AIM: To investigate the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stress-stimulated production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, and macrophage chemotactic protein (MCP)-1 by peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C and healthy controls.

METHODS: The PBMCs were separated from age-matched 72 males and 71 females with and without chronic hepatitis C, who were divided into two groups based on a mean menopausal age of 50 years. Oxidative stress was induced by hydrogen peroxide in the cells incubated in serum-free media. Cytokines in the culture supernatant were measured by an enzyme-linked immunosorbent assay.

RESULTS: The highest levels of the spontaneous production of TNF-α, IL-1β, IL-8, and MCP-1 by the unstimulated PBMCs were in the older male patients with chronic hepatitis C and the lowest levels were in the pre-menopausal female healthy controls. E2 inhibited the cytokine production by the unstimulated PBMCs from the older male and post-menopausal female patients, which was further stimulated by progesterone. The exposure to hydrogen peroxide in the PBMCs from the younger male and pre-menopausal female healthy subjects induced the production of cytokines. The change rates of the hydrogen peroxide-stimulated cytokine production were suppressed by E2 and enhanced by progesterone.

CONCLUSION: These findings suggest that E2 may play a favorable role in the course of persistent liver injury by preventing the accumulation of monocytes-macrophages and by inhibiting proinflammatory cytokine production, whereas progesterone may counteract the favorable E2 effects.

In inflammatory and oxidative liver injury, the accumulation of leukocytes and macrophages including Kupffer cells on the sites of injury and inflammation in the liver is thought to be mediated by such cytokines as chemokines, including interleukin (IL)-8 and macrophage chemotactic protein (MCP)-1. These monocytes and macrophages are in turn able to release proinflammatory cytokines such as tumor necrosis factor (TNF)-α and IL-1β, thus leading to the occurrence of persistent liver injury. It has been reported that chronic hepatitis C virus (HCV) infection tends to progress more rapidly in men than in women[12], and that the decline in estradiol (E2) production with menopause is associated with a spontaneous increase in TNF-α and IL-1β[3]. It should be noted that large increases in the amount of reactive oxygen species (ROS) lead to disturbance of prooxidant-antioxidant balance, or oxidative stress. Our previous studies have shown that hepatocytes possessed estrogen receptor (ER), and that in cultured hepatocytes in a state of oxidative stress, E2 inhibited the activation of the nuclear factor κB (NF-κB), a key transcription factor that induces multiple genes in response to inflammation and oxidative stress[4], through ER[56]. These findings suggest that E2 could enhance the anti-inflammatory activity in the injured liver by decreasing the proinflammatory cytokine production, and it might, therefore, play a cytoprotective role through ER in the liver[78]. However, regarding E2 and another female sex steroid, progesterone, there is little information about the direct effects of these sex hormones on the production of chemokines and proinflammatory cytokines by monocytes and macrophages in chronic HCV infection, although in vitro experiments in which monocytes were incubated with sex hormones have revealed conflicting results regarding monocyte cytokine production[9]. Therefore, this study investigated the effects of E2 and progesterone on the production of TNF-α, IL-1β, IL-8, and MCP-1 by the heparinized peripheral blood mononuclear cells (PBMCs), after stimulation with the ROS, hydrogen peroxide[10]. These effects were then compared between the blood samples from the age-matched male and female patients with chronic hepatitis C and the healthy controls, which were divided into two groups based on a mean menopausal age of 50 years.

In the present study, the highest levels of the spontaneous production of TNF-α, IL-1β, IL-8, and MCP-1 by the unstimulated PBMCs were found to be in the older male patients with chronic hepatitis C, and the lowest levels were in the pre-menopausal female healthy subjects, although the cytokine levels were considerably different among the individuals. The male subjects tended to produce cytokines from the unstimulated PBMCs to a much greater degree than did the age-matched female subjects. The augmented cytokine production by the PBMCs from the older male and post-menopausal female patients with chronic hepatitis C was inhibited by supplementation with E2, and was further stimulated by supplementation with progesterone through their receptors, when the unstimulated cells were cultured for an additional 6 h. The exposure to low doses of hydrogen peroxide in the PBMCs from younger male and pre-menopausal female healthy subjects incubated in the serum-free media for 6 h was observed to induce cytokine production. The change rates of the hydrogen peroxide-stimulated production of TNF-α, IL-1β, IL-8, and MCP-1 in the PBMCs were suppressed by E2, and were enhanced by progesterone through their receptors. The specificity of the E2-mediated anti-inflammatory induction through the ER and the progesterone-mediated proinflammatory induction through the PR was shown by ICI and RU, respectively, in both the unstimulated and oxidative stress-stimulated PBMCs. The inhibitory effect of E2 at a dose of 10^-8 mol/L on the unstimulated and stimulated cytokine production was blocked by ICI in both gender subjects. Treatment with the progesterone receptor antagonist RU led to a blockage of further cytokine production induced with 10^-7 mol/L progesterone by the unstimulated and stimulated PBMCs from both genders. No parameters examined in the PBMCs were found to be significantly different between the male and female subjects.

