β-Adrenergic receptor mediated increases in activation and function of natural killer cells following repeated social disruption

Abstract

Natural killer (NK) cells are specialized innate lymphocytes important in the early defense against tumor and virus bearing cells. Many factors influence the immune system's effectiveness against pathogens, including stress. Social disruption (SDR) "primes" macrophages/monocytes and dendritic cells thereby enhancing their anti-microbial function. What remains unclear is whether similar responses are evident in NK cells. Current studies investigated the cellular distribution and activation/inhibitory phenotypes of NK cells in the spleen, lung, and blood of C57BL/6 male mice following SDR. Furthermore, cytolytic activity and anti-viral cytokine production of splenic NK cells were determined. Lastly, β-adrenergic receptor (β-AR) signaling was investigated to determine possible mechanisms behind the SDR-induced NK cell alterations. Results indicated NK cells from SDR mice have increased expression of CD16 and CD69 and reduced NKG2a and Ly49a expression on splenic CD3-/DX5+ NK cells indicative of an activated phenotype, both immediately and 14h post-SDR. Administration of propranolol (10mg/kg; non-selective β-adrenergic receptor antagonist) was shown to block these "priming" effects at the 14h time-point. In the lung, SDR had similar effects on activation and inhibitory receptors 14h post-SDR, however no alterations were evident in the blood besides increased NK cells directly after SDR. Additionally, splenic NK cells from SDR mice had increased CD107a surface expression, cytolytic activity, and IFN-γ production was increased upon costimulation with IgG and IL-2 ex vivo. Collectively, these data suggest that social stress "primes" NK cells in the spleen and lung to be more proficient in their cytolytic and anti-viral/tumor effecter functions through β-adrenergic receptor dependent signaling.

Fig. 1

Mean splenic weight (A) and mean total spleen number (B) 14hrs after six consecutive cycles of social disruption (SDR) compared to home cage control (HCC) animals. * Indicates a significant difference from HCC animals (p’s≤0.01). Bars represent group means ± SEM.

Fig. 2

Representative flow cytometric dot plots for CD3-/DX5+ natural killer cells in the spleen (A and B) and lung (D and E). Average change in the percentage of CD3-/DX5+ natural killer cells in the spleen (C) and lung (F), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. * Indicates a significant difference from HCC animals (p’s≤0.05). Bars represent group means ± SEM.

Fig. 3

Mean percentage of CD3-/DX5+ natural killer cells expressing the activation marker CD16 in the spleen (A), lung (C), and blood (E), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. Mean percentage of CD3-/DX5+ natural killer cells expressing the activation marker CD69 in the spleen (B), lung (D), and blood (F), 14hrs following 6 consecutive cycles of SDR compared to HCC animals.* Indicates a significant difference from HCC animals (p’s≤0.05). Bars represent group means ± SEM.

Fig. 4

Mean percentage of CD3-/DX5+ natural killer cells expressing the inhibitory receptor NKG2a in the spleen (A), lung (C), and blood (E), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. Mean percentage of CD3-/DX5+ natural killer cells expressing the inhibitory receptor Ly49a in the spleen (B), lung (D), and blood (F), 14hrs following 6 consecutive cycles of SDR compared to HCC animals.* Indicates a significant difference from HCC animals (p’s≤0.05). Bars represent group means ± SEM.

Fig. 5

Mean percentage of splenic CD3-/DX5+ cells expressing the degranulation marker CD107a (A), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. Mean percentage of activated splenic CD3-/DX5+/CD16+ cells expressing the degranulation marker CD107a (B), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. * Indicates a significant difference from HCC animals (p’s≤0.05). Bars represent group means ± SEM.

Fig. 6

Mean total isolated DX5+ splenic cell numbers (A), 14hrs following 6 consecutive cycles of SDR compared to HCC animals. Fourteen hours post-SDR, splenic DX5+ cells were isolated, incubated for 48hrs with media, IgG, IL-2, or a combination of IgG/IL-2. Mean IFN-γ concentration was determined by ELISA (B). * Indicates a significant difference from DX5+ NK cells from HCC animals treated with IL-2/IgG and all other control groups (p’s≤0.05). + Indicates a significant difference from DX5+ NK cells from SDR-treated animals treated with IL-2/IgG and all other control groups (p’s≤0.05). Bars represent group means ± SEM.

Fig. 7

Mean percentage of CD3-/DX5+ natural killer cells (A) expressing the activation receptors CD16 (B) and CD69 (C) in the spleen immediately following the last cycle of SDR. Mean percentage of CD3-/DX5+ natural killer cells expressing the inhibitory receptors NKG2a (D) and Ly49a (E) in the spleen immediately following the last cycle of SDR. Mean percentage of CD3-/DX5+ natural killer cells expressing the degranulation marker CD107a (F) in the spleen immediately following the last cycle of SDR. * Indicates a significant difference from HCC animals (p’s≤0.05). Bars represent group means ± SEM.

Fig. 8

Mean percentage of CD3-/DX5+ natural killer cells (A) expressing the activation receptors CD16 (B) and CD69 (C) in the spleen 14hrs following SDR and/or propranolol administration. Mean percentage of CD3-/DX5+ natural killer cells expressing the inhibitory receptors NKG2a (D) and Ly49a (E) in the spleen 14hrs following SDR and/or propranolol administration. Mean percentage of CD3-/DX5+ natural killer cells expressing the degranulation marker CD107a (F) in the spleen 14hrs following SDR and/or propranolol administration. Different letters represents a statistical significance among groups (p’s≤0.05). Bars represent group means ± SEM.

Fig. 9

Mean percent cytotoxicity displayed from IL-15 stimulated splenocytes taken from SDR-treated and control animals that received 10mg/kg propranolol or vehicle injections 1hr prior to each cycle of social disruption. * Indicates a main effect of Propranolol treatment regardless of stress condition within each E:T ratio (p’s≤0.05). + Indicates a main effect of Stress within each E:T ratio when comparing HCC to SDR-treated mice receiving vehicle treatment. (p’s≤0.05). Bars represent group means ± SEM.