Light pulse induces ALA-S gene expression in the rat Harderian gland.

The rodent Harderian glands (HGs) are large paired orbital organs with highest porphyrinogenic rates. We have previously shown that continuous light exposure abolished the day/night variations of the delta-aminolevulinate synthase (ALA-S; the rate-limiting enzyme for porphyrin biosynthesis) gene expression observed under standard light: dark cycles (LD 12:12) in the rat HGs. This study was designed to examine whether the ALA-S changes were actually associated directly with light. The response of ferrochelatase (enzyme that converts protoporphyrin IX into heme) to light was also examined. Male Wistar rats were acclimatized to light: dark cycles regimen of 12:12 for 2 weeks. At the end of the 2 weeks, a 1 h-light pulse was applied in the middle of the dark phase. Animals were sacrificed immediately after the end of the light pulse. HGs were collected and stored at -80 degrees C until processed for quantitative RT-PCR. A 1 h-light pulse applied during mid-dark caused a significant increase of ALA-S gene expression (3-fold higher than in controls), whereas it was without effect on ferrochelatase gene expression. Our results suggest that light per se may regulate ALA-S gene expression in the rat HGs, and reveal that the ALA-S gene expression, and so heme biosynthesis, is under a photodynamic control.

Excessive erythrocyte PPIX influences the hematologic status and iron metabolism in patients with dominant erythropoietic protoporphyria.

Partial deficiency of the last enzyme of the heme biosynthetic pathway (namely ferrochelatase, FECH) in humans is responsible for erythropoietic protoporphyria (EPP). This disorder is characterised by painful photosensitivity, due to excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial hypotheses have been proposed to explain the hematologic and iron status of EPP patients. In the present work, we explored these parameters in 55 patients with dominant EPP recruited at the French Center of Porphyrias (Colombes, France) and confirmed by molecular analysis. Our data show that erythrocyte accumulation of PPIX in EPP patients influences hematologic and iron status. Patients studied had a mild anemia and thrombocytopenia, as shown by the downward shift of hematologic parameters, which positively correlated with the amount of erythrocyte PPIX. Interestingly, erythropoiesis did not seem to be limited by iron supply in patients, since serum iron and soluble transferring (Tf) receptor (sTfR) were normal. However, iron and Tf saturation negatively correlated with erythrocyte PPIX. Moreover, and as previously described in a mouse model of EPP, we noted a positive correlation between erythrocyte PPIX and Tf levels. Altogether, these results suggest a positive effect of PPIX on the synthesis on Tf, which could facilitate the mobilization of tissue iron stores to meet erythropoiesis requirement. Based on these observations and previous results in EPP mouse model, we propose that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen and the bone marrow.

Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats.

AIMS/BACKGROUND: Liver regeneration is a physiological mechanism which leads to restoration of the hepatic parenchyma following hepatectomy or toxic injury. This process is mediated by a wide variety of cytokines and growth factors. The aim of the present study was to evaluate the influence of hepatectomy extent on the levels of intrahepatic mRNAs for cell-cycle markers and growth factors in rats submitted to a 30%, two-third or 80% hepatectomy. METHODS: Cyclins, thymidine kinase and growth factors mRNA levels were quantitatively assessed by RT-PCR at different time points post-hepatectomy (2h, 6h, 12h, days 1, 2, 6). RESULTS: As compared with a two-third hepatectomy, cyclins and thymidine kinase mRNA levels were increased but with a delayed peak at day 2 in the 80% hepatectomy group and showed a progressive increase until day 6 in the 30% hepatectomy group; mRNA levels for HGF or TGFalpha were increased with a delayed peak at 12 h or day 2 in the 80% hepatectomy group, respectively and this delay was more pronounced in the 30% hepatectomy group with a peak at day 1 or day 6. CONCLUSION: A regenerative response occurs whatever the extent of hepatectomy but the course of regeneration and expression of growth factors differs according to the volume of resected liver. A better knowledge of these events could improve the clinical results of hepatic resection for primary or metastatic liver disease.

Induction of haem oxygenase contributes to the synthesis of pro-inflammatory cytokines in re-oxygenated rat macrophages: role of cGMP.

