Lymphocyte Mitochondria Research Articles

COMPARISON OF CELL DEATH RATE IN VARIOUS CHICKEN LYMPHOCYTES POPULATIONS IN A RESPONSE TO THREE TEMPERATURE TREATMENTS

The goal of the experiment was to evaluate susceptibility of lymphocytes populations derived from bursa of Fabricius, spleen, thymus, and blood to temperature treatments in vitro. Temperatures used in study was 37 °C (thermoneutral), 41 °C (mild heat stress) and 45 °C (severe heat stress). Cells were incubated for 8 hours and after 3 and 8 hours were measured numbers of apoptotic, necrotic and CellROX positive cells by flow cytometry. Representation of live lymphocyte phenotypes (CD3, CD4, CD8, BU1) in cell populations were also measured. At all temperature treatments, thymus lymphocytes population had the lowest (p < 0.05) percentage of apoptotic cells. On the other hand, significantly highest numbers (p < 0.05) of apoptotic cells were found in bursal lymphocytes after 8 hours of incubation. Differences in numbers of necrotic cells were more pronounced at 45 °C, where after 3 and 8 hours of incubation were significantly highest numbers (p < 0.05) of necrotic cells in spleen. Increasing temperature induce production of reactive oxygen species (ROS) in cells and highest proportion (p < 0.05) of ROS-positive cells were found in bursal and spleen cell population after 8 hours of incubation. Transmission electron microscopy showed loss of mitochondrial matrix and disrupted mitochondrial membrane in mitochondria of lymphocytes at 45 °C as initial damage caused by ROS. From individual cell phenotypes, there were significantly decreased (p < 0.05) B lymphocytes in bursa of Fabricius, CD4+ and CD8+ lymphocytes in thymus and whole T lymphocytes population in spleen at 45 °C. Various susceptibility to temperature can be predictive factor of worse function in some organs in stress conditions where body temperature increase.

High-Resolution Multicolor Imaging of Mitochondria in Lymphocytes.

The identification of cellular changes that accompany immune activation has been a long-standing interest for immunologists. Among these, alterations in the metabolic states of these cells have gained particular attention in the last decade due to the emergence of the field of immunometabolism. A thorough investigation of these metabolic changes can only be achieved with an in-depth visualization of mitochondrial organization; however, current strategies for mitochondrial imaging have been optimized in model cells with a high cytoplasm-to-nucleus ratio and thus are not readily adaptable for many immune cells. Here, we devised a multicolor high-resolution microscopy strategy to image mitochondrial morphology in lymphocytes at both their resting and activated states. Our method allowed us to stain both the mitochondrial surface (by targeting TOM-20) and the mitochondrial matrix (through the use of Mitotracker dyes) while efficiently excluding nonviable cells. Our novel imaging strategy offers a powerful tool to study changes in mitochondrial morphology and complements any research focusing on lymphocyte metabolism.

Morphometric analysis of mitochondria in lymphocytes of patients with exacerbations of chronic obstructive pulmonary disease - pilot study.

IntroductionExacerbations of chronic obstructive pulmonary disease (ECOPD) are important events in the course of the disease, negatively influencing health status and disease progression. Therefore, there is a strong need for deeper understanding of the pathology of ECOPD to elaborate new therapeutic approaches and ameliorate prognoses. Contributions of mitochondria to pathobiology of COPD are still under investigation, although growing evidence suggests their important role in this disease. The aim of our study was to assess the morphometric parameters of mitochondria in lymphocytes of patients with ECOPD.Patients and methodsLymphocytes were isolated from the peripheral blood of patients with COPD. Transmission electron microscopy was used to assess absolute number of mitochondria per cell, mitochondrial content, and morphometric parameters of individual mitochondria. We also counted indexes for elongation and interconnectivity.ResultsEighteen patients (9 with ECOPD and 9 in the stable period of the disease) were analyzed. We observed significantly lower length of mitochondrion (P=0.03) and significant decrease both in elongation (P=0.03) and interconnectivity indexes (P=0.04) in ECOPD patients.ConclusionsThe morphometric parameters of mitochondria in lymphocytes derived from patients during the early period of ECOPD requiring hospitalization are altered in comparison to patients in the stable period of the disease. This suggests their contribution to pathobiology of ECOPD. These preliminary outcomes should be further validated in larger size samples.

