Stress-induced suppression of in vivo splenic cytokine production in the rat by neural and hormonal mechanisms.

The mechanisms mediating the effects of stress on immune function have yet to be fully described. In vitro studies have demonstrated a role for both the sympathetic nervous system (SNS) and the hypothalamic pituitary adrenal axis (HPAA) in regulating immune responses following exposure to various stressors. The purpose of the present set of experiments was to determine the in vivo contribution of the HPAA and SNS in regulating the effects of stress on lipopolysaccharide (LPS) induced splenic cytokine production. For this, rats with combinations of sham surgeries, splenic nerve cuts (SNC), and adrenalectomies (ADX) were exposed to 15 min of 1.6 mA intermittent footshock immediately following the intravenous (i.v.) injection of 0.1 microg of LPS. Although footshock was immunosuppressive to most indices of cytokine production, neither SNC nor ADX alone blocked the effects of stress on splenic immune function. However the combination of these two manipulations significantly abrogated the immunosuppressive effects of stress on cytokine production. Adrenal demedullation of animals with a SNC demonstrated that the SNS, not the HPAA, was primarily responsible for the immunosuppressive effects of stress.

Contribution of the adrenal glands and splenic nerve to LPS-induced splenic cytokine production in the rat.

Both the hypothalamic pituitary adrenal axis (HPAA) and the sympathetic nervous system (SNS) can inhibit immune function and are regarded as the primary efferent pathways for neural-immune interactions. To determine if this relationship is maintained in vivo in response to an inflammatory stimulus, rats were injected intravenously (iv) with various doses of lipopolysaccharide (LPS) and splenic cytokine mRNA and protein levels were measured at several dose and time intervals post-injection. The spleen was chosen as the target organ because both the neural and hormonal inputs to the spleen can be selectively removed by splenic nerve cut (SNC) and adrenalectomy (ADX), respectively. Data from our dose response studies established that maximum levels of splenic cytokines were induced in response to relatively low doses of LPS. Minimal changes in LPS-induced splenic cytokine levels were observed in response to ADX, SNC, or a combination of the two procedures across several doses of LPS. These results suggest that there are aspects of immune regulation that are functionally removed from these central modulatory systems and that the counter-regulatory responses induced by LPS have minimal impact on the concurrent induction of cytokines by this inflammatory stimulus. The conceptual model of neural-immune regulation as an inhibitory feedback system, at least with regards to the early activational effects induced by an inflammatory stimulus, was not supported by these studies.

Arrest of B16 melanoma cells in the mouse pulmonary microcirculation induces endothelial nitric oxide synthase-dependent nitric oxide release that is cytotoxic to the tumor cells.

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only approximately 55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis.