High-affinity substance P binding sites of neurokinin-1 receptor type autoradiographically associated with vascular sinuses and high endothelial venules of human lymphoid tissues.

BACKGROUND

Factors that affect leukocyte-endothelial cell interaction in high endothelial postcapillary venules and vascular sinuses of lymphatic tissues indirectly regulate immune function. Studies in sheep demonstrated that acute infusion of substance P (SP) into cannulated popliteal lymph node afferent lymphathics produced a marked and prolonged increase in the output of lymphocytes into nodal efferent lymph. The proposed mechanism is an influence of SP on lymph vascular systems. Such functional importance of the immunoregulatory properties of SP in man is unknown.

EXPERIMENTAL DESIGN

The expression and distribution of SP receptors in human lymphoid tissue was investigated using slide-mounted fresh-frozen sections of lymph node, hyperplastic tonsillitis, and spleen for ligand binding and autoradiographic studies.

RESULTS

Specific binding of radiolabeled SP to lymph node and tonsils reached a plateau within approximately 40 minutes, being half-maximal at 20 minutes. The specific binding was between 65 and 75% of total binding. In contrast, iodinated neurokinin A under identical incubation conditions, did not significantly associate with the tissues. Neither the SP nor the neurokinin A tracer specifically associated with spleen. Binding specificity of radiolabeled SP was analyzed in binding competition experiments. Synthetic SP, SP (3-11), a selective neurokinin-1 agonist and a neurokinin-1 antagonist competed with the specific binding of SP to lymph node and tonsil sections. The half-maximal inhibition of binding was obtained at a concentration of about 0.5 nmol/liter of SP. The fragment SP (1-4) and selective neurokinin-2 ligands did not compete with the specific binding of SP. Scatchard and nonlinear algorithm analyses revealed two binding sites for SP. For lymph node and tonsil, one site showed a high affinity of about 0.4 nmol/liter and 0.7 nmol/liter and a low capacity for SP, respectively. The second site exhibited a lower affinity of 100 nmol/liter and 50 nmol/liter and a higher capacity for SP in lymph node and tonsil, respectively. Autoradiographic localization of the binding sites shows a very high concentration of silver grains when compared with controls. The reaction occurred mainly over vascular sinuses and high endothelial venules with heterogenous density. Silver grain accumulation was also noticed over the marginal sinuses of the B cell follicles.

CONCLUSIONS

The biochemical results indicate the presence of neurokinin-1 receptors in human lymph node and tonsil. We suggest that the neurokinin-1 receptors localized to vascular tissues of lymph node and tonsil mediate the affects of SP on lymphocyte traffic, and we propose that SP plays a regulatory role in lymphocyte homing in man.