Antigen-activated immune cells acutely release cytokines which, besides their effects on the immune system, increase hypothalamopituitary-adrenocortical (HPA) function to counteract the inflammatory process. The present study was designed to test, using in vitro paradigms, whether there exists a hypothalamic and/or a median eminence site of action, whereby different substances derived from the immune system could stimulate the CRH and/or the arginine-vasopressin (AVP) neuronal pathway. For this purpose, whole medial basal hypothalamus (containing the median eminence) were dissected from female rats and incubated in vitro with several concentrations of interleukin-1 (IL-1)β interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, thymosin fraction 5 (TF5) or bacterial lipopolysaccharide (LPS). After a 40-min incubation period, the amounts of CRH and AVP released into the incubation medium were measured by specific radioimmunoassays (RIAs). Additional experiments were carried out by superfusing isolated rat median eminence fragments with the different test substances; CRH and AVP released into the medium were also measured by RIAs. The results indicated that IL-1β (10^–11 to 10^–7M), IL-6 (0.06 × 10^–10 to 0.4 × 10^–10M), TNF-α(6 × 10^–9 to 6 × 10^–7M) and TF5 (5-500 µg/ml) but not LPS (1-100 ng/ml) significantly enhanced hypothalamic CRH secretion above baseline in a concentration-related fashion. Additionally, superfusion experiments demonstrated that, among all test substances, only IL-6 possesses a direct and dose-dependent CRH-releasing activity at the median eminence level. Conversely, no preparation enhanced basal AVP release in either in vitro design. For the pure cytokines, hypothalamic incubation studies further suggested a rank order of CRH releasing activity, as follows: IL-1β > TNF > IL-6; the partial purifed thymic preparation, TF5, was approximately as potent as IL-1β whereas LPS had no effect. Our results provide strong evidence for a hypothalamic site of action of several cytokines on CRH secretion. Additionally they suggest that IL-6 also contributes to stimulate HPA function by inducing CRH output from neuron terminals present at the median eminence level, where no effective blood-brain barrier is present. These data further suggest that the CRH neuronal system is one of the most important interfaces between the immune and neuroendocrine axes and that vasopressin is not involved in such an effect.

Bacterial lipopolysaccharide (LPS) stimulates the hypothalamo-pituitary-adrenal axis by a mechanism involving the release of cytokines, which activate the CRH-ACTH system and, as a result, increase glucocorticoid secretion. In the present study we investigated the possibility that endogenous sex hormones modulate the in vivo endotoxin-stimulated adrenal and immune responses in adult BALB/c mice. In preliminary experiments we determined that the maximal glucocorticoid release in response to LPS (50 micrograms, ip) administration was reached 2 h after treatment. The endotoxin effect on the adrenal and immune responses was then tested in male, randomly cycling female, 20-day-gonadectomized and 20-day-gonadectomized mice treated with either testosterone or estradiol. In addition, in vitro experiments were performed to determine whether 1) LPS exerts any direct effect on basal and ACTH-stimulated corticosterone release, and 2) adrenal function is influenced by bilateral gonadectomy and sex steroid therapy. Our results indicate that 1) randomly cycling female mice have significantly more pronounced corticosterone secretion than males 2 h after endotoxin injection, although the tumor necrosis factor responses were similar; 2) the response of the hypothalamo-pituitary-adrenal axis to endotoxin stimulation in female mice was invariable throughout the different stages of the normal estrous cycle; 3) gonadectomy leads to enhanced (P < 0.05) adrenal and immune responses to LPS stimulation compared to the responses in shams; 4) the endotoxin-elicited adrenal and immune overresponses observed in gonadectomized mice are reversed by testosterone treatment, regardless of sex; 5) LPS does not directly modify spontaneous and ACTH-stimulated adrenal corticosterone secretion; and 6) gonadectomy alone or combined with sex steroid therapy does not increase the in vitro adrenal response to ACTH stimulation. Our findings further suggest an evident neuroendocrine-immunological sexual dimorphism during the acute phase of inflammatory processes.

It is known that in vivo administration of bacterial endotoxin activates immune cells to release cytokines, these substances in turn enhancing hypothalamo-pituitary-adrenal (HPA) axis function; additional evidence supports the existence of an immune-neuroendocrine sexual dimorphism. In the present study, we investigated: (1) the in vivo response of both the HPA and the immune systems to single and repeated endotoxin administrations in mice, and (2) whether testosterone possesses a modulatory effect on neuroendocrine-immune function under endotoxemia. For these purposes, adult male BALB/c mice were orchidectomized (Odx) or sham-operated and injected s.c, on alternate days, with either corn oil alone (Odx and Sham) or containing 20 µg of testosterone (Odx+T) until animals were killed. One week after surgery, different groups of mice were treated i.p. with bacterial lipopolysaccharide (LPS; 25 µg per mouse) in a single (day 1 D1) or repeated (at 24-hour intervals for 5 consecutive days) form. Animals were decapitated (on Dl, D3 and D5 of the treatment) 2 h after the last injection of either vehicle alone or containing LPS (the two groups were run in parellel). Trunk blood was collected and the whole medial basal hypothalamus (wMBH), the anterior pituitary (AP) and adrenal glands were dissected. Plasma tumor necrosis factor-alpha (TNFα), ACTH and corticosterone (B) concentrations as well as wMBH CRH, AP ACTH and adrenal B contents were determined by specific assays. Our results indicate: (1) a significant decrease in mice body weight after repeated LPS injections, regardless of the group; (2) a sex steroid environment-independent increase in plasma ACTH, B and TNFα levels 2 h after a single LPS injection; (3) that all these responses decreased after repeated LPS administration; (4) that a significant rise in adrenal B content occurred 2 h after the first, third and fifth LPS treatments and that such an effect was significantly enhanced by Odx and fully reversed by Odx followed by T therapy, and (5) that while hypothalamic CRH and AP ACTH were not modified by endogenous sex steroid environment or endotoxin administration, Odx significantly enhanced the LPS-induced ACTH release only 2 h after a single LPS treatment. Our findings suggest that endogenous TNFα plays a mediatory role in the acute activation of HPA axis function after LPS and that under persisting endotoxemia both immune and HPA functions are decreased. Finally, testosterone has an inhibitory role on adrenal glucocorticoidogenesis during endotoxic shock.

