Ronit Avitsur

Social stress induces glucocorticoid resistance in subordinate animals.

Introducing an aggressive intruder into a cage of mice (social disruption, SDR) resulted in intense fighting and defeat of the cage residents. Defeat was accompanied by elevated levels of serum corticosterone and nerve growth factor (NGF). Repeated exposure to an intruder induced a state of glucocorticoid resistance in peripheral immune cells. The present study sought to examine the behavioral factors that mediated the development of glucocorticoid resistance following SDR. Glucocorticoid resistance developed in animals that exhibited a subordinate behavioral profile, which consisted of a low tendency for social investigation and a high level of submissive behavior in response to the intruders attacks. Glucocorticoid resistance was also linked to the presence of injuries due to fighting, but not to changes in systemic levels of either corticosterone or NGF. Since a submissive behavioral profile is associated with increased risk for injuries due to fighting, it may be that the development of glucocorticoid resistance is an adaptive mechanism that allows the inflammatory component of wound healing to occur in the presence of high levels of corticosterone. Together, these findings demonstrate that the outcomes of social stress may be modified by physiological changes associated with wounding, as well as by behavioral variables such as social status.

Social stress increases the susceptibility to endotoxic shock

The influence of social disruption stress (SDR) on the susceptibility to endotoxic shock was investigated. SDR was found to increase the mortality of mice when they were challenged with the bacterial endotoxin lipopolysaccharide (LPS). Histological examination of SDR animals after LPS injection revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen, indicating inflammatory organ damage. In situ hybridization histochemical analysis showed that the expression of the glucocorticoid receptor mRNA was down-regulated in the brain and spleen of SDR animals while the ratio of expression of AVP/CRH-the two adrenocorticotropic hormone secretagogue, increased. After LPS injection, the expression of pro-inflammatory cytokines, IL-1beta and TNF-alpha, was found significantly higher in the lung, liver, spleen, and brain of the SDR mice as compared with the LPS-injected home cage control animals. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock and suggest that the development of glucocorticoid resistance and increased production of pro-inflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.

Social Disruption, Immunity, and Susceptibility to Viral Infection: Role of Glucocorticoid Insensitivity and NGF

Abstract: Glucocorticoid (cort) responses have been shown to suppress inflammatory reactions by inhibiting the trafficking of immune cells. Recently, it was demonstrated that restraint stress (RST) and psychosocial stress (social reorganization; SRO) differentially affected the pathophysiology and survival in the mouse influenza viral infection model. While both stressors activated the HPA axis, only SRO affected survival. in RST, elevated cort diminished recruitment of inflammatory cells following intranasal challenge of C57BL/6 mice with A/PR8 virus. However, infected SRO mice developed hypercellularity in the lungs and were more likely to die from lung consolidation than controls. Since elevated cort failed to be anti‐inflammatory in SRO mice, the hypothesis that psychosocial stress induced steroid insensitivity was tested. An in vitro cort suppression test was performed by stimulating splenocytes from SRO and control mice with mitogen in the presence or absence of cort. Proliferation of ConA‐stimulated cells was inhibited by cort in a dose‐dependent fashion in controls, but splenocytes from SRO mice stimulated with ConA were resistant to cort‐induced suppression. Thus, psychosocial stress induced a state of steroid insensitivity. SRO also induced the release of nerve growth factor (NGF) from the salivary glands into circulation; plasma NGF correlated with development of steroid insensitivity. NGF has been reported to negatively regulate the expression of type II glucocorticoid receptors, and thus may be a key factor in the induction of steroid insensitivity.

Interleukin-6 and the development of social disruption-induced glucocorticoid resistance

Following social disruption (SDR) stress in male mice, corticosterone resistance of splenocytes was accompanied by enhanced LPS-stimulated interleukin (IL)-6 secretion. The present study examined the role of IL-6 in the development of corticosterone resistance. Addition of IL-6 to control splenocyte cultures did not induce corticosterone resistance. SDR also elevated IL-6 in plasma and liver, but not in spleen. IL-6 deficient mice that were exposed to SDR developed glucocorticoid resistance despite the absence of systemic IL-6. These findings suggest that although SDR enhanced IL-6 responses, IL-6 was not essential for the development of stress-induced splenocyte corticosterone resistance.

Molecular mechanisms of glucocorticoid resistance in splenocytes of socially stressed male mice.

Splenocytes from socially stressed male mice display functional glucocorticoid (GC) resistance, viz., the antiproliferative effects of GC on lipopolysaccharide (LPS)-stimulated splenocytes is absent. In this study, we investigated changes in the structure and function of the glucocorticoid receptor (GR) in socially stressed animals. Changes of GR at both DNA and RNA levels were excluded. Reduced GR function was restricted to macrophages (CD11b(+)) in association with impaired nuclear translocation of GR after GC stimulation. Consequently, GC failed to block the activation of NF-kappa B in these cells. Thus, impaired nuclear translocation of GR and the lack of transcriptional suppression of NF-kappa B by GC were identified as the molecular mechanisms responsible for the observed GC resistance in spleens of socially stressed mice.

