Transient sex-related changes in the mice hypothalamo–pituitary–adrenal (HPA) axis during the acute phase of the inflammatory process

The potential role of endogenous sex hormones in regulating hypothalamo–pituitary–adrenal (HPA) axis function was investigated after a single injection of endotoxin in adult (8 week old) BALB/c mice of both sexes. The effect of LPS on plasma ACTH, corticosterone (B), testosterone and oestradiol (E) levels and on anterior pituitary (AP) ACTH and adrenal B contents at different times after treatment was studied. The results indicate that: (a) basal B but not ACTH plasma levels were significantly higher in female than in male mice; (b) LPS significantly increased both ACTH and B plasma levels over the baseline 2 h after injection, both hormone levels being higher in female than in male mice; (c) although plasma ACTH concentrations recovered the basal value at 72 h after LPS in animals of both sexes, plasma B levels returned to the baseline only at 120 h after treatment; (d) E plasma levels significantly increased 2 h after LPS and returned to the baseline at 72 h post-treatment, in both sexes; (e) at 2 h after LPS, testosterone plasma levels significantly decreased in male mice and increased in female mice, recovering the baseline level at 120 and 72 h after LPS, respectively; (f) AP ACTH content was similar in both sexes in basal condition and it was significantly diminished 72 h post-treatment without sex difference; whereas AP ACTH returned to basal content 120 h after LPS in males, it remained significantly decreased in females; (g) basal adrenal B content was higher in female than in male mice, and it significantly increased in both sexes 2 h post-LPS, maintaining this sex difference. Whereas adrenal B returned to basal content 72 h after treatment in male mice, it remained significantly enhanced up to 120 h post-LPS in female animals. The data demonstrate the existence of a clear sexual dimorphism in basal condition and during the acute phase response as well as in the recovery of the HPA axis function shortly after infection.


Introduction
Gram-negative bacterial sepsis is still one of the major causes of morbidity and mortality in humans and other animal species. The A portion of the bacerial lipopolysaccharide (LPS) interacts in the infected host inducing the activation of immunerelated cells, which in turn release cytokines. 2 The most important cytokines released during the acute phase of the endotoxic shock are interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF); 3'4 these substances act synergistically, although through different mechanisms, to induce pathophysiological symptoms observed during infection." It has been shown in different animal models that the infusion of 1L-1 and TNF mimick the LPS induced effects during infection, [7][8][9] and that the treatment of mice with anti-TNF antibodies (C) 1993 Rapid Communications of Oxford Ltd significantly diminishes the lethal effect of LPS in these animals. 1 Accumulated evidence indicates that both cytokines activate the hypothalamo-pituitary-adrenal (HPA) function by acting at multiple levels of this axis. 1>16 There is also clinical and experimental evidence indicating that gonadal steroids can modulate the immunological function. It has been established previously that a sexual dimorphism exists in the immune response to different noxious substances. 17'18 Skin allograft rejection time in mice is longer in males than in females. 9 Male F1 N2B/N2W mice are less susceptible than females to the development of autoimmune lupus, but they 2O will die if gonadectomized. In addition, mitogen driven plaque forming cell response of Blymphocytes in vitro is inhibited by androgens. 2 This evidence is strongly supported by the fact that specific receptors for sex hormones are present in organs responsible for the immune response. 22 Taking into account the important role of the Results Sex difference in the LPS stimulated A CTH and glucocorticoid release in plasma" Figure 1 shows the time  female mice were significantly (p < 0.05) higher than those attained in male animals. As is also shown in Fig. 1, plasma ACTH levels returned to baseline 72 h after LPS injection in both groups of mice. Figure 2 shows plasma glucocorticoid levels before and at different times after endotoxin administration. Basal plasma B values were significantly (p < 0.05) higher in female than in male mice. Coinciding with the rise in plasma ACTH levels as a consequence of endotoxin administration, plasma B concentrations were significantly (p < 0.05) increased over the baseline 2 h after LPS in a dimorphic fashion, since values in female mice were significantly higher than in male animals. Figure 2 also shows that plasma B levels were still higher than baseline 72 h post-LPS and returned to basal levels 120 h after endotoxin treatment in animals of both sexes.    Table 1 shows plasma testosterone and E levels before and at different times after LPS administration in mice of both sexes. In male animals, basal plasma testosterone levels significantly (p < 0.05) decreased 2 h after LPS with minimal values at 72 h after endotoxin; then the baseline recovered at 120 h post-LPS.
Conversely, in female mice, plasma testosterone levels significantly (p < 0.05) increased over the baseline at 2 h after endotoxin and returned to basal levels at 72 h after treatment.
On the other hand, plasma E values significantly (p < 0.05) increased over the baseline 2 h after endotoxin treatment in mice of both sexes, then returned to basal levels at 72 h after treatment. As seen in Table 1, plasma E levels in basal condition were of a similar magnitude in animals of both sexes; however, 2 h post-LPS, plasma E levels were significantly (p < 0.05) higher in female than in male animals and the same occurred 72 and 120 h after endotoxin treatment.