Cytokines secreted by bacterial endotoxin-activated immune cells are substances known to stimulate the hypothalamo-pituitary-adrenal (HPA) axis function. The present study was designed to better understand the effect of different mediators of inflammation, such as cytokines and histamine, on the acute HPA axis response induced by administration of a single dose of bacterial lipopolysaccharide (LPS) in adult, male, BALB/c mice. Two different experimental designs were set up. In the first design, mice (n = 8–11 per group) were injected i.p. with LPS (90 µg/kg body weight) and killed by decapitation 2 or 6 h after treatment. Additional groups of mice were pretreated i.p. 12 h before LPS treatment with: (a) 3–4 mg IgG/kg body weight of either an anti-tumor necrosis factor-α (TNF)-α, anti-interleukin (IL)-1β- or IL-6 serum; (b) IL-1 receptor antagonist (IL-1ra) (120 µg/kg body weight) immediately before LPS and also 3 h later (when animals were killed 6 h after LPS injection), or (c) 182 µg/kg body weight of clemastine, an antagonist of H₁ histaminergic receptors, 2 h before LPS treatment; animals were killed in a similar fashion to that described for treatment with LPS alone. In the second experimental design, mice were pretreated (i.p., 10 mg/kg body weight, 30 min before administration of a similar dose of LPS) with different blockers of histaminergic pathway function such as: (a) mepyramine, another anti-H₁, (b) cimetidine, an H₂ receptor blocker, and (c) Rα-methylhistamine dihydrochloride, an H₃ presynaptic receptor agonist which inhibits histamine synthesis and output. These animals were then killed by decapitation 40 min after endotoxin treatment. After decapitation, trunk blood was collected for further determination of plasma levels of both ACTH and corticosterone (B) by specific assays. The results indicate that plasma levels of both ACTH and B were several-fold increased over baseline, 2 and 6 h after LPS administration. Two hours, the effect of LPS on ACTH output was not modified by pretreatment with anti-IL-1β IgG, anti-IL-6 IgG, anti-TNF-α IgG nor with IL-1ra, although IL-1ra treatment was able to fully block the IL-1β (35 µg/kg body weight)-stimulated HPA axis function, 1 and 2 h after cytokine administration. Six hours after LPS administration, anti-IL-1β and anti-TNF-α IgGs were both able to significantly reduce HPA axis response to the endotoxins, whereas anti-IL6 IgG had no effect. Anti-IL-1β IgG reduced only B secretion, whereas anti-TNF-α IgG decreased both ACTH and B secretion. The blockade of histaminergic pathway functions did not impede the LPS-induced ACTH and B release regardless of the product employed. The present results indicate that TNF-α, and to a lesser extent IL-1β, are the most relevant cytokines involved in HPA axis response to endotoxin administration. Our data also suggest that, in mice, HPA axis activation after infection appeared to be independent of stimualtion of the histaminergic pathway.

Various evidence suggests a bidirectional circuit between the immune and neuroendocrine systems. Because of the well-known role of the thymus in the regulation of the immune function, we designed this study to determine whether the lack of thymus may affect hypothalamo-pituitary-adrenal (HPA) axis activity by using both in vivo and in vitro paradigms in Swiss nude (athymic) and BALB/c (normal) mice. Eight-week-old female mice of both strains were used to study: (a) the in vivo response of the HPA axis to various stress stimuli acting at either hypothalamic (ether vapor inhalation, insulin administration), pituitary (CRH injection) or adrenal (ACTH treatment) level and (b) the in vitro response of pituitary and adrenal cells to CRH and ACTH stimulation, respectively. The results indicate that: (1) basal plasma ACTH levels were significantly (p < 0.05) higher in Swiss nude than in BALB/c mice, whereas basal plasma corticosterone (B) concentrations were similar in both strains of mice; (2) the stress-induced release of ACTH and B in plasma were significantly (p < 0.05) lower in Swiss nude than in BALB/c mice, regardless of the stimulus applied; (3) the in vitro pituitary response to CRH and the adrenal response to ACTH were significantly (p < 0.05) lower in Swiss nude than in BALB/c mice, whereas (4) hypothalamic CRH and pituitary ACTH contents were similar in both strains, adrenal B concentration was significantly (p < 0.05) lower in athymic mice; in addition, the nude mice adrenal glands were larger than those of BALB/c animals, due to marked hypertrophy of the zona fasciculata. In conclusion, our results indicate that athymic nude mice have a blunted HPA axis response to various stress stimuli; this defect seems to reside at both the pituitary and adrenal levels. It remains to be determined whether this impairment is directly related to the immunodeficiency of this animal model.