In vitro superfusion method for the investigation of nerve-immune cell interaction in murine spleen

Immune defence mechanisms can be modulated by brain function. To study such interactions, an in vitro method was developed to examine the release of cytokines and norepinephrine (NE) after electrical stimulation. Slices of mouse spleen were placed in chambers with a volume of 80 μl and superfused with culture medium. To characterize electrically evoked NE release and cell viability a suitable stimulation protocol was developed using of ³H]NE. As parameter for immune function, modulation of interleukin-6 (IL-6) release by the spleen cells brought about by electrical stimulation was investigated. Splenic ³H]NE overflow was calcium-dependent, tetrodotoxin-sensitive and elicited by 54 mM potassium. Electrically evoked ³H]NE release at 22 h was about 80% of the release at 5.3 h. Electrical stimulation substantially reduced IL-6 secretion at 6 h (control: 143.4 ± 14.3 vs. electrical: 71.3 ± 7.9 pg/ml/10⁶ leukocytes, P = 0.0001). This effect was inhibited in a concentration-dependent manner by the β-adrenergic antagonist propranolol (P = 0.0298, EC₅₀approx. 10⁻⁷M). In conclusion, this new technique allows long-term investigation of cell function in slices of murine spleen. In addition, these are the first in vitro data indicating the presence of a functional neuroimmunological link in murine lymphoid tissue.