Macrophage activation and the resulting inflammatory response may be a major component of tissue injury upon hypoxia and re-oxygenation. Activation of the haem oxygenase (HO)/carbon monoxide (CO) pathway may be an important regulator of the inflammatory response, through production of cyclic 3', 5'-monophosphate (cGMP). We have assessed whether HO contributes to the increased production of the pro-inflammatory cytokines TNF-alpha and IL-6 in re-oxygenated rat peritoneal macrophages.Hypoxia/re-oxygenation markedly increased levels of HO-1 mRNA and cGMP. The increase in cGMP was reduced by the HO-1 inhibitor tin-protoporphyrin (SnPP-9) given during re-oxygenation. Hypoxia and re-oxygenation also increased IL-6 and TNF-alpha mRNA expression, as well as IL-6 and TNF-alpha concentrations in the cell supernatant. These increases were nullified by SnPP-9 and by Methylene Blue, an inhibitor of guanylate cyclase, but were not affected by L-NNA, an inhibitor of NO synthesis. The inhibitory effect of SnPP on the synthesis of cytokines was reversed by co-administration of the stable analogue of cGMP, 8-Br-cGMP. Our results indicate that activation of haem oxygenase and of the CO/cGMP pathway is a major stimulus for the synthesis and release of pro-inflammatory cytokines in re-oxygenated macrophages. This pathway may play a central role in pathological situations in which local tissue hypoxia/re-oxygenation triggers a systemic inflammatory response, for example in patients with shock.

Heme and acute inflammation role in vivo of heme in the hepatic expression of positive acute-phase reactants in rats.

Acute-phase protein synthesis in the liver during inflammation is regulated via cytokines and glucocorticoids. Using quantitative reverse transcription (RT)-PCR analysis and immunoassay, we explored, in the rat, the response of the acute-phase protein, alpha-2 macroglobulin (A2M), after systemic inflammation induced by lipopolysaccharide (LPS) or localized inflammation induced by turpentine oil (TO). The results indicate that synthesis of A2M is higher following TO-induced inflammation than LPS-induced inflammation and is not correlated with interleukin (IL)-6 or glucocorticoid levels. We studied the putative role of heme in this differential A2M expression following localized vs. systemic inflammation; addition of heme during LPS-induced inflammation can boost the expression of A2M, whereas blocking heme synthesis (by succinyl acetone) or enhancing its consumption in parallel biosynthetic pathways (cytochrome P450 induction by phenobarbital) decreases A2M expression. This decrease was abolished by exogenous heme supplementation. Finally, we demonstrate that heme supplementation is also able to increase the A2M response in female rats to a level similar to that in male rats providing a new insight into the puzzling sexual dimorphism observed previously during localized inflammation. We propose that heme should be considered a new regulatory element in controlling liver A2M expression during inflammation.

Nitric oxide synthase inhibition and the induction of cytochrome P-450 affect heme oxygenase-1 messenger RNA expression after partial hepatectomy and acute inflammation in rats.

OBJECTIVES: a) To evaluate in vivo, in rat liver, heme oxygenase-1 (HO-1) messenger RNA (mRNA) expression level and the regulation of 3',5'-cyclic guanosine monophosphate (cGMP) production during hepatic regeneration, localized inflammation, and systemic inflammation; and b) to investigate the effect of the induction of cytochrome P-450 and nitric oxide synthase (NOS) inhibition on HO-1 mRNA level and cGMP production in the liver. DESIGN: Experimental, comparative study. SETTING: Biochemical and molecular biology laboratory. SUBJECTS: Six-wk-old male Sprague-Dawley rats (n = 60). INTERVENTIONS: We randomly divided the rats into four groups: a) saline controls; b) animals receiving lipopolysaccharide (600 microg/kg) for systemic inflammation; c) animals receiving turpentine oil (5 mL/kg) for localized inflammation obtained by sterile abscess; and d) partially hepatectomized animals (two-thirds removal of the parenchyma) for hepatic regeneration. MEASUREMENTS AND MAIN RESULTS: Hepatic regeneration induced HO-1 mRNA expression, as shown by quantitative reverse transcription-polymerase chain reaction analysis. The time course of liver HO-1 mRNA induction after partial hepatectomy and localized and systemic inflammation showed a similar and gradual increase, with a maximum at 6 hrs and a return to a minimal level 48 hrs after treatments. Liver HO-1 mRNA was overexpressed during localized vs. systemic inflammation. This overexpression was not correlated with either serum IL-6 or corticosterone concentrations, but is related to increased cGMP production. The administration of phenobarbital, a cytochrome P-450 inducer and of nitro-L-arginine methyl ester, a NOS inhibitor, prevented cGMP production and abolished the overexpression of HO-1 mRNA. CONCLUSIONS: The results of this study indicate that HO-1 mRNA is induced during hepatic regeneration with a similar time course to that observed during acute inflammation. In addition, we demonstrated that: a) HO-1 mRNA is overexpressed during localized vs. systemic inflammation; b) this overexpression is not correlated with IL-6 or corticosterone concentrations but is related to intrahepatic cGMP production; c) induction of cytochromes P-450 and/or inhibition of NOS both reduce liver cGMP production and HO-1 mRNA expression. These results suggest that in rat liver, a cGMP-transducing pathway may control HO-1 mRNA expression. Thus, there may be a role for HO-1 mRNA in the modulation of the hepatic stress response.

