Mitochondrial redox impairment and enhanced autophagy in peripheral blood mononuclear cells from type 1 diabetic patients.

Type 1 diabetes (T1D) involves critical metabolic disturbances that contribute to an increased cardiovascular risk. Leukocytes are key players in the onset of atherosclerosis due to their interaction with the endothelium. However, whether mitochondrial redox impairment, altered bioenergetics and abnormal autophagy in leukocytes contribute to T1D physiopathology is unclear. In this study we aimed to evaluate the bioenergetic and redox state of peripheral blood mononuclear cells (PBMCs) from T1D patients in comparison to those from healthy subjects, and to assess autophagy induction and leukocyte-endothelial interactions. T1D patients presented lower levels of fast-acting and total antioxidants in their blood, and their leukocytes produced higher amounts of total reactive oxygen species (ROS) and superoxide radical with respect to controls. Basal and ATP-linked respiration were similar in PBMCs from T1D and controls, but T1D PBMCs exhibited reduced spare respiratory capacity and a tendency toward decreased maximal respiration and reduced non-mitochondrial respiration, compared to controls. The autophagy markers P-AMPK, Beclin-1 and LC3-II/LC3-I were increased, while P62 and NBR1 were decreased in T1D PBMCs versus those from controls. Leukocytes from T1D patients displayed lower rolling velocity, higher rolling flux and more adhesion to the endothelium versus controls. Our findings show that T1D impairs mitochondrial function and promotes oxidative stress and autophagy in leukocytes, and suggest that these mechanisms contribute to an increased risk of atherosclerosis by augmenting leukocyte-endothelial interactions.

Nanoparticles in Medicine: A Focus on Vascular Oxidative Stress.

Nanotechnology has had a significant impact on medicine in recent years, its application being referred to as nanomedicine. Nanoparticles have certain properties with biomedical applications; however, in some situations, they have demonstrated cell toxicity, which has caused concern surrounding their clinical use. In this review, we focus on two aspects: first, we summarize the types of nanoparticles according to their chemical composition and the general characteristics of their use in medicine, and second, we review the applications of nanoparticles in vascular alteration, especially in endothelial dysfunction related to oxidative stress. This condition can lead to a reduction in nitric oxide (NO) bioavailability, consequently affecting vascular tone regulation and endothelial dysfunction, which is the first phase in the development of cardiovascular diseases. Therefore, nanoparticles with antioxidant properties may improve vascular dysfunction associated with hypertension, diabetes mellitus, or atherosclerosis.

Oxidative and endoplasmic reticulum stress is impaired in leukocytes from metabolically unhealthy vs healthy obese individuals.

BACKGROUND: Oxidative stress and inflammation are related to obesity, but the influence of metabolic disturbances on these parameters and their relationship with endoplasmic reticulum (ER) stress is unknown. Therefore, this study was performed to evaluate whether metabolic profile influences ER and oxidative stress in an obese population with/without comorbidities. SUBJECTS AND METHODS: A total of 113 obese patients were enrolled in the study; 29 were metabolically healthy (MHO), 53 were metabolically abnormal (MAO) and 31 had type 2 diabetes (MADO). We assessed metabolic parameters, proinflammatory cytokines (TNFα and IL-6), mitochondrial and total reactive oxygen species (ROS) production, glutathione levels, antioxidant enzymes activity, total antioxidant status, mitochondrial membrane potential and ER stress marker expression levels (glucose-regulated protein (GRP78), spliced X-box binding protein 1 (XBP1), P-subunit 1 alpha (P-eIF2α) and activating transcription factor 6 (ATF6). RESULTS: The MAO and MADO groups showed higher blood pressure, atherogenic dyslipidemia, insulin resistance and inflammatory profile than that of MHO subjects. Total and mitochondrial ROS production was enhanced in MAO and MADO patients, and mitochondrial membrane potential and catalase activity differed significantly between the MADO and MHO groups. In addition, decreases in glutathione levels and superoxide dismutase activity were observed in the MADO vs MAO and MHO groups. GRP78 and CHOP protein and gene expression were higher in the MAO and MADO groups with respect to MHO subjects, and sXBP1 gene expression was associated with the presence of diabetes. Furthermore, MAO patients exhibited higher levels of ATF6 than their MHO counterparts. Waist circumference was positively correlated with ATF6 and GRP78, and A1c was positively correlated with P-Eif2α. Interestingly, CHOP was positively correlated with TNFα and total ROS production and GRP78 was negatively correlated with glutathione levels. CONCLUSIONS: Our findings support the hypothesis that both inflammation and oxidative stress are involved in the induction of ER stress signaling pathways in the leukocytes of metabolically unhealthy obese vs healthy obese subjects.

