Lipid Peroxidation and Changes in T Lymphocyte Subsets and Lymphocyte Proliferative Response in Experimental Iron Overload

This study was undertaken to investigate the hypothesis that lipid peroxidation might be associated with immunological abnormalities in experimental hemosiderosis. The correlation between the degree of plasma and spleen lipid peroxidation with lymphocyte proliferative response and with the proportion of T lymphocyte subsets was studied in normal and iron overloaded male Sprague Dawley rats. The iron-loading protocol consisted of a total dose of iron-dextran (1.5 mg/Kg body weight) divided in daily i.m. injections over twenty consecutive days. Lipid peroxidation was measured by the thiobarbituric acid assay in plasma and in homogenates of spleen. Plasma lipid peroxide level increased rapidly after i.m. administration of iron-dextran and decreased sharply at 48 h after the last injection. Conversely, a progressive increase of lipid peroxidation in homogenates of spleen was observed in the course of the iron overload protocol, remaining high even at 50 days after initiation of iron-dextran injections. The increase of spleen lipid peroxide levels was associated with decreased lymphocyte proliferative response to Con A in iron overloaded rats. The addition of superoxide dismutase and catalase to lymphocyte cultures reversed the inhibition of the proliferative response, implicating reactive species of oxygen as the causative agents of these alterations. These effects may be related with the enhanced membrane and DNA damage occurring during intracellular and extracellular peroxidation. Negative correlations between helper/cytotoxic ratio and malondialdehyde levels were obtained in blood and spleen during iron administration. These results supports the hypothesis that lipid peroxidation plays a role in the immunological abnormalities observed in experimental hemosiderosis.     

T Lymphocytes Subsets in Experimental Iron Overload

Several abnormalities of the immune system have been reported in association with clinical and experimental iron overload. To dissect further such abnormalities, changes in lymphocyte subsets were evaluated in iron-loaded male Sprague-Dawley rats. The iron-loading protocol consisted of a total dose of iron-dextran (1.5 mg/Kg body weight) divided in daily intramuscular injections over twenty consecutive days. At days 0, 20, and 50 after initiation of iron injections lymphocyte subsets in blood, spleen and mesenteric lymph nodes were estimated by indirect immunofluorescence using monoclonal antibodies recognizing T cells (W3.13), the subset of helper T cells in (W3.25), and the subset of cytotoxic T cells (OX.8). By day 20, there was no change in the number of W3.25+ T cells in the blood of iron-loaded animals as compared to the controls, but the OX.8 + T cells were significantly elevated. At this time, the ratio W3.25 ⁺/OX.8⁺ cells was significantly decreased (0.5 in experimental rats vs 2.0 in controls). Similar results were obtained at day 50. In the spleen, there was a decrease in the proportion of W3.25 ⁺T cells and an increase in OX.8⁺ T cells at day 20. However, these values returned to normal by day 50. A negative correlation between W3.25 ⁺/OX.8⁺ ratio and serum ferritin was observed in blood and spleen during iron administration. These changes were associated with abnormalities in lymphocyte proliferative response. No changes in W3.25 ⁺/OX.8⁺ ratio were observed in mesenteric lymph nodes. These results demonstrate that iron overload alters the distribution of T lymphocytes in various compartments of the immune system.     

Effect of Hepatic Isoferrttins from Iron Overloaded Rats on Lymphocyte Proldzerative Response: Role of Ferritin Iron Content

Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a dose-dependent manner in all assays in which spleen cells were incubated with rat and horse isoferritins. However, isoferritins with higher iron contents (rat isoferritin obtained by ultracentrifugation and horse ferritin) caused a greater decrease of proliferative response at 5 and 25 μ/ml than the others. Rat and horse apoferritins showed no inhibitory effect on lymphocyte proliferative response, suggesting that the effect is due to iron probably through the damaging effect of reactive oxygen species generated by iron released by the isoferritins on lymphocyte functions.

