Low interleukin-10 production by monocytes of patients with a self-limiting hepatitis C virus infection.

Host factors seem to be crucial for the spontaneous clearance of hepatitis C virus (HCV). Monocytes play a pivotal role in innate immunity and help regulate adaptive responses. This study assesses the characteristics of monocytes from patients with self-limiting HCV infections. We studied 35 consecutive patients [11 with a self-limiting HCV infection, 16 chronically infected with HCV and sustained virological responders (SVR) following antiviral therapy, and eight chronically infected HCV but untreated] and eight healthy donors (HD). The production of interleukin (IL)-10, tumour necrosis factor-alpha (TNF-alpha) and IL-12p40 by monocytes stimulated with lipopolysaccharides(LPS) or HCV Core protein was measured by enzyme-linked immunoassay. Monocyte surface markers were analysed by flow cytometry. LPS and Core protein triggered IL-10 and TNF-alpha production, but monocytes from self-limiting infection patients produced significantly less IL-10 and TNF-alpha than those of SVR, chronically infected or HD (P < 0.05), while IL-12p40 production was unchanged. This cytokine production profile did not appear to be due to expansion of the CD14(+) CD16(+) monocyte subset or to a classical or alternative activation monocyte profile. Monocytes from self-limiting infection patients had more CCR7 than those from SVR or chronically infected patients (P < 0.05). Monocytes of self-limiting infection patients appear to produce little IL-10 and TNF-alpha in response to viral or unspecific stimulation and to have a higher CCR7 expression. This profile seems to be independent to a particular monocyte subset or activation state. Low IL-10 production may help establish an effective immune response and spontaneous HCV clearance.

Effect of continuous irradiation with a very low dose of gamma rays on life span and the immune system in SJL mice prone to B-cell lymphoma.

Ionizing radiation has been shown to have dose- and dose-rate-dependent carcinogenic effects on the hematopoietic and lymphoreticular systems. We report here that continuous exposure to a low dose of gamma rays influences the course of spontaneous B-cell lymphoma in SJL mice. We studied the biological effects of 10 cGy year(-1) gamma rays on the life span of 560 4-week-old SJL/J female mice and on various parameters of the cell-mediated immune response. Life span was slightly prolonged. The mean survival was 397 days for controls and 417 days for irradiated mice that died with lymphoma (P = 0.34). In lymph nodes and spleen, lower percentages of CD4+ and CD8+ T cells were observed in irradiated mice before 32 weeks. Interestingly, the percentages of CD49+ NK cells were increased in the spleens of irradiated mice at 28 weeks (0.61 +/- 0.08% compared to 0.43 +/- 0.12% in controls, P = 0.01) and at 32 weeks (0.62 +/- 0.24% compared to 0.33 +/- 0.09%, P = 0.02), while NK cell activity remained unchanged in exposed mice. These results provide further support for the absence of harmful effects of a continuous very low dose of radiation on life span and incidence of lymphoma in SJL mice.

Treatment of experimental murine pancreatic peritoneal carcinomatosis with fibroblasts genetically modified to express IL12: a role for peritoneal innate immunity.

BACKGROUND: Peritoneal carcinomatosis from pancreatic cancer has a poor prognosis with a median survival of 3.1 months. This is mainly due to lack of effective treatment. Interleukin 12 (IL12) is a proinflammatory cytokine that has a potent antitumoral effect by stimulating innate and adoptive immunity. AIM: To examine the antitumoral effect and toxicity of intraperitoneal delivery of IL12 using an ex vivo gene therapy approach in a murine model of pancreatic peritoneal carcinomatosis. METHODS: Peritoneal carcinomatosis was generated by direct intraperitoneal inoculation of the pancreatic cancer cell line Capan-1 in athymic mice. Syngenic fibroblasts were genetically modified in vitro to secrete IL12 using a polycistronic TFG murine IL12 retroviral vector coding for both p35 and p40 murine IL12 subunits. Ex vivo gene therapy involved injection of the genetically modified fibroblasts intraperitoneally twice a week for 4 weeks. RESULTS: Treatment of pre-established peritoneal carcinomatosis with fibroblasts genetically modified to express IL12 induced a marked inhibition of tumour growth as measured by comparison of the weights of the intraperitoneal tumour nodules in the treated and control animals (3.52 (SD 0.47) v 0.93 (SD 0.21) g, p<0.05) and improved survival. This effect was associated with infiltration of the peritoneal tumour nodules with macrophages. Peritoneal lavage confirmed enhancement of the innate peritoneal inflammatory activity, with an increased number of activated macrophages and natural killer cells. Moreover, macrophages harvested from animals with peritoneal carcinomatosis and treated with IL12-expressing fibroblasts expressed an activated proinflammatory antitumoral M1 phenotype that included strongly enhanced reactive oxygen species and nitric oxide production. There was no treatment-related toxicity. CONCLUSION: Multiple injections of genetically modified fibroblasts to express IL12 is an effective and well-tolerated treatment for experimental murine pancreatic peritoneal carcinomatosis via activated innate immunity and in particular activated M1 macrophages.