Mitochondrial (dys)function - a factor underlying the variability of efavirenz-induced hepatotoxicity?

BACKGROUND AND PURPOSE: The non-nucleoside analogue reverse transcriptase inhibitor efavirenz is associated with hepatic toxicity and metabolic disturbances. Although the mechanisms involved are not clear, recent evidence has pinpointed a specific mitochondrial action of efavirenz accompanied by the induction of an endoplasmic reticulum (ER) stress/unfolded protein response in human hepatic cells. The aim of this study was to further investigate the involvement of this organelle by evaluating efavirenz's effects in cells lacking functional mitochondria (rho°) and comparing them with those of the typical mitotoxic agent rotenone, a standard complex I inhibitor, and the ER stress inducer thapsigargin. EXPERIMENTAL APPROACH: Hep3B rho(+) and rho° cells were treated with clinically relevant concentrations of efavirenz, then mitochondrial function and cytotoxicity were studied using standard cell biology techniques. KEY RESULTS: Efavirenz-treated rho° cells exhibited a substantial reduction in parameters indicative of mitochondrial interference, such as increased superoxide production, mitochondrial mass/morphology alterations and enhanced expression of LONP, a highly conserved mitochondrial protease. In line with these results, the cytotoxic effect (cell number, chromatin condensation, cell cycle alterations and induction of apoptosis) of efavirenz was less pronounced in Hep3B respiration-depleted cells than in wild-type cells. The effect of efavirenz was both similar and different from those of two distinct mitochondrial stressors, thapsigargin and rotenone. CONCLUSIONS AND IMPLICATIONS: Cells lacking normal mitochondria (rho°) are less vulnerable to efavirenz. Our results provide further evidence that the hepatic damage induced by efavirenz involves acute interference with mitochondria and extend our knowledge of the response of mitochondria/ER to a stress stimulus.

Relation between lipoprotein subfractions and TSH levels in the cardiovascular risk among women with subclinical hypothyroidism.

OBJECTIVE: Subclinical hypothyroidism (SCH) is a common condition associated with increased cardiovascular risk. A standard treatment is yet to be established, as there is no consensus on the TSH cut-off values which should be used as indicators. Thus, the aim of this study was to assess cardiovascular risk in patients with SCH and to differentiate it according to TSH levels. DESIGN: This was an observational study conducted in an academic medical centre. PATIENTS: The study population consisted of 95 middle-aged women recently diagnosed with SCH and 65 euthyroid controls. MEASUREMENTS: We measured anthropometric parameters, lipid cardiovascular risk markers and lipoprotein subclasses of HDL and LDL. RESULTS: Patients with SCH exhibited a significant increase in triglycerides and atherogenic index of plasma and a significant reduction in HDL-cholesterol with respect to the control group after adjusted by age and BMI. A similar lipid profile was observed in both SCH groups. However, patients with TSH levels higher than 10 mIU/l showed a significant reduction in LDL particle size, which was associated with a higher prevalence of atherogenic pattern B. CONCLUSIONS: Our findings indicate that cardiovascular risk is affected in patients with TSH levels over 10 mIU/l, who have a lipid profile characteristic of atherogenic dyslipidemia.

Serum lipid responses to phytosterol-enriched milk in a moderate hypercholesterolemic population is not affected by apolipoprotein E polymorphism or diameter of low-density lipoprotein particles.