Additionally, the role of serum ferritin level on proliferative response was studied in an experimental model of iron overload in rats. An inverse relationship between the proliferative response and serum ferritin levels was observed.

Our results suggest that the inhibitory effect of the isoferritins on lymphocyte proliferative response is due, at least partially, to the iron content of this protein and not exclusively to variation in pi as suggested by other authors. These results are in agreement with the possible immunosuppressor role of ferritin in vivo.     

Effect of Hepatic Isoferrttins from Iron Overloaded Rats on Lymphocyte Proldzerative Response: Role of Ferritin Iron Content

Abstract

Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a dose-dependent manner in all assays in which spleen cells were incubated with rat and horse isoferritins. However, isoferritins with higher iron contents (rat isoferritin obtained by ultracentrifugation and horse ferritin) caused a greater decrease of proliferative response at 5 and 25 μ/ml than the others. Rat and horse apoferritins showed no inhibitory effect on lymphocyte proliferative response, suggesting that the effect is due to iron probably through the damaging effect of reactive oxygen species generated by iron released by the isoferritins on lymphocyte functions.

Additionally, the role of serum ferritin level on proliferative response was studied in an experimental model of iron overload in rats. An inverse relationship between the proliferative response and serum ferritin levels was observed.

Our results suggest that the inhibitory effect of the isoferritins on lymphocyte proliferative response is due, at least partially, to the iron content of this protein and not exclusively to variation in pi as suggested by other authors. These results are in agreement with the possible immunosuppressor role of ferritin in vivo.     

Betanodavirus as a novel transneuronal tracer for fish

Deferoxamine (DF) is an antioxidant molecule because of its ability to chelate iron. This study compared the ability of DF alone or in combination with melatonin, 5-methoxytryptophol or pinoline in preventing lipid peroxidation due to hydrogen peroxide (H(2)O(2)) in rat brain homogenates. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the homogenates were measured as indices of lipid peroxidation. Incubation of homogenates with DF reduced, in a dose-dependent manner, MDA+4-HDA formation due to H(2)O(2). When melatonin, 5-methoxytryptophol or pinoline were added to the incubation medium, the efficacy of DF in preventing lipid peroxidation was enhanced. These cooperative effects between DF, melatonin, and related pineal products may be important in protecting tissues from the oxidative stress due to iron overload.     

The effect of parenteral iron dextran, with or without desferrioxamine, on the development of experimental pseudotuberculosis in the domestic chicken.

The development of disease following oral challenge with Yersinia pseudotuberculosis (serotype II) was compared in four groups of five chickens treated with a combination of 10 mg parenteral iron-dextran, 10 mg of the chelating agent desferrioxamine or 10 mg of dextran, 2 days before infection. The chickens pretreated with iron-dextran, with or without desferrioxamine, developed diarrhoea and were lethargic 2 days following bacterial challenge. Chickens not given iron-dextran showed no clinical signs of disease. Histological examination of selected tissues indicated that chickens pre-treated with iron-dextran had significantly more intestinal lesions, but fewer lesions in the spleen, than chickens in groups not treated with iron-dextran. In contrast to control chickens given iron dextran, but not challenged with bacteria, there was no stainable iron in the livers of chickens challenged with Y. pseudotuberculosis 10 days after an injection of 10 mg of iron-dextran. This suggests that chickens challenged with Y. pseudotuberculosis utilised exogenously administered iron during infection.     