Hepatic and extra-hepatic transcription of inter-alpha-inhibitor family genes under normal or acute inflammatory conditions in rat.

The expression and level of the mRNAs for the five genes that code for a set of plasma proteins collectively referred to as the inter-alpha-inhibitor family have been studied in rat under a normal condition or in the course of a turpentine-induced, systemic inflammation. In healthy rats, all five mRNAs [H1, H2, H3, H4, and alpha1-microglobulin/bikunin precursor (AMBP)] are expressed primarily in liver and two of them (H2 and H3) are found to a lower extent in brain. By in situ hybridization onto sections of a normal brain, the H3 mRNA has been precisely localized to the hypothalamus, amygdala, pontine area, optic tectum, and cerebellum. By reverse transcriptase-polymerase chain reaction of total RNAs obtained from a panel of organs, low amounts of one or more mRNA(s) could be detected in other locations (e.g., intestine and stomach). Furthermore, the extrahepatic expressions of several of these genes are up- or downregulated at 20 h after the start of a turpentine-induced inflammation. In liver, the contents of H3 and H4 mRNA are upregulated, whereas those of AMBP and H2 are downregulated during the acute phase. This is accounted for by changes in gene transcription, the kinetics of which is gene-specific. This behavior of H1, H2, H3, H4, and AMBP mRNAs in rat liver is in keeping with more limited analyses made at mRNA and/or protein levels in other species (human, pig) suffering from an acute inflammation. Therefore, the inflammation-associated regulation of these five genes that is conserved between species indicates that the inter-alpha-inhibitor family members are likely to be important partners of the acute phase response.

Induction and modulation of acute-phase response by protein malnutrition in rats: comparative effect of systemic and localized inflammation on interleukin-6 and acute-phase protein synthesis.

The acute-phase protein (APP) response is regulated by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor (TNF), but may also be influenced by malnutrition. The aims of this study were as follows: 1) to determine in rats the effect of a protein-deficient diet on IL-6 mRNA expression in intestine, liver and peripheral blood mononuclear cells (PBMC), and on alpha-1 acid glycoprotein (AGP) and alpha-2 macroglobulin (A2M) serum levels and hepatic mRNA expression; 2) to compare, in protein-deficient rats, the IL-6 and APP responses after a turpentine (TO)- or a lipopolysaccharide (LPS)-induced inflammation; and 3) to determine the effect of a protein malnutrition on IL-6 mRNA expression in rat PBMC treated ex vivo with LPS. Interleukin-6 mRNA was present in intestine and PBMC but not in the liver of malnourished rats, and was absent in any tissue or cells of controls. A2M was present in the serum from malnourished rats but not after refeeding. AGP mRNA expression was not influenced by protein malnutrition. In malnourished rats, IL-6 serum level peaked later than in controls after TO and LPS treatment. In malnourished TO-treated rats, A2M mRNA increased earlier than in controls and remained detectable later than in controls. AGP mRNA expression after TO was not influenced by protein malnutrition. In PBMC of malnourished rats, LPS-induced IL-6 mRNA expression occurred earlier and lasted longer than in controls. Our results indicate that protein malnutrition by itself induces IL-6 and A2M expression, and that it modulates the APP response to inflammation.