Pilot study of interferon-alpha-ribavirin-interleukin-2 for treatment of nonresponder patients with severe liver disease infected by hepatitis C virus genotype 1.

The aim of this randomized prospective study was to assess the efficacy and safety of a triple therapy with interferon-alpha (IFN-alpha)-ribavirin-interleukin-2 (IL-2) for the treatment of patients with genotype 1 infection and high viral load nonresponsive to primary IFN-ribavirin therapy. Twenty hepatitis C virus (HCV) genotype 1 patients with high viral load and Metavir fibrosis score >or=2 without HIV co-infection who were previously nonviral responders to standard treatment with IFN plus ribavirin were intensively re-treated with IFN-alpha2a (3 millions (M) IU every 2 days) combined with ribavirin (1000-1200 mg/day) for a 24-week period. Patients were randomized to receive four cycles of subcutaneous injection of IL-2 (3 MIU/day, 5 days a week every 3 weeks) during either the first 12 weeks (group 1, n = 10) or the last 12 weeks (group 2, n = 10) of combination therapy. At the end of triple therapy, six patients (30%) achieved a biochemical response and 4 (20%) a viral response followed by a relapse after triple therapy withdrawal. After 12 weeks of therapy, no difference in viral load was observed between the groups. The decrease in viral load in group 2 was not raised after the addition of IL-2 to IFN plus ribavirin combination therapy. No serious adverse effects were observed. In conclusion, in patients with poor predictive factors of response, the addition of IL-2 to IFN ribavirin combination therapy does not exert a favourable impact on HCV treatment.

Ex vivo and in vitro impairment of CD36 expression and tumor necrosis factor-alpha production in human monocytes in response to Plasmodium falciparum-parasitized erythrocytes.

Severe malaria is associated with the failure of host defenses to control parasite replication, with the excessive secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), and with the sequestration of parasitized erythrocytes (PEs) in the microcirculation of vital organs. The scavenger receptor CD36, known as a major sequestration receptor, has also been identified as an important factor in mediating nonopsonic phagocytosis of PEs by monocytes and macrophages. The specific consequence of this phagocytosis is a decrease in parasite-induced TNF-alpha secretion. We evaluated the variations in CD36 level and in lipopolysaccharide (LPS)-induced TNF-alpha production in monocytes from Plasmodium falciparum-infected patients and in vitro in the presence of PEs. Both the monocytes from infected patients and from in vitro culture showed a decrease of CD36 expression and a reduced production of TNF-alpha induced by LPS. Using incubation assays with no contact between monocytes and PEs, or in the presence of a soluble supernatant obtained from the incubation of monocytes and PEs, this study shows that decreased CD36 expression was posttranscriptional and not directly related to PEs phagocytosis. In addition, these culture models suggest that the reduced capacity of TNF-alpha production occurred in 2 phases. The early phase (24 hr) appeared to be CD36 dependent and the second phase (48 hr) was due to a soluble factor produced by PEs. These observations suggest that the control of the TNF-alpha production in malaria by monocytes was not entirely dependent on the phagocytosis of PEs by CD36 and that soluble factors produced by PEs could play a role in this process.

Life span, cancer and non-cancer diseases in mouse exposed to a continuous very low dose of gamma-irradiation.