Increased lipid peroxidation in tissues of nickel chloride-treated rats

Parenteral administration of nickel chloride (NiCl2) to rats enhanced lipid peroxidation in liver, kidney, and lung (but not in brain, heart, spleen, or testis), as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) and related chromogens in fresh tissue homogenates. After sc injection of NiCl2 (0.75 mmol per kg body wt), MDA concentrations in liver and kidney became significantly increased by nine h and reached peak values at 48 h. For example, in nine rats killed 48 h after the NiCl2 injection, hepatic MDA concentrations averaged 2.5 +/- 1.0 mumol per g dry wt (P less than 0.001 versus 0.5 +/- 0.3 mumol per g in 30 controls). Dose-effect relationships for lipid peroxidation in liver and kidney were observed with NiCl2 dosages ranging from 0.12 to 0.75 mmol per kg, sc. Intrarenal administration of a carcinogenic nickel compound, nickel subsulfide (Ni3S2, 0.36 mmol per kg body wt), did not affect MDA concentrations in the injected kidneys of rats killed one to 20 days post-injection. The results of this study implicate lipid peroxidation as a molecular mechanism for cell injury in acute NiCl2 poisoning, but they do not furnish any evidence that lipid peroxidation is involved in the initiation of nickel carcinogenesis.     

Effect of streptozotocin on glutathione and lipid peroxide levels in various tissues of rats.

In this study, the effect of streptozotocin (STZ) on lipid peroxidation and glutathione (GSH) content was investigated in the liver, pancreas and kidney of rats. Lipid peroxide levels were significantly increased in homogenates and mitochondrial fractions of the liver, kidney and pancreas after STZ administration. GSH levels in hepatic and pancreatic tissues were decreased but unchanged in the kidney of diabetic rats. GSH content in hepatic mitochondrial fraction was also decreased compared to control group. On the other hand, the destruction of pancreatic beta-cells was also observed histopathologically. Our results indicate that oxidative stress may play an important role in STZ induced diabetes and mitochondrial fraction may be the target in this toxicity.     

The effect of iron (Fe3+) on the cloning efficiency of human memory T4+ lymphocytes.

The effect of iron (Fe3+) and normal human liver ferritin on the proliferative response of normal human lymphocytes to tetanus toxoid was examined. This proliferative response involved memory T4+ lymphocytes as shown by a selective depletion study. Limit dilution analysis revealed that iron, present as ferric citrate, affected the initiation of clone development, and that concentrations of ferric citrate from 30 microM to 1 nM were able to reduce significantly the cloning efficiency of precursor T cells (up to 90% reduction). The reduced cloning frequency was not due to immunological suppression. Clone size was also reduced when iron was present during culture. In contrast, the presence of normal human liver ferritin during culture (concentration range: 300 micrograms/1-10,000 micrograms/1) had no effect on lymphocyte proliferation. The data indicate that low molecular weight iron (as ferric citrate) in concentrations similar to those which have been reported in the serum of patients with iron overload diseases, can interfere with antigen-specific lymphocyte responses and this may have implications for the development of infections and neoplasia in diseases of iron-overload.     

Modulation of exudate inflammation parameters in rat carrageenan-induced granuloma by modification of exudate iron levels

We have used the carrageenan-induced pouch-granuloma in rats to investigate how changes in low-molecular-mass iron chelate levels in the exudate, induced by iron loading (iron-dextran) or chelation (desferrioxamine) influence cellular and systemic inflammatory parameters.In the iron-treated group we observed a rapid decrease in the number of leukocytes and exudate volume; there was also an increase in ferritin iron and low-molecular-mass iron chelates, and on the eighth day a systemic response. In the desferrioxamine-treated group we detected a decrease in low-molecular-mass iron chelates, ferritin iron, and an increase in the number of leukocytes. We describe the protective effects of desferrioxamine against the deleterious effects of ferrous iron and relate this to its chelating and scavenging activity.The results suggest that the levels of low-molecular-mass iron chelates modulate the inflammatory response, possibly through their contribution to the oxygen free radical generation, which is responsible for the cell membrane damage and subsequently its death. The modulatory action of iron-dextran and desferrioxamine support our hypothesis.     

The effect of experimental iron-overload on splenic T cell function: analysis using cloning techniques.

