Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice.

Although elevated activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) has been proposed to mediate comorbid depression in inflammatory disorders, its causative role has never been tested. We report that peripheral administration of lipopolysaccharide (LPS) activates IDO and culminates in a distinct depressive-like behavioral syndrome, measured by increased duration of immobility in both the forced-swim and tail suspension tests. Blockade of IDO activation either indirectly with the anti-inflammatory tetracycline derivative minocycline, that attenuates LPS-induced expression of proinflammatory cytokines, or directly with the IDO antagonist 1-methyltryptophan (1-MT), prevents development of depressive-like behavior. Both minocycline and 1-MT normalize the kynurenine/tryptophan ratio in the plasma and brain of LPS-treated mice without changing the LPS-induced increase in turnover of brain serotonin. Administration of L-kynurenine, a metabolite of tryptophan that is generated by IDO, to naive mice dose dependently induces depressive-like behavior. These results implicate IDO as a critical molecular mediator of inflammation-induced depressive-like behavior, probably through the catabolism of tryptophan along the kynurenine pathway.

Decreased brain tryptophan availability as a partial determinant of post-partum blues.

BACKGROUND: The post-partum blues is a transient mood alteration affecting most women a few days after delivery. Its stereotypic pattern of symptoms and time course, peaking on post-partum day 3-5, is suggestive of biological determinants superimposed on psycho-social factors. This study was designed to evaluate the possible role of the serotonin system during this period through assessment of brain tryptophan availability. METHODS: Blood samples from 50 women were collected just before (D0) and 3 days after (D3) delivery. Based on plasma concentration of tryptophan, amino acids competing with tryptophan for transport across the blood-brain barrier and on their respective affinities for this transporter, a brain tryptophan availability index (BTAI) was calculated and its variation correlated with the intensity of post-partum blues evaluated through the Kennerley and Gath score at D3. RESULTS: The BTAI showed a -15% decrease between D0 and D3 (p < 0.01, paired t-test). This decrease was not supported by a drop in plasma tryptophan since its level rather increased (+19%). There was no evidence for change in placental indoleamine-2,3-dioxygenase activity since the variation in plasma l-kynurenine (+12%) paralleled the change in tryptophan level. The decreased BTAI appeared the consequence of a dramatic increase in plasma levels of most amino acids, particularly the competitor aminoacids leucine, isoleucine, valine and tyrosine, during the early post-partum. This decrease in brain tryptophan availability was concomitant to the post-partum blues, whose intensity significantly correlated with the amplitude of BTAI variation (Pearson's coefficient -0.283, p < 0.05). CONCLUSION: This study suggests that generalized, large amplitude metabolic and/or nutritional changes occurring in the early post-partum result in a transient decrease in brain tryptophan availability, partly accounting for the mood alteration referred to as the post-partum blues, a model for the triggering of puerperal mood disorder in vulnerable women.

The enzyme indoleamine 2,3-dioxygenase is induced in the mouse brain in response to peripheral administration of lipopolysaccharide and superantigen.

The essential amino-acid, L-tryptophan, is the precursor of serotonin. Its availability in the brain is controlled by indoleamine 2,3-dioxygenase (IDO). This enzyme is inducible by cytokines such as interferon-gamma (IFN-gamma) and is the first and rate-limiting enzyme of the catabolism pathway of tryptophan. Since induction of IDO has been proposed to mediate the influence of cytokines on mood in patients with various somatic disorders, the present study aimed at analyzing the relationships between changes in brain IDO activity and serum IFN-gamma levels in response to peripheral immune stimulation by lipopolysaccharide (LPS) and superantigen in mice. Each of these treatments induced an increase in serum IFN-gamma at 6 h post-treatment followed 24 h later by a two-fold increase in IDO activity in the brain. These results support the involvement of peripheral IFN-gamma in the control of L-tryptophan catabolism in the brain.