PURPOSE: To analyse the life-span and pathologies of mice living under a continuous very low-dose gamma-irradiation. MATERIAL AND METHODS: We exposed 300 C57B1/6J female mice, 3 weeks old, to 10 cGy year(-1) gamma-rays while 300 control mice lived in the same room. Irradiation was delivered continuously by thorium nitrate. We kept all the animals until natural death and performed autopsy. RESULTS: No difference was observed in life-span (mean lifespan +/-SE: 805.2 +/- 9.62 days for controls and 815 +/- 9.57 days for irradiated mice), weight curves or food intake. At autopsy, cancer was present in 40.9% of controls and 37.9% of irradiated mice. They were mainly represented by lymphomas (23.7 and 24.9%) and histiocytic sarcomas (12.6 and 8.7%, respectively, for controls and irradiated mice). Vascular diseases occurred in 24.1% of controls and 23% of irradiated mice. Infections were present at autopsy in 14.1 and 12.3%, respectively, of controls and irradiated animals. No statistical difference was observed at the end of the experiment for cancer or non-cancer diseases between the two groups. CONCLUSION: Continuous 10 cGy year(-1) gamma-irradiation had no adverse effect on malignant or non-malignant diseases in this strain of mouse.

Increased expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in rat peritoneal macrophages during ovalbumin-induced sensitization.

BACKGROUND: Macrophages are involved in immediate hypersensitivity reactions by their ability to release leukotrienes involved in the symptomatology of allergy. To date it is unknown whether this ability to secrete leukotrienes has been favoured by modifications, occurring during the sensitization phase, of the enzymes involved in leukotriene metabolism. OBJECTIVE: We used ovalbumin-sensitized rats to study the expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in peritoneal macrophages during active sensitization. We compared basal and challenged (PMA, A23187 and allergen) arachidonic acid (AA) metabolism of macrophages from control (cPM) and sensitized (sPM) rats. Then we tested, in cultured cPM, whether IL-4, the predominant cytokine of sensitization process, could reproduce the enzymatic modifications occurring in macrophages during sensitization. METHODS: cPLA2, 5-LO and FLAP expression was assessed by Western blotting. The arachidonic acid (AA) metabolism study was performed after incorporation of tritiated AA in macrophages and analysis of secreted tritiated eicosanoids. RESULTS: Ovalbumin-sensitization of rats increased cPLA2, 5-LO and FLAP expression in peritoneal macrophages. These increased expressions were not paralleled by modifications of basal and PMA- or A23187-stimulated AA metabolism of sPM. However, when macrophages encountered the specific allergen for a second time, sPM secreted higher levels of leukotrienes than cPM. IL-4 induced FLAP expression in cPM but had no effect on cPLA2 and 5-LO expression. CONCLUSION: Active sensitization of rats induces an increase, in peritoneal macrophages, of the enzymes involved in leukotriene metabolism. The increased leukotriene secretion of sPM in response to ovalbumin challenge may be favoured by this increased expression of cPLA2, 5-LO and FLAP that, however, is not able to lead to modifications of macrophage AA metabolism in any circumstance. Our results also suggest that IL-4 is not the major element originating the enzymatic modification induced by sensitization in peritoneal macrophages.

Influence of continuous, very low-dose gamma-irradiation on the mouse immune system.

PURPOSE: To investigate whether continuous, very low-dose gamma-irradiation (10 cGy/year) modifies immune parameters in mice. MATERIAL AND METHODS: C57BL/6 female mice, 4 weeks old, were irradiated for 24 months and compared with control mice living in the same room. B- and T-cell subsets were evaluated by flow cytometry before and after stimulation with lectins; subclasses of immunoglobulins were determined by ELISA 2, 4, 6, 8, 12, 18 and 24 months after the beginning of the irradiation. RESULTS: No difference was found in the percentage of CD4(+) and CD8(+) cells in the thymus and the spleen, or in the reactivity of T-cells to lectins. While the number of B-cells in the spleen remained unchanged, a significant decrease of IgG1, IgG2b and IgG2a was observed after respectively 12, 18 and 24 months of irradiation. CONCLUSION: The parameters of cellular immunity studied were not affected by this chronic low-dose of irradiation, but this dose rate is probably too low to induce the hormetic effect previously described. Further investigations are necessary to assess whether the decline of immunoglobulin secretion is indicative of a lower rate of infectious diseases or a defect in B-cell function.

Contrasting effect of interleukin-4 on the expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in peritoneal macrophages from control and ovalbumin-sensitized rats.