The effect of iron-overload on cell-mediated immunity was examined in C57 mice. Two methods of iron-loading were used: (i) dietary carbonyl iron which produced iron-loading primarily of parenchymal cells or (ii) intraperitoneal administration of iron-dextran which produced iron-loading predominantly of Kupffer cells. Both methods of iron-loading resulted in a diminished capacity of spleen cells to generate an allo-specific cytotoxic response in the absence of exogenous interleukin 2 (IL-2). Exogenous IL-2, however, restored the ability of spleen cells from iron-loaded mice to generate allo-specific cytotoxicity in bulk culture. Clonal assays for the precursor cells of cytotoxic T lymphocytes (CTL-P), performed in the presence of added IL-2, demonstrated that iron-loaded mice contained normal numbers of CTL-P. However, cultures of spleen cells from carbonyl iron-loaded mice generated less IL-2 following Concanavalin A stimulation, apparently as a result of a reduction in the number of IL-2-secreting cells amongst the spleen cell population. This work presents further evidence that iron-overload is associated with defective immunoregulatory control.     

Lysosomotropic Agents Ameliorate Macrophage Dysfunction Following the Phagocytosis of IgG-Coated Erythrocytes: A Role for Lipid Peroxidation

Phagocytosis of IgG-coated erythrocytes (EIgG) can depress several macrophage functions. Our previous studies have suggested that this macrophage dysfunction may be due to an oxidative stress caused by the interaction of hemoglobin-derived iron with superoxide and/or hydrogen peroxide. Since lysosomotropic agents are capable of altering iron handling by macrophages, the present study evaluated the ability of these agents to prevent the macrophage dysfunction and lipid peroxidation caused by a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages showed a depression of PMA-stimulated hydrogen peroxide production, calcium ionophore-stimulated arachidonate release and Fc receptor-mediated phagocytosis. The lysosomotropic agents; chloroquine, quinacrine, ammonium chloride and methylamine all prevented the depression of hydrogen peroxide production and arachidonate release but did not alter the depression of phagocytic function. These agents also prevented the increase in lipid peroxidation products caused by a phagocytic challenge with EIgG. These results suggest that the ability of lysosomotropic agents to prevent some aspects of macrophage dysfunction after a phagocytic challenge may be due to their ability to block the oxidative stress caused by the challenge.     

Intravenous iron-dextran: studies on unsaturated iron-binding capacity

A method is described for measuring the plasma unsaturated iron-binding capacity in the presence of very high concentrations of iron as iron-dextran. The procedure utilizes (59)Fe to label the apotransferrin with subsequent separation of ionic iron from transferrin-bound iron on an ion exchange or Sephadex G.25 column.The unsaturated iron-binding capacity has been measured in rabbits and dogs after intravenous injection of iron-dextran and in human subjects after total dose infusion of iron-dextran. No evidence of saturation of the unsaturated iron-binding capacity was found even when the plasma iron values were greater than 40,000 mug Fe/100 ml.     

Effects of magnetic fields on the accumulation of thiobarbituric acid reactive substances induced by iron salt and H2O2 in mouse brain homogenates or phosphotidylcholine

In this study, we examined the effects of magnetic fields (MFs) on the generation of thiobarbituric acid reactive substances (TBARS) in the mouse brain homogenates or phosphotidylcholine (PC) solution, incubated with FeCl₃ and/or H₂O₂. Active oxygen species were generated and lipid peroxidation was induced in mouse brain homogenates by incubation with iron ions, resulting in the accumulation of TBARS. Lipid peroxidation was induced in PC by incubation with iron ions and H₂O₂. Exposure to sinusoidal MFs (60 Hz, 0.2–1.2 mT), symmetric sawtooth-waveform MFs (50 Hz, 25–600 mT/s), rectangular MFs (1/0.4–1/16 Hz, 3.3 mT) and static MFs (1, 5–300 mT) had no effect on the accumulation of TBARS in brain homogenates induced by FeCl₃. In contrast, when the homogenates were incubated with FeCl₃ in static MFs (2–4 mT), the accumulation of TBARS was decreased. However, this inhibitory effect disappeared when EDTA was added to the homogenate and incubated with H₂O₂. The accumulation of TBARS in PC solution incubated with FeCl₃ and H₂O₂ was also inhibited by the static MF. These results indicate that only static MFs had an inhibitory effect on iron-induced lipid peroxidation and the effectiveness of this magnetic field on iron ion-induced active oxygen species generation is restricted to a so called ‘window’ of field intensity of 2–4 mT.     

