alterations in the expression of NGF, the key neurotrophic factor that supports growth and maintenance of peripheral sympathetic fibers (Levi-Montalcini, 1987
; Farinas, 1999
), we assayed expression of NGF
mRNA using quantitative RT-PCR. Results showed significantly greater concentration of NGF
mRNA in lymph nodes from LS animals (2.3-fold elevation relative to HS animals, p
<.0001) (). NGF
mRNA concentrations emerged as a quantitatively plausible mediator of Sociability-related differences in innervation in multivariate statistical mediation analyses () (Baron and Kenny, 1986
; Hoyle and Kenny, 1999
), with results indicating that NGF
variation alone could potentially account for 94.7% of Sociability-related differences in lymphoid innervation density (p
=.0099 by Sobel’s test). NGF
concentrations were strongly related to increased innervation density in analyses controlling for Sociability (p
=.0427), and could account for all significant Sociability-related differences in innervation (i.e., no significant residual effect of Sociability remained after control for NGF
NGF gene expression and Sociability-related differences in lymph node innervation
Immunoregulatory gene expression
To assess the potential immunoregulatory correlates of Sociability-related differences in lymph node innervation, we assayed lymph node expression of genes encoding the Th1 cytokine interferon-γ (IFNG) and the Th2 cytokine IL-4 (IL4) (). Relative to HS animals, tissues from LS animals showed slightly greater expression of IFNG (1.35-fold elevation, p =.0340) and significantly greater expression of IL4 (2.48-fold, p =.0002), resulting in a net reduction in the overall equilibrium of Th1/Th2 signaling (.55-fold, p =.0401). Similar results were observed in analyses treating Sociability as a continuous measure of individual differences in relationship to all lymph nodes examined (correlation with IFNG, r = −.526, p =.0058; correlation with IL4, r = −.683, p <.0001), and in relationship to mean gene expression levels within each animal (). The density of parenchymal catecholaminergic varicosities was not significantly correlated with IFNG (r = +.353, p =.1268), but did show a significant positive relationship to IL4 (r = +.488, p =.0291).
IFNG and IL4 mRNA expression as a function of Sociability.
Sociability and IgG response to tetanus vaccination
Antibody response to vaccination
To define the functional impact of Sociability-related differences in lymph node innervation, we assayed secondary IgG responses to a tetanus toxoid booster vaccination. At baseline, tetanus-specific IgG levels varied substantially, and did not differ significantly as a function of Sociability (p =.427) (). Peak tetanus-specific IgG levels were also similar at 2 weeks post-vaccination (p =.835), but LS animals showed significantly lower IgG levels at a 9-month follow-up (p =.018). Similar results emerged when Sociability was analyzed as a continuous measure of individual differences, with nonsignificant associations observed at baseline (r = +.332, p =.446) and during peak response (r = +.116, p =.805), followed by a positive relationship between Sociability and plasma IgG levels at 9 months (r = +.707, p =.048; ). Thus, Sociability-related differences in immunobiology do not appear to substantially affect the acute phase of a secondary antibody response, but LS animals do show poorer long-term maintenance of such responses.
Consistent with the possibility that differences in catecholaminergic innervation might mediate differential antibody responses, parenchymal varicosity density was inversely correlated with tetanus toxoid-specific IgG levels at 9 months post-vaccination (r =−.553, p =.0497). Quantitative mediation analyses estimated that 37.9% of that total relationship could be attributable to differential innervation (p =.0080; ). However, a significant residual effect of Sociability also remained after control for lymph node innervation (p =.0070), suggesting that other factors also contribute. Tetanus toxoid IgG levels at 9 months post-vaccination showed a significant positive correlation with IFNG/IL4 expression ratios (r = +.422, p =.0316).