Central injection of interleukin-13 potentiates LPS-induced sickness behavior in rats.

Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) has profound depressive effects on behavior that are mediated by the inducible expression of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) in the brain. To assess the regulatory effects of the anti-inflammatory cytokine IL-13 on LPS-induced sickness behavior, rats injected i.p. with LPS were administered rat recombinant IL-13 i.c.v. IL-13 (300 ng) potentiated the behavioral effects of LPS (125 microg/kg) when both molecules were co-injected. Administration of IL-13 (300 ng) 12 h prior to LPS (150 microg/kg) did not block the depressing effects of LPS on social exploration. These results indicate that IL-13 acts as a proinflammatory cytokine in the brain.

Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide.

Little is known on the forms of interleukin-1beta (IL-1beta) that are produced by microglial cells in the nervous system. Mixed glial cell cultures of rats produced IL-1beta in response to lipopolysaccharide (LPS). Using Western blot, pro-IL-1beta was found to be localized both intracellularly and in the supernatant, whereas mature IL-1beta was found only in the supernatant but in lower quantities than pro-IL-1beta. Immunocytochemistry confirmed that microglial cells are the exclusive source of IL-1beta. Blockade of the IL-1beta-converting enzyme (ICE) by Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO) decreased the levels of mature IL-1beta but had no effect on pro-IL-1beta. Release of pro-IL-1beta was not associated with cell death nor with the extracellular release of ICE. Using gelatin zymography, glial cells were found to express constitutive matrix metalloproteinases (MMP) in the form of MMP-2. Exposure to LPS induced MMP-9 expression in a time-dependent manner similar to the pro-IL-1beta expression profile. MMP activation and inhibition experiments indicated a possible role of MMPs in the cleavage of pro-IL-1beta but not in the generation of mature IL-1beta. Microglial cells share with macrophages the ability to release large amounts of pro-IL-1beta of which the extracellular role remains to be determined.

Characterization of interleukin-1 receptor antagonist isoform expression in the brain of lipopolysaccharide-treated rats.

The endogenous interleukin-1 receptor antagonist is the natural inhibitor of the biological effects of interleukin-1 during inflammation. Interleukin-1 receptor antagonist refers to three isoforms: one secreted and two intracellular forms (types I and II). The objective of the present study was to investigate the expression of interleukin-1 receptor antagonist isoforms in the rat brain in vivo in response to an i.p. injection of lipopolysaccharide. The interleukin-1 receptor antagonist was studied at the messenger and protein levels by reverse transcription-polymerase chain reaction and western blot analysis, respectively. Interleukin-1 receptor antagonist messenger RNA was constitutively expressed in the brain and its expression increased in response to lipopolysaccharide. The three interleukin-1 receptor antagonist protein isoforms were up-regulated after lipopolysaccharide treatment in a time-dependent manner. Their relative expression differed according to the isoform and brain region studied. Double immunofluorescence staining revealed interleukin-1 receptor antagonist positive neurons and microglia in hippocampus 24h after lipopolysaccharide stimulation. These results demonstrate for the first time that brain cells are able to produce interleukin-1 receptor antagonist isoforms in response to a peripheral immune challenge with a predominance of the secreted over intracellular forms.

Production of interleukin-1 receptor antagonist isoforms by microglia in mixed rat glial cells stimulated by lipopolysaccharide.

Although the natural interleukin-1 receptor antagonist (IL-1Ra) has been shown to be produced by microglial cells in response to immune stimuli, nothing was known about the ability of these cells in primary culture to produce the different isoforms of IL-1Ra. Using RT-PCR, we first confirmed that mixed glial cell cultures from newborn rats respond to the cytokine inducer, lipopolysaccharide, by synthesizing IL-1Ra mRNA. Using double immunostaining, we showed that IL-1Ra was detected in microglia but not in astrocytes. Using Western blotting, we finally demonstrated that the IL-1Ra1 isoform was secreted in the supernatant of mixed glial cell cultures, and its production increased in response to lipopolysaccharide. The three different IL-1Ra isoforms were constitutively expressed in cell lysates and their levels increased after lipopolysaccharide treatment, except for IL-1Ra3. These results point to the ability of microglial cells in primary culture to produce the different isoforms of IL-1Ra.