Immunological consequences of innate resistance and susceptibility to BCG

The immunological consequences of genetically controlled innate resistance and susceptibility to Mycobacterium bovis (BCG) infection in mice were investigated. The susceptible (BcgS) BALB/c and the congenic resistant BALB/c.Bcgr mouse strains were employed to test for differences in the specific immune response to BCG. Antigen-specific lymphocyte proliferation and production of interleukin 2 (IL-2) to purified protein derivative (PPD) in vitro were measured following infection of congenic mice with low (10(4)) and high (10(6)) doses of BCG. The lymphocyte subsets were identified by testing the ability of spleen cells to respond after separation of T and B cells and after cytotoxic depletion of T cell subsets. In addition, fluorescence activated cell sorter (FACS) analysis with anti-T and -B cell monoclonal reagents was used to enumerate lymphocyte populations in BCG-infected spleens. The results indicated that the innately resistant mice displayed antigen-specific T helper cell function three weeks following the injection of BCG. The susceptible animals were found to be T cell-unresponsive since they lacked both proliferative and IL-2 secreting specific T cells. No evidence for suppression of IL-2 production or proliferation was detected in BALB/c (susceptible) spleen cultures in cell mixing experiments. The results provide evidence for a regulatory role of the Bcg gene on the generation of lymphocyte responses to BCG.     

Spleen immunotoxicities induced by intra-testicular injection of magnetic nanoparticles and the role of Echinacea purpurea extract: a histological and immunohistochemical study

Recently, magnetic nanoparticles (MNP) have been used in several medical applications. High accumulations of MNP can be found in spleen and liver after different injection routes. Immunotoxicities produced by MNP in spleen, a lymphoid organ, need more investigations. This study investigated biodistribution, histological and immune responses to MNP in spleen after intra-testicular injection. Echinacea purpurea (EP) extract has immune stimulation and anti-oxidant properties. Investigation was done on the role of EP reduction of immunotoxicities produced by MNP in the spleen. After intra-testicular injection, MNP reached spleen parenchyma through blood circulation. Long term EP administration induced mild iron accumulation resulting from red blood cell (RBC) lysis. The opposite was observed when MNP treatment during EP administration significantly reduced lysed RBC iron in the spleen as well as MNP iron. It is possible the chelating properties of EP molecules possibly explained the presence of inert iron complexes from RBC in the spleen after EP administration only. The presence of MNP induced severe splenic cell degeneration and hemorrhage. The treatment with MNP combined with EP administration reduced these abnormalities. Additionally, anti-oxidant properties of EP and presence of MNP iron in the spleen had a role in activating macrophages to recycle all splenic iron. However, F4/80⁺, immunoglobulin A (IgA)⁺ and Ulex europaeus agglutinin I (UEAI)⁺ expressions significantly decreased when MNP were injected without EP. Otherwise, long term EP treatment maintained normal function of these cells.     

Monoclonal Antibodies against Murine Neonatal and Pregnancy-Associated Natural Suppressor Cells Induce Resorption of the Fetus

Pregnant and neonatal/fetal mice have been shown to harbour naturally occurring inhibitory cells of both T and non-T type. Non-T suppressor cells present in the spleen of primiparous pregnant and newborn animals inhibit proliferative responses in autologous and allogeneic mixed lymphocyte reactions. Such cells can be positively selected for by agglutination with the B cell-specific lectin soybean agglutinin (SBA). We generated rat IgG monoclonal antibodies against unique cell surface structures on the non-T inhibitory cells, and cylotoxic pretreatment of spleen cells from pregnant or neonatal/felal mice largely abrogates their suppressive activity on proliferative responses. Furthermore, in vivo administration of such antibodies to pregnant inbred and outbred mice results in termination of the pregnancy or decreased litter size. Since injection of anti-T cell IgG monoclonal antibodies does not interfere with the delivery of normal sized litters it is evident from these studies that the non-T immunoregulatory cells, in contrast to T-inhibitory cells, are of great importance in ensuring immunological homeostasis in the fetal-placental environment during pregnancy in mice.     