There is a large body of evidence indicating that the decline in the ovarian function with menopause is associated with spontaneous increases in TNF-α, IL-1β, and IL-6[3]. E2, at physiological concentrations (10^-11-10^-8 mol/L), has been reported to inhibit the spontaneous secretion of these proinflammatory cytokines in whole blood cultures[12] or PBMCs[13]. The unstimulated production of TNF-α and IL-1β in PBMCs has been reported to be higher in patients with chronic hepatitis C than in healthy subjects[14]. These findings were consistent with the present data. The in vivo treatment with E2 transdermally in postmenopausal women has been reported to decrease the spontaneous IL-6 production by PBMCs after 12 mo of the therapy[13]. One preliminary study also showed the hydrogen peroxide-induced TNF-α and MCP-1 expressions to be attenuated by E2 in the peritoneal macrophages of female mice[15]. Furthermore, E2 is able to attenuate IL-1β in ER expressing HepG2 cells[16], and to ameliorate the burn-induced increase in the serum TNF-α levels in rats[17]. These findings suggest that E2 may exert a hepatoprotective action against inflammation and oxidative stress, at least in part, by preventing accumulation of monocytes and macrophages and by inhibiting the production of proinflammatory cytokines.

As far as the in vitro studies with female sex hormones on the production of TNF-α and IL-1β by monocytes are concerned, however, conflicting data have been published[9], varying from some[18–20] to no[1221] effect of E2 or progesterone on cytokine production. E2 has been reported to suppress the TNF-α production in unstimulated PBMCs, but not in endotoxin-stimulated PBMCs, from postmenopausal females with osteoporosis[22]. An inhibition of IL-1β production in endotoxin-stimulated monocytes by E2 or progesterone at physiological concentrations has also been reported[23]. The results of the reported studies did not correlate with the present data. These conflicting results may possibly be due to the handling of the cells during the in vitro research, different experimental methods used, and/or differences in the subjects employed in the studies.

In the premenopausal female subjects with and without chronic hepatitis C enrolled herein, the blood samples were taken during the luteal phase of the menstrual cycle. During the luteal phase, the serum concentration of endogenous progesterone rises up to a maximum of about 10^-7 mol/L, which can be ten to a hundred times higher than E2. Higher blood levels of TNF-α have been observed during the luteal phase in comparison to the follicular phase[24]. In males, a higher percentage of IL-1β producing stimulated monocytes has been demonstrated in comparison to females in the follicular phase[19]. The male sex hormone testosterone has some structural and functional similarities to progesterone[25]. Judging from these findings and the present data showing that treatment with E2 (10^-8 and 10^-7 mol/L) and progesterone (10^-7 mol/L) significantly affected the change rate of the cytokine production in hydrogen peroxide-stimulated PBMCs, E2 may, therefore, exert an anti-inflammatory action against both inflammation and oxidative stress in the mononuclear cells from the chronic hepatitis C patients, whereas progesterone may counteract the favorable effects of E2.

HCV infections are recognized to be a major causative factor in the development of liver injury leading to cirrhosis[2627] The HCV core protein has been reported to enhance the signaling pathway of NF-κB activation in human hepatoma HuH-7 and cervical cancer HeLa cells, and the HCV core protein is triggered by TNF-α-related cytokines[28]. Damage to the parenchymal cell membranes and liver mitochondria could produce ROS derived from lipid peroxidative processes, which constitute a general feature of a sustained inflammatory response and liver injury[29]. In comparison to other types of ROS, hydrogen peroxide is more stable and membrane permeable leading to the hypothesis that it acts as a second messenger in regulating the signaling events, including the mitogen-activated protein kinase (MAPK) activation. We have already reported that E2 inhibited the prooxidant-induced lipid peroxidation in rat liver mitochondria[5], attenuated ROS generation and NF-κB activation in cultured rat hepatocytes in a state of prooxidant-induced oxidative stress[6], while also suppressing the hydrogen peroxide-induced activation of MAPKs and transcription factors including NF-κB in cultured rat hepatic stellate cells[30]. In the present study, hydrogen peroxide exposure resulted in an increase in the TNF-α, IL-1β, IL-8, and MCP-1 levels in the cultured mononuclear cells from the male and female healthy subjects. The oxidative stress-stimulated cytokine expression was attenuated by E2 and augmented by progesterone in a dose-dependent manner without any significant difference between the males and females. These effects of E2 and progesterone were blocked by their receptor antagonists ICI and RU, indicating that ER and PR could mediate female sex hormone action in the oxidative stress-stimulated monocytes and macrophages.

Finally, the current data suggest that E2 may play a favorable role in the course of persistent liver injury, at least in part, by preventing the accumulation of monocytes and macrophages and by also inhibiting the proinflammatory cytokine production through ER, whereas progesterone may counteract these positive E2 effects by enhancing the accumulation of inflammatory cells and their cytokine production through PR.