Central injection of IL-10 antagonizes the behavioural effects of lipopolysaccharide in rats.

Peripheral (i.p.) and central (i.c.v.) injections of lipopolysaccharide (LPS) have been shown to induce brain expression of proinflammatory cytokines and to depress social behaviour in rats, increase duration of immobility and induce body weight loss. To determine if the anti-inflammatory cytokine, interleukin-10 (IL-10) is able to modulate these effects, recombinant rat IL-10 was injected in the lateral ventricle of the brain (30, 100, 300 ng/rat) prior to i.p. or i.c.v. injection of LPS (250 micrograms/kg or 60 ng/rat, respectively). Social exploration was depressed for 6 h after i.p. LPS injection. This effect was attenuated by IL-10 (30 and 100 ng) 2 h after injection, whereas the highest dose of IL-10 blocked the depression of social interaction for 6 h after LPS injection. IL-10 produced the same effects on the increase of immobility although the results did not reach significance. Social exploration was depressed 3 h after i.c.v. LPS injection, and this was accompanied by increased immobility. These effects were totally blocked by i.c.v. IL-10 (300 ng/rat). Rats lost body weight after i.c.v. LPS, and this effect was attenuated by i.c.v. IL-10. These results indicate that IL-10 is able to modulate the production and/or action of central proinflammatory cytokines.

Apoptosis occurs independently of the release of interleukin-1 beta in the anterior pituitary of end-lactating rats.

The mechanism of Interleukin-1 beta (IL-1 beta) release remains unknown. Because of the absence of typical peptide signal on the precursor, IL-1 beta is not secreted through the classical pathway. The aim of this study was to determine whether IL-1 beta is released during apoptosis, as has been reported for activated macrophages. We chose anterior pituitary cells of end-lactating rats because of their capacity to produce IL-1 beta spontaneously and because this organ undergoes cellular degeneration. The combination of two techniques, reverse haemolytic plaque assay (RHPA) and terminal transferase dUTP nick end labelling (TUNEL), allowed us to observe simultaneously the release of IL-1 beta and apoptosis. Our results show that in these conditions apoptosis is not the mechanism of IL-1 beta release.

Expression of galactose-1-phosphate uridyltransferase in the anterior pituitary of rat during the estrous cycle.

Biological activity of the follicle-stimulating hormone (FSH) is dependent on its pattern of glycosylation and is altered during galactosemia, a genetic disease characterized by deficient activity of galactose-1-phosphate uridyltransferase (GALT). To assess the role of this enzyme in the synthesis of FSH, the expression of GALT at the mRNA and protein levels was measured in the whole anterior pituitary during the estrous cycle of rat. GALT was maximally expressed during the proestrous and estrous phases of the estrous cycle. The expression pattern of GALT was associated with gonadotropin-expressing cells. This close association is in accordance with the postulated role of GALT in modulating biological activity of FSH.

Identification of epidermal growth factor-secreting cells in the anterior pituitary of lactating female rats.

Epidermal growth factor (EGF) is synthesized and secreted by mammalian anterior pituitary cells. It stimulates GH and prolactin (PRL) secretion, but the cellular origin of EGF is relatively unexplored. The objective of this study was to characterize the cells that secrete EGF in the anterior pituitary of lactating rats. An EGF reverse haemolytic plaque assay (RHPA) was used to identify EGF-secreting cells and this RHPA was combined with immunofluorescence using antibodies to the six major adenohypophysial hormones (i.e. PRL, GH, LH, FSH, TSH and ACTH). Approximately 20% (20.33 +/- 2.96%) of the cells in the pituitary of lactating rats secrete EGF. The EGF-secreting cell population was composed of the following labelled cells: PRL (27%), GH (20%), LH (18%), FSH (14%), TSH (14%) and ACTH (5%). The present study showed that EGF is released by a subpopulation of anterior pituitary cells composed of all the classic hormone-containing cells.