BACKGROUND/OBJECTIVES: The importance of both low-density lipoprotein cholesterol (LDLc) size and the apolipoprotein E (Apo E) in the atherogenic process is known, but there is little information with regard to the effect of phytosterols (PS) on these parameters. The aim of this study was to evaluate the influence of PS on lipid profile and LDLc size according to Apo E genotype. SUBJECTS/METHODS: This was a randomized parallel trial employing 75 mild-hypercholesterolemic subjects and consisting of two 3-month intervention phases. After 3 months of receiving a standard healthy diet, subjects were divided into two intervention groups: a diet group (n=34) and a diet+PS group (n=41) that received 2 g/day of PS. Total cholesterol (TC), triacylglycerols, LDLc, high-density lipoprotein cholesterol (HDLc), non-HDLc, Apo A-I and B-100, LDLc size and Apo E genotype were determined. RESULTS: Patients receiving PS exhibited a significant decrease in TC (5.1%), LDLc (8.1%), non-HDLc (7.4%) and Apo B-100/Apo A-I ratio (7.7%), but these effects did not depend on Apo E genotype. No significant changes were found in lipid profile according to Apo E genotype when patients following dietary recommendations were considered as a whole population or separately. No variations in LDLc size were observed in any of the intervention groups. CONCLUSION: The results of this study show that Apo E genotype does not have an impact on the lipid response to PS as a cholesterol-lowering agent in mild-hypercholesterolemic patients. Furthermore, the evidence obtained confirms that LDLc particle size is not modified when PS are added to a standard healthy diet.

The effects of aging on dopaminergic neurotransmission: a microPET study of [11C]-raclopride binding in the aged rodent brain.

Rodent models are frequently used in aging research to investigate biochemical age effects and aid in the development of therapies for pathological and non-pathological age-related degenerative processes. In order to validate the use of animal models in aging research and pave the way for longitudinal intervention-based animal studies, the consistency of cerebral aging processes across species needs to be evaluated. The dopaminergic system seems particularly susceptible to the aging process, and one of the most consistent findings in human brain aging research is a decline in striatal D2-like receptor (D2R) availability, quantifiable by positron emission tomography (PET) imaging. In this study, we aimed to assess whether similar age effects can be discerned in rat brains, using in vivo molecular imaging with the radioactive compound [(11)C]-raclopride. We observed a robust decline in striatal [(11)C]-raclopride uptake in the aged rats in comparison to the young control group, comprising a 41% decrement in striatal binding potential. In accordance with human studies, these results indicate that substantial reductions in D2R availability can be measured in the aged striatal complex. Our findings suggest that rat and human brains exhibit similar biochemical alterations with age in the striatal dopaminergic system, providing support for the pertinence of rodent models in aging research.

Oxidative stress and endothelial dysfunction in cardiovascular disease: mitochondria-targeted therapeutics.

Functional impairment of endothelial activity (endothelial dysfunction) precedes the development of cardiovascular diseases (CVD). This condition is a result of a reduced bioavailability of nitric oxide (NO), a well known vasodilator, which is mainly due to increased NO degradation caused by its reaction with reactive oxygen species (ROS). Although there are several conditions that contribute independently to endothelial dysfunction, such as hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia, increased oxidative stress seems to play a key role. In addition to their original pharmacological properties, drugs used clinically at present, including anti-hypertension reagents, angiotensin receptor blockers and anti-hyperlipidemic reagents such as statins, protect various organs via anti-oxidative stress mechanisms. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, have been used to eradicate the oxidative stress associated with CVD. The results of the clinical trials employing anti-oxidative stress reagents in patients with CVD are contradictory, which could be a result of inadequate study design or selected targets. This review considers the process of endothelial dysfunction and CVD from a mitochondrial perspective and evaluates strategies currently under development for the targeted delivery of antioxidants or NO to mitochondria. It endorses the idea that selectively targeting specific antioxidants and NO donors to mitochondria is an effective strategy for modulating mitochondrial respiration and ROS production and protecting mitochondria against oxidative stress.

Oxidative stress and mitochondrial dysfunction in atherosclerosis: mitochondria-targeted antioxidants as potential therapy.

Chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions forms an integral part of the development of cardiovascular diseases (CVD), and in particular atherosclerosis. These ROS are released from different sources, such as xanthine oxidase, lipoxygenase, nicotinamide adenine dinucleotide phosphate oxidase, the uncoupling of nitric oxide synthase and, in particular, mitochondria. Endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not clear, oxidative stress seems to play an important role. In general, ROS are essential to cell function, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects of excessive ROS production. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions. This review considers the process of atherosclerosis from a mitochondrial perspective, and assesses strategies for the targeted delivery of antioxidants to mitochondria that are currently under development. We will provide a summary of the following areas: the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as atherosclerosis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondrially-targeted antioxidants that concentrate on the matrix-facing surface of the inner mitochondrial membrane in order to protect against mitochondrial oxidative damage, and their therapeutic potential as a treatment for atherosclerosis.

Targeting antioxidants to mitochondria: a potential new therapeutic strategy for cardiovascular diseases.

Mitochondria produce large amounts of free radicals and play an important role in the life and death of a cell. Thus, mitochondrial oxidative damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions including ischemia-reperfusion injury, sepsis, diabetes, atherosclerosis and, consequently, cardiovascular diseases (CVD). In fact, endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not yet clear, oxidative stress seems to play an important role. This review considers the process of CVD from a mitochondrial perspective. Accordingly, strategies for the targeted delivery of antioxidants to mitochondria are being developed. In this review, we will provide a summary of the following areas: the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as CVD; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; recent developments in mitochondrially-targeted antioxidants that concentrate on the matrix-facing surface of the inner mitochondrial membrane and therefore protect against mitochondrial oxidative damage, and their therapeutic potential for future treatment of CVDs. More pre-clinical and clinical studies, however, are necessary in order to evaluate the effectiveness and toxicity of mitochondrially-targeted antioxidants.

Several functions of immune cells in mice changed by oxidative stress caused by endotoxin.

We have studied natural killer (NK) activity, lymphoproliferative response, the release of several cytokines (IL-2, TNF alpha and IL-1 beta) and the ROS production in peritoneal leukocytes obtained 0, 2, 4, 12 and 24 h after lipopolysaccharide (LPS) injection. Lethal septic shock (100 % mortality occurred at 30 h after LPS administration) was caused in female BALB/c mice by intraperitoneal injection of 100 mg/kg of E. coli LPS. Cytotoxicity and lymphoproliferation assay were preformed together with the measurement of IL-1 beta, IL-2 and TNF alpha production, and quantification of ROS. Natural killer activity, spontaneous lymphoproliferative response, IL-2, TNF alpha, IL-beta release and ROS production were increased after LPS injection. In conclusions, ROS and proinflammatory mediators produced by immune cells in response to LPS are involved in the oxidative stress of endotoxic shock. This oxidative state alters some functional characteristics of leukocytes (proliferation and NK activity).

Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress.

Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS.

Comparative study of several lymphocyte functions in two strains of mice with different models of endotoxic shock.

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.

The amount of thiolic antioxidant ingestion needed to improve several immune functions is higher in aged than in adult mice.

With aging there is an increase of oxidative stress due to an imbalance between the oxidant production and the antioxidant levels in favor of the former. Since immune cell functions are specially linked to reactive oxygen species (ROS) generation, the oxidant/antioxidant balance is essential for these cells. Although low levels of antioxidants cause a decrease in immune function, very high levels of antioxidant compounds could show prooxidant effects. In the present work, we have studied the effect of diet supplementation, for 4 weeks, with two different doses of two thiolic antioxidants, namely thioproline (TP) and N-acetylcysteine (NAC), at 0.1% (w/w) and 0.3% (w/w, of each antioxidant) on the main immune system cells, i.e.: macrophages, lymphocytes and natural killer (NK) cells of adult (33+/-1 week old) and aged (75+/-1 week old) female Swiss mice. Two groups of animals, adult and aged mice, fed standard diet were used as controls. The results show that the ingestion of 0.1% doses of thiols improves, in the adult mice, several immune functions such as the chemotaxis capacity of both macrophages and lymphocytes, the phagocytosis of macrophages, the lymphoproliferative response to the mitogen Con A and the NK activity. Moreover, no change was observed in adherence capacity of immune cells, and superoxide production was decreased. By contrast, in aged mice the ingestion of these amounts of antioxidants did not change the immune functions studied with the exception of NK activity, which was stimulated. The ingestion of 0.3% of antioxidants by adult mice only increased some immune functions such as adherence and superoxide production, which are markers of oxidative stress. Other functions such as chemotaxis or lymphoproliferative response decreased. However, the ingestion of these very high amounts of thiols by aged animals increased the phagocytosis, the NK activity and specially the lymphoproliferative response to the mitogen, a function that is very depressed with aging.

