Diazepam treatment of pregnant rats differentially affects interleukin-1 and interleukin-2 secretion in their offspring during different phases of postnatal development

Treatment of pregnant Long Evans rats with benzodiazepines was found to cause alterations in cellular immune responses in their offspring. We now report on changes in interleukin-1 (IL-1) and IL-2 secretion which were analyzed in rats from birth until 12 weeks. Time-pregnant rats were treated with diazepam (1.25 mg/kg/day subcutaneously) from gestational day 14 to 20. Lipopolysaccharide-stimulated release of macrophage-derived IL-1 by spleen cells, determined on D10.G4.1 cells, remained in the control range during the preweaning period (postnatal day 6-28), then decreased in prenatally diazepam-exposed offspring, significantly in males during the postweaning period (postnatal day 34-61) and in both sexes in adults (postnatal day 62-83). Concanavalin A-stimulated release of T lymphocyte-derived IL-2 from spleen cells, determined on CTLL-2 cells, was reduced in male and female offspring during preweaning (postnatal day 3-28) and postweaning (postnatal day 33-55) periods and normalized in adulthood (postnatal day 60-84). The percentage of IL-2 receptor expressing (CD25+) cells was unaffected. From these and our earlier data it is evident that prenatal exposure to low doses of benzodiazepines can result in long-lasting alterations of the cytokine network, as indicated by reduced release of TNF-alpha, IL-1, IL-6, IL-2 and interferon-gamma. The concomitant reduction of peripheral type benzodiazepine receptors on macrophages is discussed as a possible link between prenatal treatment and disturbed function.     

Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function

Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b+ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation.     

Is HIV infection a risk factor for advanced cervical cancer?

TNF-alpha plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-alpha from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-alpha. The presence of lipopolysaccharide (LPS, 0.1 microg/mL) in the culture medium significantly increased TNF-alpha release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 beta-estradiol in the culture medium also increased TNF-alpha release. LPS significantly stimulated TNF-alpha release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-alpha on prolactin release. The presence of TNF-alpha (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-alpha and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-alpha could affect pituitary hormone release during endotoxemia.     

Fetal alcohol exposure augments the blunting of tumor necrosis factor production in vitro resulting from in vivo priming with lipopolysaccharide in young adult male but not female rats

We previously reported altered responses of thymocytes and splenocytes to mitogen stimulation in fetal alcohol-exposed (FAE) male Sprague-Dawley rats. We also reported enhanced neuroendocrine responses to stressful stimuli in these animals. The experiments we describe herein aimed at testing whether young adult FAE rats manifest a notable dysregulation in the neuroendocrine-immune response to pathogen administration. We tested the effect of in vivo priming of the animal with a low dose of endotoxin [lipopolysaccharide (LPS), 5 micrograms/kg], considered to be suboptimal from the perspective of mounting detectable levels of circulating monokines several hours after administration, upon the production of immunoreactive tumor necrosis factor (TNF-alpha) in response to a further in vitro challenge of peripheral blood mononuclear cells with 2.5 micrograms/ml of LPS 90 min after priming. We show that the response to the LPS pathogen in vitro after priming is significantly blunted (p < 0.01) in male rats exposed prenatally to alcohol, compared with control male animals. FAE female rats and FAE ovariectomized female rats do not show significant differences in the priming response, compared with control animals. We also show that there is no correspondence between plasma corticosterone levels and TNF-alpha production after priming in any of the groups tested.     

Determination of distances from tyrosine D to QA and chlorophyllZ in photosystem II studied by ''2+1'' pulsed EPR

The immunomodulating effect of a new polysaccharide-peptide complex from culture mycelia of Lentinus edodes (LE) was studied for elucidation of the mechanism of augmentation of cell-mediated immunity. RNA samples were isolated from the untreated and treated murine splenocytes and human peripheral blood mononuclear cells. RT-PCR was used to analyze the cytokine gene expression and bioassay was used to analyze the cytokine production. By administration of LE, the expression levels of IL-2 and TNF-alpha genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-alpha were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-alpha were augmented in the treated human peripheral blood mononuclear cells. These results suggest that LE may induce Th immune responses.     