EGF release by rat gonadotroph cells: characteristics and effects of LHRH.

The ability of anterior pituitary cells of immature female rats to secrete epidermal growth factor (EGF) was studied using the reverse hemolytic plaque assay. An average of 22% of cells (22.12 +/- 0.49%) spontaneously released low amount of EGF, as measured by the small area of plaques surrounding cells (1230 +/- 67 micron2). To determine the cellular origin of EGF from the rat anterior pituitary gland, a combination of immunocytochemistry and reverse hemolytic plaque assay was used. Most of the EGF-secreting cells were identified as luteinizing hormone (LH) containing cells (72.14 +/- 2.97%). Treatment with luteinizing hormone releasing hormone (LHRH; 10 nM) significantly increased the plaque area formed around EGF-secreting cells (2438 +/- 114 micron2) without altering the number of EGF-secreting cells. These results indicate that EGF is mostly secreted by gonadotrophs and that this EGF release is enhanced by LHRH.

Estrogen increases the release of epidermal growth factor from individual pituitary cells in female rats.

Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17 beta (10 nM) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290 +/- 58 to 2566 +/- 57 microns 2, P < 0.01) and the percentage of EGF plaque-forming cells (from 20.57 to 28.19%; P < 0.01) to a level as high as observed in proestrous cultures (basal mean area 2171 +/- 157 microns 2, and basal percentage of EGF plaque-forming cells 22.71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon.

Epidermal growth factor: a potential paracrine and autocrine system within the pituitary.

Epidermal growth factor (EGF) secretion and EGF receptor (EGFR) expression were studied by a combination of reverse hemolytic plaque assay (RHPA), which identifies the EGF secreting cells, and immunocytochemical detection of EGFR. Our results showed that 19% of total rat anterior pituitary (AP) cells secrete EGF and 48% of total AP cells possess EGFR. In addition, most of the EGF-secreting cells (73%) possess EGFR. By measuring the size of plaques surrounding EGF-secreting cells, we found that EGF secretion per cell is significantly higher in cells that did not express EGFR (1930 +/- 92 microns2) than in EGFR-expressing cells (1551 +/- 89 microns2). Taken together, these results strongly suggest that EGF acts by both an autocrine and paracrine fashion in the rat anterior pituitary gland.

Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism.

To assess the possibility that lipopolysaccharide (LPS) induces sickness behaviour by activating primary afferent nerves, the effects of LPS (1.25 mg kg-1, intraperitoneally) were compared in vagotomized and sham-operated rats. Subdiaphragmatic vagotomy blocked the LPS-induced depression of social investigation but had no effect on LPS-induced increases in levels of IL-1 beta in plasma and peritoneal macrophages and on LPS-induced changes in dehydrogenase activity of peritoneal macrophages.

Evidence for an alpha 2-macroglobulin with complement-inhibiting activity in rat serum.

alpha 2-Macroglobulin (alpha 2M) was purified both from the serum of male rats developing an acute turpentine-induced inflammatory reaction where its concentration is greatly increased (3-4 mg/ml) and from the serum of healthy males where it is present at low levels (15-30 micrograms/ml). A three-step purification procedure involving gel filtration, anion exchange chromatography on DEAE cellulose and negative immunoaffinity was used. A pure native alpha 2M, as assessed by biochemical and immunological tests, was obtained. This alpha 2M differed from other subforms in terms of its electric charge and its complement-inhibiting activity in a complement-dependent immune haemolysis test. Moreover, this inhibitory activity was not affected by complexing with trypsin or modification by interaction with methylamine showing that this newly described property is not linked to the well known antiproteinase function of alpha 2M.