Leukocyte function and life span in a murine model of premature immunosenescence.

Aging associates with a decline of physiological functions, including the function of the nervous and the immune system. These aged-related changes occur in various degrees in different members of a mouse outbred population. Accordingly, we have proposed a model of premature immunosenescence in mice, based on the demonstration of premature decline in the behavioral response in a simple T-maze and in several immune functions in Swiss outbred mice. Those mice with a worst (slow) performance in this test (linked to a higher emotional response to stress) show a shorter life span and a decreased immune function when compared to fast mice. In order to provide biomarkers of "biological aging" related to health and survival, the present longitudinal study includes the analysis of several immunological parameters such as, proliferative response to mitogen Con A, NK activity and cytokine (TNFalpha, IL-1beta and IL-2) release by peritoneal leukocytes from female Swiss mice. Slow mice showed a lower proliferative response to Con A, IL-2 and IL-1beta release, an impaired NK activity and an increased TNFalpha production as compared to fast mice. Moreover, the age-associated decline of these functions is more strikingly slow than in fast mice. In summary, we propose the above immunological parameters, that change with aging at a different rate in members of a same population, as useful biomarkers to asses the rate of biological aging in mice.

Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress.

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.

Neuropeptide Y effects on murine natural killer activity: changes with ageing and cAMP involvement.

Changes in the bidirectional interaction between the nervous and the immune systems have been proposed as a cause of ageing. Neuropeptides, such as neuropeptide Y (NPY), could show different effects on immune function with age. In the present work, we have studied the in vitro action of a wide range of NPY concentrations, i.e. from 10(-13) to 10(-7) M, on natural killer (NK) activity, a function which decreases with age. Spleen, axillary nodes, thymus and peritoneum leukocytes from mice of different ages: young (12+/-2 weeks), adult (24+/-2 weeks), mature (50+/-2 weeks) and old (72+/-2 weeks) were used. Stimulation by NPY of NK activity was observed in adult and mature animals in axillary nodes and thymus, and an inhibition in the spleen from young mice. The specificity of the NPY effect on cytotoxic activity was confirmed using a C-terminal fragment of NPY. Furthermore, cAMP levels in leukocytes were found to be decreased by NPY in adult mice, suggesting an involvement of this messenger system in the NK modulation by this neuropeptide.

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress.

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.

Comparative study of peritoneal macrophage functions in mice receiving lethal and non-lethal doses of LPS.

In previous studies, we have observed changes in several functions of peritoneal macrophages from female BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli O55:B5 lipopolysaccharide (LPS; 100 mg/kg), which were associated with a high production of superoxide anion and tumor necrosis factor alpha (TNF-alpha). In the present work, both a lethal dose (250 mg/kg) and a non-lethal dose (100 mg/kg) of LPS were used in female Swiss mice. In peritoneal macrophages, the following functions were studied at 2, 4, 12 and 24 h after LPS injection: adherence to substrate, chemotaxis, ingestion of particles, and superoxide anion and TNF-alpha production. In both groups, the results showed a stimulation of adherence, ingestion and superoxide production as well as a decrease of chemotaxis, whereas TNF-alpha could not be detected in either of the two groups. These effects were more evident with the 250 mg/kg dose, especially as regards superoxide anion production, which was higher in the animals treated with a lethal dose of LPS.