Effects of high doses of diazepam on carrageenin-induced paw edema in rats

Benzodiazepine (BDZ) receptor sites play a relevant role in immune/inflammatory reactions. Acute BDZ treatments were shown not only to suppress cell proliferation in rat thymus but also to decrease TNF-alpha, IL-1 and IL-6 release from adult mouse macrophages. In the present investigation the effects of acute (10.0 and 20.0 mg/kg) and long-term (10.0 mg kg-1 day-1, for 21 days) diazepam treatment on carrageenin-induced paw edema were studied in rats. The results showed that acute treatment with high doses of diazepam decreased paw edema volume in a dose-dependent manner, and this effect was observed as early as 1 h after the administration of the 20.0 mg/kg dose and continued until the last measurement was performed (8 h). In contrast, long-term diazepam administration did not modify the phlogistic-induced edema. Taken together, these data show that 1) acute diazepam treatment with high doses decreases the volume of the acute inflammatory paw edema developed by the organism as a response to carrageenin-induced injury, and 2) long-term diazepam treatment induces tolerance to this effect. These results are discussed in the light of a possible effect of diazepam on the components of the rat cellular and humoral immune/inflammatory reaction such as T lymphocytes and/or interleukins.     

Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages

Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB). Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood. The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process. As a control, the effect of E. coli LPS was also investigated. TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent. All of the toxins stimulated the release of TNF-alpha from macrophages. TxB was again the most active in inducing this response. The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter. NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma. In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB. Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS. TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms. TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB. NO is not involved in the killing action of any of these toxins.     

Monoamniotic twin pregnancy and cord entanglement: a clinical dilemma

Although isoflurane inhibits TNF-alpha and IL-1 beta release from human monocytes stimulated by LPS in dose dependent fashion, it is unclear whether sevoflurane has the same effects. Therefore, we investigated whether sevoflurane could inhibit TNF-alpha and IL-1 beta secretions from human monocytes stimulated by LPS in dose dependent fashion in vitro. Human monocytes stimulated by LPS were cultured for 3 h in the presence of sevoflurane 1% or 5%. Another group of human monocytes were cultured in the absence of sevoflurane. TNF-alpha and IL-1 beta increased after stimulation of LPS and these increases were not inhibited by sevoflurane in a dose dependent fashion. We conclude that sevoflurane does not inhibit TNF-alpha and IL-1 beta release from monocytes stimulated by LPS.     

Distinct tumor necrosis factor-alpha responses in alveolar and peritoneal macrophages are associated with local levels of endotoxin

Tumor necrosis factor alpha (TNF-alpha) responses of alveolar macrophages (AMs) and peritoneal macrophages (PMs) were studied in rats after intravenous injection of lipopolysaccharide (LPS). High levels of plasma TNF-alpha, increased pulmonary myeloperoxidase activity, and leukopenia occurred within 2 h after LPS injection. Alveolar spaces exhibited a strict compartment property, as manifested by only slightly increased LPS and TNF-alpha levels in alveolar lavage fluid and an unchanged capacity of AMs to produce TNF-alpha. By contrast, the peritoneal cavity had greatly increased local LPS and TNF-alpha levels and a diminished PMs TNF-alpha response to LPS. The amount of LPS in the alveolar spaces was less than 0.2% of the level in peritoneal fluid. These results indicate that activation of resident macrophages is dependent on the amounts of local LPS and, in addition, suggest that resident AMs neither participate in the plasma TNF-alpha response nor contribute to neutrophil sequestration in the lung during the early stages of endotoxemia.     

In vitro IL-1 beta and TNF-alpha release from THP-1 monocytes in response to metal ions