Characterization of one alpha 2-macroglobulin with anticomplementary activity from pregnant women.

In 15 pregnant women during the third term of pregnancy, the immunomodulatory property of alpha 2-macroglobulin (alpha 2M) was initially detected by measuring the inhibitory effect on immune complement-dependent haemolysis of serum alpha 2M fractions obtained by gel filtration. By a two-step chromatography procedure consisting of gel filtration followed by anion-exchange chromatography, different sub-forms of alpha 2M in serum were separated. Amongst them, it was shown that the inhibition of complement activity was almost exclusively linked to one particular subform. Additional studies revealed that the observed effect was not due to proteases bound to alpha 2M during clotting since, by using protease-specific inhibitors, no change was observed in complement inhibition. This subform, though present at very low levels in control sera, appeared in strikingly increased amounts during the third trimester of pregnancy (35 mg/l) and comprised between 3 and 5% of the total alpha 2M. Results show that the increase of alpha 2M anticomplementary activity is linked to the increase in alpha 2M levels in serum.

The phagocytic activity of Lumbricus terrestris leukocytes is enhanced by the vertebrate opsonins: IgG and complement C3b fragment.

The effect of the vertebrates opsonins: IgG and complement C3 fragments on phagocytic activity of Lumbricus terrestris leukocytes towards sheep erythrocytes was studied. Sheep erythrocytes were previously sensitized with specific IgG or IgM and coated with one of the third component fragments (C3b, C3bi, C3d) of human complement. Our results show that leukocyte phagocytosis was enhanced by vertebrate IgG and C3b complement fragments but not by IgM and fragment C3d. Because opsonization in vertebrates is related to the presence of receptors on the surface of phagocyte membrane, our results suggest that similar receptors exist on earthworm leukocyte surfaces. These new data strengthen the arguments in favour of the presence of components in Lumbricus terrestris which partially share common structures and functions with components of vertebrate humoral immune reactions.

Evidence of synthesis by Lumbricus terrestris of specific substances in response to an immunization with a synthetic hapten.

Lumbricus terrestris (Annelid, Oligocheta) is capable of cellular- and humoral-specific reactions against natural antigens. Is this earthworm able to elaborate a response of antibody type against a synthetic hapten? L. terrestris have been immunized with conjugates made of one of two different synthetic haptens (400 mw) and a carrier protein: bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). The presence of anti-hapten substances in coelomic fluid was tested against each iodinated hapten derivative (125I-hapten). The 125I-hapten-substance complexes were separated from the free derivatives by polyethylene glycol (PEG) 6000 precipitation or by gel filtration. The experiments showed that L. terrestris synthesized specific substances against the immunizing hapten. The importance of the response depended on the carrier protein and on the amount of introduced immunogen. A kinetic study of first and second immunization showed that these substances, elaborated in response to the immunization with the synthetic hapten, were synthesized by cells which kept the immunological memory. These data are discussed in relation to the humoral protection mechanisms already established in the invertebrates.

[Substances contained in the coelomic fluid of Lumbricus terristris having functions in common with those of some human complement components]. / Mise en évidence de substances contenues dans le liquide coelomique de Lumbricus terrestris, possédant des fonctions communes avec celles de certains composants du complément humain.

In the present work, it was demonstrated for the first time that the coelomic fluid of Lumbricus terrestris contained a substance recognized by human classical convertase. This substance permitted an immune, complement-dependent agglutination response of sheep erythrocytes carrying C3-convertase (EAC142). In addition, substances secreted by coelomic leukocytes possessed an inhibitory activity against human complement: the component C3 was cleaved into a C3b fragment. This result suggests the presence of a system the function of which is C3-convertase-like. We have thus shown that some complement functions, with their natural inhibitors, appear early in evolution. Certain of these functions and structures might be preserved throughout evolution.