Interleukin-4 (IL-4), which has been widely described as an anti-inflammatory cytokine, can also exert proinflammatory effects. In this study, we extend these findings to demonstrate, in an allergic model, the dual effect of IL-4 on arachidonic acid (AA) metabolism in macrophages. In peritoneal macrophages from control rats (cPM), IL-4 had no effect on cPLA2 and 5-LO expression, but increased FLAP expression without affecting basal and A23187- or PMA-challenged arachidonic acid (AA) metabolism. In contrast, in peritoneal macrophages from ovalbumin-sensitized rats (sPM), IL-4 decreased cPLA2, 5-LO and FLAP expression and PMA-challenged eicosanoid production. A23187-challenged AA metabolism of sPM was not affected by IL-4 pretreatment. Thus, IL-4 acts differently on cPLA2, 5-LO and FLAP expression and AA metabolism in peritoneal macrophages depending on their resident or sensitization-induced differentiated status.

Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase.

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47(PHOX) phosphorylation and translocation showed that NADPH oxidase was activated and involved in ROS production induced by NS3. In a second experiment, NS3 inhibited the oxidative burst induced by phorbol 12-myristate 13-acetate. These results indicate that NS3 activates NADPH oxidase and modulates ROS production, which may be involved in the natural history of hepatitis C infection.

[Calcium oscillations induced by lindane in peritoneal macrophages of mice: control by the maturation stage of the macrophage]. / Oscillations calciques induites par le lindane chez les macrophages péritonéaux de souris: contrôle par l'état de maturité du macrophage.

Mouse resident peritoneal macrophages loaded with Fluo-3 were examined for changes in cytosolic calcium concentration ([Ca2+]i) after stimulation with gamma-hexachlorocyclohexane (Lindane or gamma-HCCH). These studies, realized on macrophage populations, or single cells, by digital imaging microscopy, sought to determine the role of calcium influx on cyclical changes according to maturation stages of macrophages. Single cell analysis of [Ca2+]i changes in macrophages, after gamma-HCCH exposure in 600 microM extracellular calcium, demonstrated that: 1) these [Ca2+]i variations were asynchronous oscillations with the same frequency (1.7 min-1), and 2) these [Ca2+]i variations in macrophages were not at the same [Ca2+]i level. This heterogeneity could be correlated to a cell size partition of the macrophage population (10.1 +/- 0.44 and 11.45 +/- 0.43 microns). In the presence of 100 microM calcium, gamma-HCCH induced a calcium influx into the two subpopulations, but the calcium oscillations appeared only in small macrophages. In the largest ones, [Ca2+]i slowly decreased back down to the basal level. The cell size variation could be correlated to a phenotypic heterogeneity, linked to the differenciation stage of the cell. Peroxydase activity showed that small macrophages were in fact exudate macrophages and the largest ones were resident macrophages. Inhibition of the oscillatory patterns by a decrease in the extracellular calcium concentration ([Ca2+]ext) or by lanthanum chloride (LaCl3) addition is indicative of the important role of calcium influx in the triggering of oscillations. The calcium influx was transient and induced inositol phosphate (InsP3) production in macrophages. The maintainance of these calcium oscillations depended on calcium mobilization from intracellular calcium stores by InsP3, since neomycin and 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) abolished the oscillations. gamma-HCCH induced a transient calcium entry which triggered phospholipase C activation and the associated [Ca2+]i oscillations. However, we showed that differences in cell responses were observed in relationship with the differentiation stage of the mouse peritoneal macrophages, and with the extracellular calcium concentration.

Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques.

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

Increased cyclooxygenase-2 and 5-lipoxygenase activating protein expression in peritoneal macrophages during ovalbumin immunization of mice and cytosolic phospholipase A(2) activation after antigen challenge.

The present study investigates phenotypic and functional differentiation of peritoneal macrophages during ovalbumin-induced subcutaneous immunization of mice. For the first time we show that, in mouse peritoneal macrophages, ovalbumin immunization induces an increase in cyclooxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP) expression whereas it inhibits cytosolic phospholipase A(2) (cPLA2) expression. The study of arachidonic acid (AA) metabolism in peritoneal macrophages from control (cPM) and ovalbumin-immunized (iPM) mice shows that the reduced cPLA2 expression is correlated to a reduced basal AA metabolism, but is not a limiting factor for the opsonized zymosan-, PMA-, or A23187-triggered AA metabolism. We also show that in vitro ovalbumin challenge induces, only in iPM, cPLA2 activation through phosphorylation of serine residues, via a mechanism involving MAP kinases, and through increased intracellular calcium concentrations, leading to eicosanoid production. In parallel, we report that, in peritoneal macrophages, ovalbumin immunization induces the expression of CD23, the low affinity receptor for IgEs known for its involvement in allergic diseases. Thus, the modified expression of the enzymes involved in AA metabolism and the difference of response of cPM and iPM toward the antigen are important elements to understand the underlying mechanisms of ovalbumin-induced allergic responses.