OBJECTIVES: Previous studies have shown that biomaterials can activate macrophages to produce cytokines and promote an inflammatory response. Although the toxicity of many metal ions has been extensively investigated, little is known about the ability of these ions to alter cytokine release from macrophages. Yet the release of these ions from biomaterials has been well documented. Previous studies indicated that alterations in cytokine release might be expected because metal ions alter protein production in macrophages at sub-toxic concentrations. Thus, the hypothesis of this study was that metal ions can alter the secretion of cytokines from macrophages at sub-toxic concentrations. METHODS: The release of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from macrophages was investigated when the macrophages were exposed to metal ions, with or without lipopolysaccharide (LPS), a component of dental plaque. Human THP-1 macrophages were exposed to ions of Ag, Au, Cu, Hg, and Ni for 24 h. In half of the cultures, LPS was added for the last 4 h. The release of IL-1 beta and TNF-alpha into the medium was measured using enzyme-linked immunosorbent assays. ANOVA and Tukey multiple comparison intervals were used to compare the various experimental conditions. RESULTS: None of the metal ions elevated the IL-1 beta or TNF-alpha levels after 24 h, but Ni ions significantly elevated the IL-1 beta and TNF-alpha levels after 72 h. With LPS added, Ag, Cu, and Ni significantly amplified the LPS-induced production of IL-1 beta but only Ni amplified the TNF-alpha response. These alterations in cytokine response occurred with metal ion concentrations which have been previously shown to be released from dental alloys in vitro and in vivo. SIGNIFICANCE: It appeared plausible that macrophage-cytokine mediated inflammatory responses may be altered by the presence of some metal ions in tissues, particularly Ni.     

Tumour necrosis factor-alpha as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved (mostly through the activation of inducible nitric oxide synthase) in the pathogenesis of circulatory shock. On the other hand, melanocortin peptides are potent and effective in reversing haemorrhagic shock, both in animals (rat, dog) and in humans. This prompted us to study the influence of the melanocortin peptide ACTH-(1-24) on the blood levels of TNF-alpha in haemorrhage-shocked rats and on the in vitro production of TNF-alpha by lipopolysaccharide (LPS)-activated macrophages. Plasma levels of TNF-alpha were undetectable before starting bleeding and greatly increased 20 min after bleeding termination in saline-treated rats. In rats treated with ACTH-(1-24) the almost complete restoration of cardiovascular function was associated with markedly reduced levels of TNF-alpha 20 min after bleeding termination. On the other hand, ACTH-(1-24) did not influence TNF-alpha plasma levels in sham-operated, unbled rats. In vitro, ACTH-(1-24) (25-100 nM) dose-dependently reduced the LPS-stimulated production of TNF-alpha by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF-alpha overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.     

Gamma interferon cooperates with lipopolysaccharide to activate mouse splenic macrophages to an antihistoplasma state

Inhibition of the intracellular growth of Histoplasma capsulatum by murine resident red pulp splenic macrophages was examined. Splenic macrophages, unlike resident peritoneal macrophages, required a prolonged preincubation (18 h) with recombinant murine gamma interferon (rMuIFN-gamma) for activation. To be fully activated, the splenic macrophages required incubation with rMuIFN-gamma in combination with 0.1 microgram of lipopolysaccharide (LPS) per ml. Splenic macrophages stimulated with rMuIFN-gamma, LPS, or rMuIFN-gamma and LPS produced tumor necrosis factor alpha (TNF-alpha), but recombinant murine TNF-alpha (rMuTNF-alpha) did not activate macrophages when used alone or as a second signal with rMuIFN-gamma. Anti-TNF-alpha antibody did not block IFN-gamma-LPS activation of splenic macrophages to any significant extent. One hundred micromolar ferrous sulfate antagonized IFN-gamma-LPS activation of splenic macrophages, indicating that iron was involved in the fungistatic activity of cytokine-stimulated phagocytes. Our results indicate that (i) splenic macrophages differ significantly from peritoneal macrophages in their requirements for activation and (ii) the mechanism by which splenic macrophages exert their antifungal effects involves iron.     

Exogenous bovine surfactant suppresses tumor necrosis factor-alpha release by murine macrophages stimulated by genital mycoplasmas

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that appears to play a significant role in the development of neonatal chronic lung disease (CLD). Inflammation and CLD are also associated with respiratory tract colonization with genital mycoplasmas. The possible protective roles of surfactant in mitigating the inflammatory response to these microbes were investigated. Murine RAW 264.7 macrophages were preincubated with an exogenous surfactant and exposed overnight to sterile media, lipopolysaccharide (LPS), Mycoplasma hominis, or Ureaplasma urealyticum. Macrophages released TNF-alpha in response to challenge with LPS, U. urealyticum, and M. hominis in a concentration-dependent fashion. Surfactant suppressed LPS and M. hominis induced TNF-alpha production in a dose-dependent manner but suppressed U. urealyticum-mediated TNF-alpha production only at the higher dose tested. Similar effects were seen in hyperoxia (95% O2). Thus, exogenous bovine surfactant significantly inhibits the production of TNF-alpha by murine macrophages stimulated with genital mycoplasmas and bacterial LPS.     

Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan

Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.     

Increase in medial palisade zone dopamine activity after long-term castration in the male rat

Administration of D-penicillamine (50 mg/kg/day orally) for 4 days increased the uptake of 3H-thymidine (3H-TdR) in unstimulated and concanavalin-A-stimulated unseparated lymph node and spleen cells from Lewis rats. Increased 3H-TdR incorporation was also found in cultures depleted of adherent cells. D-Penicillamine treatment did not increase the incorporation of 3H-TdR in lymph node and spleen cells from rats concomitantly treated with the selective macrophage toxin silica. In contrast, treatment with D-penicillamine during the last 4 days of silica treatment sometimes resulted in a marked decrease in 3H-TdR incorporation. It is suggested that D-penicillamine treatment in vivo is able to enhance the responsiveness of the lymphocytes, dependent on the presence of functionally intact macrophages. The increased response vanished after 2-3 weeks, even with continuous administration of D-penicillamine.     

All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134+/-119 IU/L and 72.7%) compared with the control group (713+/-411 IU/L and 18.2%; P< 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-alpha (TNF-alpha) and superoxide anions produced by activated macrophages. Serum levels of TNF-alpha 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5+/-7.0 ng/mL) as compared with the control group (105.2+/-39.3 ng/mL; P< 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-alpha synthesis has been studied in vitro and in vivo. The synthesis of TNF-alpha in J774 macrophages stimulated with LPS (0.1 microgram/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 microM) and reduced by either L-arginine (3-30-300 microM) or the NO donor SIN-1 (1-10-100 microM). The level of TNF-alpha in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L-arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-alpha synthesis suggesting an important regulatory link between NO and TNF-alpha in pathological processes.     

Maternal stress induces synaptic loss and developmental disabilities of offspring

Mild prenatal stress affects the serotonergic system in the hippocampus of rat offspring. Pregnant rats were daily exposed to mild stress treatments (consisting of crowding and saline injection) during days 15 to 21 of pregnancy. Their offspring were assessed by a series of biochemical, histological and behavioral tests. On 35 days after birth, 5-hydroxytryptamine (5-HT) level was decreased by 17% (P < 0.05), whereas 5-hydroxyindolacetic acid (5-HIAA) level was increased by 18% (P < 0.05) in the offspring of prenatally stressed rats. The metabolic rate (5-HIAA/5-HT) was increased by 49% (P < 0.01). Synaptic density in the hippocampus of prenatally stressed offspring was also decreased by 32% (P < 0.0001) on postnatal day 35. There was no significant group difference in the spatial learning acquisition test of the Morris water maze; however, in the reversal task, prenatally stressed 5-week old rats spent more time than control animals searching for the platform of the pool. Escape latency in the cued test showed no significant difference. Together with data in our previous studies, that have shown 5-HT to facilitate synapse formation and maintenance in the central nervous system, synaptic loss is suggested to occur in relation to changes of 5-HT system in the hippocampus of prenatally stressed offspring. This may be associated with reported changes in behavior and learning ability in prenatally stressed offspring.     

Interleukin-4 and -13 inhibit tumor necrosis factor-alpha mRNA translational activation in lipopolysaccharide-induced mouse macrophages

The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-stimulated macrophages can be markedly inhibited by the two closely related cytokines, interleukin (IL)-4 and IL-13. To investigate the molecular mechanism of this inhibition, we analyzed the effect of the two cytokines on TNF-alpha production and TNF-alpha mRNA accumulation in the mouse macrophage cell lines RAW 264.7 and J774 stimulated by LPS. Whereas LPS-induced TNF-alpha production is strongly suppressed by both cytokines, TNF-alpha mRNA accumulation is not significantly affected, indicating that IL-4 and IL-13 induce a translational repression of TNF-alpha mRNA. Transfection of reporter gene constructs containing different regions of the TNF-alpha gene revealed that the inhibitory action of IL-4 and IL-13 is mediated by the UA-rich sequence present in the TNF-alpha mRNA 3'-untranslated region.