Gut ischemia and mesenteric synthesis of inflammatory cytokines after hemorrhagic or endotoxic shock.

The intestine plays a major role in the pathophysiology of multiorgan failure. Although the systemic inflammatory response might be induced by endotoxin released through bacterial translocation, other factors such as intestinal ischemia might be implicated. We investigated the relationship between intestinal ischemia-reperfusion and cytokine release in rat models of hemorrhagic or endotoxic shock. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), lactate, and endotoxin, as well as macrophage TNF-alpha and IL-6 mRNA expression, were assessed at the end of shock and resuscitation. Hemodynamic changes and lactate levels suggested the presence of intestinal ischemia in both models. Mesenteric levels of TNF-alpha and IL-6 were increased by hemorrhage and further increased after saline resuscitation. Similar results were obtained with mRNA cytokine gene expression in macrophages. Endotoxin was not detectable in the hemorrhagic group. Endotoxic shock also increased production of cytokines, which, in contrast to hemorrhage, was not further increased by resuscitation. These results suggest that intestinal ischemia-reperfusion upon hemorrhage and resuscitation may be a major trigger for cytokine gene expression in the absence of endotoxin.

Cytokine gene expression in liver following minor or major hepatectomy in rat.

Interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and transforming growth factors alpha and beta (TGF-alpha and TGF-beta) are important mediators which play a pleiotropic role in both inflammatory and hepatic regeneration processes. It has also been proposed that a major hepatectomy impairs the liver-related host defence mechanisms. The aim of this study was to evaluate the influence of minor (30%) vs major (80%) hepatectomy on cytokines, growth factors and acute-phase proteins both at the protein and mRNA levels in rat. For that purpose, rats were submitted to either 30% or 80% hepatectomy and sacrificed at intervals up to day 14 post-hepatectomy to collect liver and blood samples. Serum levels of IL-6 and acute-phase proteins (APPs) were determined after RNA extraction, cytokine and acute-phase proteins gene expression were evaluated using a quantitative RT-PCR method. The results demonstrate that liver mRNA levels for IL-6 were early unregulated after a 80% resection only, whereas liver mRNA levels for IL-1 slowly increased following 30 or 80% hepatectomy. For TNF-alpha, no significant changes were observed between groups. Growth factor expression differed according to the extent of hepatic resection. Moreover, plasma levels of alpha2-macroglobulin (alpha2M) and alpha1 acid glycoprotein (AGP), two major APPs which respond differently to combination of cytokines, were significantly lowered after a major resection whereas levels of serum IL-6 showed no significant changes between groups. Paradoxically, in the 80% hepatectomized group, alpha2M mRNA expression was strongly increased at 4 h and 6 h post-hepatectomy as compared with the 30% hepatectomized group. Taken together, these results suggest that, although an increased level of hepatic IL-6 expression was observed following a major resection, the liver's capacity to synthesize normal levels of APPs was impaired. Moreover, these specific changes of cytokine gene expression seen in the liver following major hepatectomy might reflect a preferential activation of the IL-6-dependent APPs.

Differential expression of cytokine genes in monocytes, peritoneal macrophages and liver following endotoxin- or turpentine-induced inflammation in rat.

Pro-inflammatory cytokines are produced after systemic or local inflammation by a wide variety of cell types including monocytes, macrophages, Kupffer and endothelial cells. Previous studies have shown that IL-6 gene expression does not occur in liver from rats undergoing an acute phase response after turpentine injection or controls. These data do not rule out the possibility that delivery of a pathogen to the liver via the portal circulation could directly activate the Kupffer cells. Rats were injected either intravenously or intraperitoneally with LPS, or subcutaneously with turpentine oil. The changes in IL-1 beta, IL-6, and TNF mRNA levels in monocytes (collected from portal vein or caval cein), peritoneal macrophages and liver over a 3-hour period post-treatment were examined. The kinetics of LPS-vs turpentine-induced cytokine mRNAs in these various cell types were compared by quantitative reverse transcription and polymerase chain reaction (RT-PCR). Our data demonstrate that an intrahepatic expression of cytokines in the non parenchymal cells was induced by an LPS challenge but not by a turpentine-induced inflammation. This process could act as a paracrine mechanism in the acute-phase response and play a role in the modulation of hepatic regeneration.