Discussion
The present study clearly demonstrates a sexual dimorphism in the HPA response during both the acute and the recovering phases of endotoxic shock. This observation is in agreement with previous data indicating the existence of a sexual dimorphism in the immune response to different stimuli. 17'18 Although it has recently been reported that LPS treatment enhances HPA axis function in a sexually dimorphic fashion, 26 to our knowledge this is the first observation indicating a sex hormone basis for the transient changes in the neuroendocrine immune response during acute inflammation.
Our results indicate that" (a) there exists a sexual dimorphism in the mice HPA axis status in basal condition, such as higher plasma levels and adrenal content of corticosterone in female than in male animals; (b) the LPS induced ACTH and B secretion in plasma is higher in female than in male mice; (c) whereas ACTH plasma levels returned to baseline 72 h post-LPS, plasma B values remained elevated at this time post-treatment, regardless of the sex of the animals; (d) a significant decrease in AP ACTH was found in animals of both sexes at 72 h after LPS and this parameter returned to baseline at 120 h after treatment in males but not in females; (e) adrenal B content significantly increased 2 h after LPS treatment in animals of both sexes and with maintained sex difference, and returned to baseline values at 72 h after endotoxin administration in male but not in female animals (which only recovered basal adrenal glucocorticoid content 120 h after LPS).
Besides the above mentioned sex-related differences in the HPA axis function of the mice, an acute increase in plasma ACTH and B levels 2 h after LPS was observed, which is in agreement with earlier reports 27 and clearly indicates a stage of transient stress. As well as other stressor induced ACTH release, a decrease in AP ACTH 72h after endotoxin was found with values returning to baseline at 120 h or more after treatment. 23 They probably reflect a stage of transient increase in AP ACTH release and thus adrenal B output (2 h after LPS) as a result of secreted cytokines from LPS activated immune cells. 2 Cytokines then could enhance hypothalamic CRH secretion 16'28'29 and thus stimulate AP ACTH synthesis to recover basal values (120 h or more). This possibility is even more clear in female mice, which showed a higher amount of ACTH and B secreted in plasma after endotoxin treatment, with AP ACTH still decreased up to 120 h after stress. The delayed recovering of AP ACTH after LPS treatment in female mice fully agrees with data obtained in adrenalectomized rats of both sexes, which indicate that 2 and 14 days after surgery AP ACTH was significantly lower in female than in male animals (unpublished results). Thus, a sex dependent change in the HPA axis response to this particular stress is indicated.
With respect to the sex related HPA axis response during the endotoxic shock, it has recently been reported 26 that bilateral gonadectomy influences the LPS stimulated TNF release in plasma 2 h after treatment; thus, a sexual difference in the stimulatory effect of TNF, or any other released cytokine after injury, on the different levels of the mice HPA axis must not be discarded.
Regarding the effect of endotoxin on adrenal glucocorticoid content, it is important to point out that at a very short time interval after LPS administration (2 h), which is coincident with the highest plasma ACTH and B levels, we found an enhanced adrenal B content in animals of both sexes, showing higher absolute values in female than in male mice. These data coincide with preliminary results from our group which indicate that orchidectomy leads to enhanced adrenal B content after LPS treatment when compared with the endotoxin effect on intact male mice. It could be speculated that the rapid LPS induced ACTH 3 or other POMC related peptide release from pituitary and/or extrapituitary 31'32 origin could be responsible for increased adrenal glucocorticoid synthesis and release. Although it has been shown that LPS did not affect in vitro adrenal B output, 26 it is not pogsible to exclude an in vivo effect of LPS on adrenal steroid synthesis.
With respect to the transient changes in circulating sex steroid levels after endotoxin administration, decreased testosterone and increased E plasma levels were found in male mice 2 h post-LPS. These data fully agree with other previous reports 33'34 and further suggest an increased conversion of testicular testosterone to E (aromatization) as a consequence of endotoxin administration. 3s It has recently been reported 26 that testosterone plays an important role in inhibiting LPS stimulated HPA immune axis function. Thus, it could be speculated that in males, having high basal plasma testosterone concentration, the increased testicular aromatization of the androgen takes place as a body's defence mechanism in order to allow an enhanced immune response shortly after infection.
To the authors' knowledge there are no data in the literature regarding plasmatic androgen and oestrogen levels during septic shock in female animals; however, it has been reported that an increase in androgen production resulted after different types of stress. 36 We found that endotoxin administration rapidly (2 h) increased plasma testosterone and E levels in female mice and these parameters were restored to the respective baseline 72 h post-treatment. It is already known that in normal females ovarian androgen production is very low and the adrenal gland substantially contributes to the total androgen production by a peripheral conversion of androstenedione to testosterone. LPS indirectly stimulates adrenal steroidogenesis 34 and thus could induce an increase in plasma testosterone levels 2 h after treatment; then, testosterone from both peripheral and ovarian (thecal) origin reaches the ovarian granulosa cell compartment to be further converted to E by LPS stimulated aromatase activity.
In summary, this study demonstrates a sexual dimorphism in the HPA axis function in both basal and LPS stimulated conditions. Endotoxin treatment induces transient changes in the HPA axis which are of a similar fashion to those described 1,6 Mediators of Inflammation. Vol 2.1993 after several types of stress, and are of a quite different magnitude when male and female parameters are compared. These results strongly suggest that endogenous sex steroid hormones play an important regulatory role in HPA axis function under stress. The exact mechanism of action whereby sex steroid hormones modulate the neuroendocrine immune response shortly after infection remains to be determined.