Respective roles of inflammation and axonal breakdown in the regulation of peripheral nerve hemopexin: an analysis in rats and in C57BL/Wlds mice.

We have previously demonstrated that one of the peripheral nerve responses to injury is the overexpression of hemopexin (HPX). Here, we demonstrate that Wallerian degeneration is required for this response, since HPX does not increase in C57BL/Wlds mice, which display a severely impaired Wallerian degeneration. We also show that HPX synthesis is dramatically increased in macrophages during their activation or after IL-6 stimulation. However, IL-6-driven HPX overexpression occurs in vivo and in vitro in the absence of substantial macrophage invasion. We conclude that, after nerve injury, HPX overexpression occurs first in Schwann cells as a result of axotomy and is subsequently regulated by inflammation. Furthermore, our results and those already described suggest that IL-6, synthesized by the various cell types producing HPX, control nerve HPX expression via paracrine and autocrine mechanisms.

Inhibitory activity of anti-interleukin-4 and anti-interleukin-10 antibodies on Toxoplasma gondii proliferation in mouse peritoneal macrophages cocultured with splenocytes from infected mice.

Cocultures of splenocytes from Toxoplasma gondii-immunized mice or from naive mice, separated by a transwell membrane from naive macrophage layers, induced a decrease in T. gondii proliferation in macrophages in comparison with cultures without splenocytes or cocultures with splenocytes from infected mice. Interleukin 4 (IL-4) and IL-10 levels increased in cocultures of splenocytes from infected mice with naive macrophages. In contrast, the levels of these cytokines decreased in cocultures with splenocytes from immunized mice. No correlation was found between the release of interferon-gamma (IFN-gamma) and the inhibition of parasite multiplication. Cocultures with splenocytes from immunized mice induced an increase in tumor necrosis factor-alpha (TNF-alpha) levels. In contrast, in cocultures with splenocytes from infected mice, TNF-alpha production decreased. In cocultures with splenocytes from infected mice, T. gondii proliferation in macrophages was neutralized by anti-IL4 or anti-IL10 antibodies and was associated with increased TNF-alpha production. Moreover, this study demonstrates the significant combined effect of IL-4 and IL-10 on the down-regulation of macrophage-effector functions. A soluble positive signal was given by splenocytes to induce the production of TNF-alpha by macrophages. This signal was inhibited by IL4 and IL10. This process is biologically relevant in the regulation of T. gondii proliferation.

Reactive oxygen intermediates and eicosanoid production by kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4.

OBJECTIVE AND DESIGN: The aim of the present study was to characterize during acute and chronic liver injury induced by CCl4, macrophage phenotypes and whether a change in reactive oxygen intermediates (ROI) and eicosanoids production by Kupffer cells (KC) was observed. MATERIAL AND METHODS: Liver steato-necrosis and cirrhosis were induced in rats after 3 weeks and 9 weeks of CCl4 intoxication, respectively. Monocytes and tissue macrophages were identified by immunohistochemical study using monoclonal antibodies ED-1 and tissue macrophages using the antibody ED-2. The release of ROI and eicosanoids in response to the phorbol ester TPA (protein kinase activator) and to the calcium ionophore A23187 was assessed in cultivated cells. RESULTS: As compared to healthy controls, livers of rats with steato-necrosis or cirrhosis exhibited a significant increase of ED-1 and ED-2 positive cells. Only KC from rats with liver steato-necrosis were found to have higher A23187, TPA + A23187 or opsonized zymosan induced ROI production than healthy controls (p < 0.01). After TPA + A23187 or opsonized zymosan stimulation, KC from both rats with steato-necrosis or cirrhosis produced more TxB2 and leukotrienes and less PGE2 as compared to healthy controls (p <0.05). CONCLUSIONS: These results suggest an influx of monocytes into the liver during acute and chronic injury induced by CCl4. Functional changes of this inflammatory infiltrate have been demonstrated with an increase of ROI production only in the early stage of liver injury whereas a rise in KC leukotriene production and an imbalance between cytoprotective and cytotoxic prostanoids were observed at all stages of liver disease.