A novel multicolor stimulated emission depletion (STED) microscopy strategy to visualize mitochondrial morphology in lymphocytes

Abstract Immunometabolism is an emerging field that studies the activation-induced reprogramming required to alter immune cell function and meet new energetic demands. In-depth investigations of the metabolism-related cellular changes necessitate high-resolution visualization of mitochondria. However, current strategies for imaging mitochondria are optimized for model cells with high cytoplasm-to-nucleus ratios and are not readily adaptable for many immune cells. Here, we devised a multicolor STED microscopy method to assess B lymphocyte mitochondria in resting and activated states. By staining specimens with TOM-20 and MitoTracker dyes, as well as DAPI and a viability dye, our strategy allowed us to assess the morphology and volumes of mitochondria while efficiently excluding dead cells. Our analyses showed that stimulation of B lymphocytes via toll-like receptor 9 (TLR9) and/or B cell receptor (BCR) led to notable mass and volume increases in individual mitochondria within 24 hours. Furthermore, TLR9-stimulated cells demonstrated more tubular and connected mitochondrial networks consistent with their higher metabolic demands. In addition, collecting images of the same specimens side-by-side on AiryScan and standard confocal laser microscopes showed that STED microscopy performed significantly better in resolving changes in 3D mitochondrial morphology. However, none of the light microscopy imaging strategies could resolve activation-induced structural alterations in cristae or mitochondrial matrix. In conclusion, our novel STED-based multicolor imaging approach offers a powerful super-resolution tool to study immunometabolism and mitochondrial reorganization in lymphocytes at the whole cell level.

Lymphocyte mitochondria: toward identification of peripheral biomarkers in the progression of Alzheimer disease

Alzheimer disease (AD) is an age-related neurodegenerative condition. AD is histopathologically characterized by the presence of three main hallmarks: senile plaques (rich in amyloid-β peptide), neuronal fibrillary tangles (rich in phosphorylated tau protein), and synapse loss. However, definitive biomarkers for this devastating disease in living people are still lacking. In this study, we show that levels of oxidative stress markers are significantly increased in the mitochondria isolated from lymphocytes of subjects with mild cognitive impairment (MCI) compared to cognitively normal individuals. Further, an increase in mitochondrial oxidative stress in MCI is associated with MMSE score, vitamin E components, and β-carotene. Further, a proteomics approach showed that alterations in the levels of thioredoxin-dependent peroxide reductase, myosin light polypeptide 6, and ATP synthase subunit β might be important in the progression and pathogenesis of AD. Increased understanding of oxidative stress and protein alterations in easily obtainable peripheral tissues will be helpful in developing biomarkers to combat this devastating disorder.

Pathology of Bursae of Fabricius in Methionine-Deficient Broiler Chickens

The purpose of this 42-day study was to investigate the effects of methionine (Met) deficiency on immune function by determining the relative weight, morphological and ultrastructural changes of bursae of Fabricius, cell cycle, and apoptosis of bursa cells. One hundred and twenty one-day-old avian broilers were randomly divided into two groups and fed on a control diet (starter diet, Met 0.50%; grower diet, Met 0.40%) and Met-deficient diet (starter diet, Met 0.26%; grower diet, Met 0.28%) for six weeks. The relative weight of bursae was decreased with Met deficiency when compared to that of the control group. Lesions were also observed in the Met-deficient group. Histopathologically, the numbers of lymphocytes in the follicles were decreased. Ultrastructurally, the mitochondria of lymphocytes were swollen in the Met-deficient group. As measured by flow cytometry, bursal cells in the G0G1 phase were significantly higher (P < 0.01), and bursal cells in the S, G2M phases and proliferating index were obviously lower (P < 0.01) with Met deficiency than in the control group. Moreover, the percentage of apoptotic cells in the bursae were significantly increased in Met-deficient birds (P < 0.01). It was concluded that Met deficiency restrained the development of the bursae of Fabricius and affected the humoral immunity of the chickens.

Basal and T3-induced ROS Production in Lymphocyte Mitochondria is Increased in Type 2 Diabetic Patients

Mitochondrial function, including production of reactive oxygen species (ROS), is important in the pathogenesis of diabetes and its complications. Thyroid hormones are major regulator of these processes. Hence, the aim of this study was to examine the thyroid hormone regulation of ROS production in human lymphocytes in patients with diabetes mellitus type 2 (T2DM). Lymphocytes from 10 controls and 10 persons with T2DM were examined. Mitochondrial membrane potential (MMP) was examined by flow cytometry after staining with MitoTracker Green (MTG). Similarly ROS was measured following staining with carboxy-H₂DCFDA. MMP was increased in T2DM patients and T₃ stimulation increased MMP in controls [1398 a.u. (979-4094) vs. 2156 a.u. (1611-15189), p=0.04, median and quartiles] as well as in T2DM patients [9167 a.u. (7387-11746) vs. 20274 a.u. (17183-27839 p=0.004, median and quartiles]. Basal ROS concentration was increased in lymphocytes from T2DM and T₃ significantly stimulated ROS concentration in controls [3691 a.u. (2584-6396) vs. 5650 a.u. (3001-7802) p=0.013, median and quartiles] and in T2DM patients [19271 a.u. (6288-25282) vs. 23178 a.u. (10004-28857) p=0.013, median and quartiles]. The ratio of ROS production related to MMP was significantly higher in T2DM, unstimulated as well as T₃-stimulated in T2DM. Unstimulated and T₃ stimulated ROS production and MMP were higher in lymphocytes from diabetic patients. An altered balance between ROS production and MMP, favoring ROS production in T2DM patients, was found suggesting that an increased mitochondrial sensitivity for T₃ may be a significant factor responsible for increased ROS activity in diabetic patients.