Impairment of T lymphocyte functions in mice with motor end-plate disease.

The present paper reports complex immunological anomalies associated with motor end-plate disease (Med) in mice. Motor end-plate disease is a severe neuromuscular disorder which leads to death (around the 25th of life) in the Medj/Medj mutant, while the heterozygotes quickly recover from mild manifestations. Medj/Medj and Medj/ + mice share some of the immunological aberrations: reduced PFC response to SRBC in 14-16 day old mice, with reduced suppressor cell function and precocious maturation of the cytotoxic response to allogeneic cells in 21-23 day old mice. The diminished PFC response is corrected in adult Medj/ + mice but persists in the small group of Medj/Medj which escape death and which were studied between the 6th and 16th week of life. In addition, the thymus and spleen of Medj/Medj mice are greatly reduced in size, a symptom which appears with the onset of the clinical disease. Also, a reduction in the NK activity in the small group of older, surviving mice was noted. T and B lymphocyte proportions and the proliferative responses to T cell mitogens were not impaired in 14-16 day old mice. The role of these abnormalities in the pathogenesis of the disease is not known. Since some of these anomalies are shared by Medj/Medj and Medj/ +, the latter of which present no or mild and transient neurological manifestations, there is no clear link between the immunological and neuromuscular disorders.     

Diphenyl diselenide behaves differently than ebselen under different pH media in rat's liver preparations

The anti-oxidant potential of diphenyl diselenide and ebselen against low pH-mediated peroxidation processes in rat's liver preparation are unknown. For this purpose, we have determined anti-oxidant activities of both compounds at a range of various pH (7.4–5.4) values. Low pH increased the rate of lipid peroxidation in the absence of Fe⁺² (20 μmol) in liver homogenates. This higher extent of lipid peroxidation can be explained by the mobilized iron, which may come from reserves where it is weakly bound. The addition of iron (Fe) chelator desferoxamine (DFO, 1 mmol) to reaction medium completely inhibited the peroxidation processes at all studied pH values. Diphenyl diselenide (0–100 μmol) significantly protected lipid peroxidation at all studied pH values, while ebselen (0–100 μmol) did not offer any protection. The differences in activities of diphenyl diselenide and ebselen have been explained with reference to the active selenol formation using their catalytic reactions. The anti-oxidant potential of diphenyl diselenide is confirmed against acidosis-catalyzed oxidative stress in rat liver homogenates.     

Diminished suppressor cell function in patients with asbestosis.

Epidemiological and immunological studies of asbestos workers documented abnormalities in humoral and cell-mediated immunity which could result from defective immunoregulation. This study tests this hypothesis with comparison of lymphocyte function in age-, sex- and smoking-matched subjects with asbestosis. In vivo measure of delayed hypersensitivity (i.e. skin test response) was significantly depressed to two recall antigens, SKSD and Candida, in asbestosis patients. Skin reaction to dinitrochlorobenzene (DNCB) was not depressed, although lymphocytes of patients giving positive skin test reactions demonstrated a significantly lower (P less than 0.001) proliferative response to dinitrobenzene sulphonic acid (DNBSO3) in vitro. T cell counts (E-rosettes) were normal in patients with asbestosis, although a subset of T cells, those forming sheep erythrocyte rosettes after prolonged incubation (Elate), were significantly depressed (P less than 0.003). This population has been equated with 'suppressor' cells (Grossi et al., 1978). Numbers of B cells were increased nearly two-fold over controls. Mitogen response of lymphocytes was normal except at suboptimal doses of mitogens where the response is known to be influenced by suppressor cell activity, which was significantly elevated. Suppressor cell function, as determined by stimulation of peripheral blood lymphocytes preincubated with concanavalin A, was also significantly decreased in asbestosis patients (P less than 0.01).