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

IL-13 induces serine phosphorylation of cPLA2 in mouse peritoneal macrophages leading to arachidonic acid and PGE2 production and blocks the zymosan-induced serine phosphorylation of cPLA2 and eicosanoid production.

In a recent investigation, we demonstrated that long-term treatment of macrophages with IL-13 enhances cPLA2 expression and modulates zymosan-stimulated AA mobilization. In the present study, we examine the ability of IL-13 to modify the cPLA2 activity and the AA mobilization of macrophages after a short-period of treatment. We demonstrate that in resting macrophages, IL-13 induces, through a MAP kinase-dependent process, (1) an increase of free AA release within 15 min, followed by increased PGE2 production and (2) a time-dependent serine phosphorylation of cPLA2. Conversely, in macrophages stimulated by zymosan, IL-13 added 30 min before zymosan inhibited the AA release and the serine phosphorylation of cPLA2 induced by the phagocytic agonist. In conclusion, these findings show for the first time that a Th2-type cytokine can upregulate cPLA2 activity and downregulate zymosan-induced AA metabolism. Thus, establishment of the connection between these two events may help to understand the complex regulatory role of IL-13 on the macrophage AA metabolism.

Activation of mitogen-activated protein kinase by fumonisin B(1) stimulates cPLA(2) phosphorylation, the arachidonic acid cascade and cAMP production.

Activation of mitogen-activated protein kinase (MAPK) results in pleiotropic effects such as modulation of the transcription and activation of enzymes involved in signal transduction. One such enzyme is the cytoplasmic phospholipase A(2) (cPLA(2)), which releases arachidonic acid (AA). AA is the precursor of prostaglandins and leukotrienes, two inflammatory mediators, which regulate gene expression and protein kinase (PK) activity. Fumonisin B(1) (FB(1)) was shown to increase PKC translocation and stimulate MAPK. We have investigated the effect of FB(1) on the AA cascade in a human epithelial cell line and the signal transduction pathway regulating PLA(2) activation. We observed that FB(1) stimulated cPLA(2) activity and increased AA release by a mechanism independent of PKC activation and that the activation of cPLA(2) is a two-step process: the first is phosphorylation of cPLA(2) by MAPK; the second is a consequence of the increase in sphingosine inside and outside the cells after 2 h, which is known to induce a rise in intracellular free calcium. Overall, this suggests that the effect of FB(1) on cells is partially dependent on the action of FB(1) on the enzymes involved in the cell cycle, such as MAPK and PKA, and on bioactive fatty acids, such as the prostaglandins and leukotrienes, and also on disruption of sphingolipid metabolism. In addition, we have observed down-regulation of cPLA(2) activity and AA metabolism by a mechanism involving prostaglandin production, cAMP synthesis and PKA activation.

Roles of cyclooxygenase and lipoxygenases in ochratoxin A genotoxicity in human epithelial lung cells.

The roles of constitutive prostaglandin-H-synthetase (PGHS) and lipoxygenases in ochratoxin A (OTA) genotoxicity, as reflected by DNA adduct formation, have been investigated in vitro: (1) in culture of human epithelial cells and (2) by incubation in presence of pig seminal vesicle microsomes. Indomethacin (0.1 µM), which inhibits PGHS and significantly increases leukotriene C(4) production by enhancement of lipoxygenases, enhanced formation of OTA-DNA adducts tenfold. At highest dose of 10 µM, indomethacin inhibited all pathways (PGHS and lipoxygenases) and thus prevented OTA-DNA adduct formation. Nordihydroguaiaretic acid, which inhibits lipoxygenases, suppressed OTA-DNA adduct formation. The OTA metabolites formed were analysed by HPLC. OTα, 4[R]- and 4[S]-hydroxy-OTA and a unidentified derivative were formed in control cells. After pre-incubation with indomethacin (0.1 µM), further unidentified metabolites were obtained. They were similar to those obtained in presence of pig seminal vesicle microsomes. These data demonstrate that OTA is biotransformed into genotoxic metabolites via a lipoxygenase, whereas PGHS decreases OTA genotoxicity.