Social disruption-induced glucocorticoid resistance: kinetics and site specificity

Social disruption (SDR) of male mice has been shown to induce a state of functional glucocorticoid (GC) resistance in splenocytes. The present study demonstrated that GC resistance developed following repeated, but not acute exposure to SDR. GC resistance was long-lasting and persisted for at least 10 days after stress. In contrast, SDR did not alter cytokine secretion from peritoneal mononuclear cells treated with corticosterone. These findings suggest that SDR-induced GC resistance may be restricted to specific sites such as the spleen.

Expression of glucocorticoid resistance following social stress requires a second signal

Stimulation of splenocytes from socially stressed mice [social disruption (SDR)] with Gram‐negative bacterial lipopolysaccharide (LPS) revealed a state of functional glucocorticoid (GC) resistance. LPS‐stimulated splenocytes were less sensitive to the inhibitory effects of corticosterone. This study demonstrated that activation signals were required for the expression of splenic GC resistance. The results demonstrated that six cycles of SDR induced splenomegaly and increased the number of CD11b‐positive monocytes. SDR also increased the viability of cultured, nonstimulated splenocytes, and addition of corticosterone reduced the viability of these cells in a dose‐dependent manner. However, following stimulation with LPS, the sensitivity of SDR splenocytes to GC was reduced. Similar results were obtained using lipid A, a fraction of the LPS molecule that binds to Toll‐like receptor (TLR)4. Furthermore, C3H/HeJ mice that do not possess a functional TLR4 molecule responded to SDR with an increased number of CD11b‐positive monocytes in the spleen and increased viability of nonstimulated splenocytes. However, neither LPS nor lipid A stimulation resulted in the expression of GC resistance. Together, these findings suggest that the expression of GC resistance in response to SDR requires a second signal that can be provided by ligation of TLR4.

Social stress alters splenocyte phenotype and function

Social stress of group-housed male mice induced a state of functional glucocorticoid (GC) resistance in splenocytes. The following studies examined the effects of paired-fighting (PF) stress on immune cell distribution and function in spleens of male mice. Following six daily PF stress sessions, splenic monocytes and neutrophils increased and lymphocytes decreased. PF also altered the distribution of CD62L and CD11b positive monocytes. Additionally, PF augmented proliferation and lowered the sensitivity of LPS-stimulated splenocytes to the antiproliferative effects of corticosterone, suggesting that PF induced a state of GC resistance in splenocytes. Together, these findings indicate that social stress altered phenotype and function of splenic immune cells. These findings may have implications for the healing of bite wounds that are often associated with social stress in rodents.

Physical defeat reduces the sensitivity of murine splenocytes to the suppressive effects of corticosterone

Social disruption (SDR) in male mice reduces the sensitivity of their splenocytes to the actions of glucocorticoids. To determine whether physical defeat is necessary for the development of this reduced sensitivity, a modification of the SDR paradigm was employed in which mice were exposed to fighting conspecifics in the presence or absence of physical contact. This was accomplished by dividing a cage of 5 resident male C57BL/6 mice in half with a wire mesh partition so that 2 of the mice in the cage (SDR Physical Contact mice) fought and were defeated by an aggressive male C57BL/6 intruder that was placed into the cage for 2h for up to 6 days, while the remaining 3 resident mice (SDR Sensory Contact mice) were on the opposite side of the partition and thus prevented from physically interacting with the intruder. Although both the SDR Physical Contact and the SDR Sensory Contact mice had significantly elevated corticosterone levels and displayed submissive postures toward the intruder, only the SDR Physical Contact animals developed functional glucocorticoid resistance. The viability of LPS-stimulated splenocytes cultured from the SDR Physical Contact mice was not affected by pharmacological doses of corticosterone, whereas splenocyte viability was significantly reduced by corticosterone in cultured cells from SDR Sensory Contact and control mice. This study indicates that exposure to a stressful environment in the absence of physical attack does not reduce the sensitivity of murine splenocytes to the suppressive effects of corticosterone.

Experimental Models of Stress and Wound Healing

Experimental animal models have been used to examine stress-induced interactions among the nervous, endocrine, and immune systems. Generally, the response to stress results in a body-wide set of physiologic adaptations, mediated through the activation of neuroendocrine pathways that intersect and modulate inflammatory and immune responses. These interacting responses modulate diverse physiological processes including the initiation of tissue repair and wound healing. Two different stressors were used to activate neuroendocrine responses to study their impact on wound healing: restraint (RST) and social disruption (SDR). Previous studies showed that both stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, resulting in elevated plasma levels of the corticosterone (cort) response and modulation of pro-inflammatory cytokine response. To test the effects of stress-induced HPA activation on inflammatory responses and wound healing, a 3.5-mm cutaneous punch biopsy wound was placed on the dorsal surface of control and stressed (RST or SDR) C57BL/6 male mice and the kinetics of wound healing were studied over 10 days. RST slowed wound healing in inbred C57BL/6 mice, whereas the wounds on SDR mice healed in a fashion similar to the non-stressed, home cage controls.