Selective depletion of macrophages prevents pituitary-adrenal activation in response to subpyrogenic, but not to pyrogenic, doses of bacterial endotoxin in rats.

The mechanisms by which bacterial endotoxin [lipopolysaccharide (LPS)] stimulates the hypothalamo-pituitary-adrenal axis (HPAA) have not been elucidated. The present study was designed to investigate the involvement of macrophages in plasma ACTH and corticosterone responses to LPS administration in rats using selective in vivo macrophage depletion. Intraperitoneal administration of subpyrogenic doses of LPS to normal rats resulted in elevated plasma ACTH and corticosterone concentrations, measured 2 h later. The response showed a remarkable steep dose relationship, with minimal effective doses between 0.5-1.5 micrograms (ACTH) and 0.5 micrograms or less (corticosterone)/kg BW. Plasma PRL, LH, and catecholamine (norepinephrine, epinephrine) levels were not significantly changed under the conditions used. Only at 6 h after LPS administration was a small elevation of norepinephrine noted. To deplete macrophages, rats were injected with liposomes encapsulated with dichloromethylene diphosphonate (Cl2MDP). Histochemical (acid phosphatase) and immunocytochemical techniques (monoclonal antibodies to rat macrophages coded ED1 and ED3) were applied to examine the efficiency of macrophage elimination by the Cl2MDP liposomes in cytospins of peritoneal exudates and in sections of the liver and spleen. Since cells of the macrophage lineage are considered to be the main source of IL-1 in the circulation, we also measured circulating levels of immunoreactive interleukin-1 beta (IL-1) concentrations in control and Cl2MDP liposome-treated rats by the use of a newly developed RIA. Reduced numbers of macrophages were seen in peritoneal lavages of Cl2MDP liposome-treated animals, whereas the morphological appearance and numbers of mast cells, granulocytes, and T-cells were unaffected. Similarly, macrophages were effectively eliminated in the spleen, mesenteric lymph nodes, and liver, as inferred from the reduction of macrophage staining in these organs. Plasma IL-1 concentrations could only be detected in response to a pyrogenic (2.5 mg/kg, iv) and not to a subpyrogenic (0.025 mg/kg, ip) dose of LPS. The increase in plasma IL-1 concentrations in response to the pyrogenic dose of LPS, reaching levels of 20-40 ng/ml in control rats, was blunted in animals treated with the Cl2MDP liposomes. Macrophage depletion by Cl2MDP liposomes did not affect either resting plasma corticosterone levels or the corticosterone response to ether exposure. At subpyrogenic doses of LPS, plasma ACTH and corticosterone responses were completely prevented by macrophage depletion. In contrast, at pyrogenic doses of LPS, plasma ACTH and corticosterone responses were not significantly affected by depleting macrophages. These data demonstrate that activation of the HPAA by a subpyrogenic dose of LPS is macrophage dependent. However, macrophage-independent mechanisms mediate activation of the HPAA in response to a pyrogenic dose of LPS.