Effect of Methionine Deficiency on the Thymus and the Subsets and Proliferation of Peripheral Blood T-Cell, and Serum IL-2 Contents in Broilers

The purpose of this study was to investigate the effects of methionine deficiency on cellular immune function by determining morphological and ultrastructural changes of thymus, thymic cell cycle and apoptosis, peripheral blood T-cell subsets, T-cell proliferation function and the serum interleukin-2 (IL-2) contents. 120 1-d-old broilers were randomly divided into two groups (6 replicates in each group and 10 broilers in each replicate) and fed on a control diet or methionine deficient diet for 42 d. Lesions were observed in experiment. Histopathologically, lymphopenia and congestion were observed in the medulla of thymic lobule. Ultrastructurally, there were more apoptosis lymphocytes, and the mitochondria of lymphocytes were swelled in thymus of methionine deficiency. The G0/G1 phase of the cell cycle of the thymus was much higher (P<0.01), and the S, G2+M phases and proliferating index (PI) were lower (P<0.01) in methionine deficiency than in control group. And the percentage of apoptotic cells in the thymus was significantly increased in methionine deficiency (P<0.01). The percentage of CD4+ and CD8+ T-cells was decreased in methionine deficiency compared with control group. Meanwhile, the proliferation function of peripheral blood T-cell was decreased in methionine deficiency. Also, the serum IL-2 contents were decreased in methionine deficiency. It was concluded that methionine deficiency could cause pathological and ultrastructural changes of thymus, reduce the T-cell population, serum IL-2 contents and the proliferation function of T-cells, and induce increased percentage of apoptotic cells. The cellular immune function was finally impaired in broilers.

Pathology of spleen in chickens fed on a diet deficient in methionine

The purpose of this 42-day study was to investigate the effects of methionine deficiency on spleen by determining the relative weight, morphological changes of spleen, cell cycle and apoptosis of splenocytes, and oxidative markers of serum and spleen. One hundred and twenty one-day-old avian broilers were randomly divided into two groups and fed on a control diet (starter diet, methionine 0.50%; grower diet, methionine 0.40%) and methionine deficient diet (starter diet, methionine 0.26%; grower diet, methionine 0.28%) for 6 weeks. The relative weight of spleen was lighter in methionine deficiency than control group. Histopathologically, lymphopenia and congestion were observed. Ultrastructurally, there were more apoptosis lymphocytes in spleen and the mitochondria of lymphocytes were swelled in methionine deficiency. By flow cytometry, the G0/G1 phase of the cell cycle of the spleen was much higher (P < 0.01), and the S, G2+M phases and proliferating index were lower (P < 0.01) in methionine deficiency than in control group. And the percentage of apoptotic cells in the spleen was significantly increased in methionine deficiency (P < 0.01).The superoxide dismutase and glutathione peroxidase activities, and abilities to inhibit hydroxyl radicals were greatly decreased while the malondialdehyde contents were markedly increased in methionine deficiency. It was concluded that methionine deficiency could restraine the development of the spleen by cell cycle arrest and increased apoptosis, cause splenic lesions and reduce splenic antioxidant function. The splenic function should be finally impaired and then the immune function could be impacted in chickens.

S14.5 Does aging influence lymphocyte mitochondria respiration in trained people?

S14.5 Does aging influence lymphocyte mitochondria respiration in trained people?

Impaired platelet mitochondrial activity in Alzheimer’s disease and mild cognitive impairment

Mitochondrial abnormalities are found in Alzheimer’s disease (AD), but previous reports have not examined at-risk groups. In subjects with AD, mild cognitive impairment (MCI), and non-demented aged controls, platelet and lymphocyte mitochondria were isolated and analyzed for Complexes I, III, and IV of the electron transport chain. Western blots were used to control for differential enrichment of samples. Results demonstrated significant declines in Complexes III and IV in AD, and a significant decline in Complex IV in MCI. This report confirms mitochondrial deficiencies in AD, extends them to MCI, and suggests they are present at the earliest symptomatic stages of disease.

O3-01-05: Aconitase activity in lymphocyte mitochondria from AD, MCI and cognitively normal subjects

O3-01-05: Aconitase activity in lymphocyte mitochondria from AD, MCI and cognitively normal subjects

A Novel Potassium Channel in Lymphocyte Mitochondria

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance.

Biological mechanochemiemission and bioenergetics

Currently three conceptions on the mechanism of the ATP synthesis in mitochondria (MT) exist: chemical, chemiosmotic and conformational relaxation. But a quantitative interpretation of the proposed theories comes across the significant difficulties. In this work, the mechanical energy dissipation in mitochondria of lymphocytes in a steady state and swollen condition was studied by means of a registration, the biological mechanochemiemission (BMCHE)—the mechanoluminescence (ML) in the optical region (200–650 nm) and the radioemission (RE) in the radio range (100 Hz to 0.1 MHz) of the electromagnetic spectrum, with the subsequent processing of the results in a microcomputer system. The ATP concentration determination in mitochondria was performed by the bioluminescent method. The results obtained show that parameters of ML and RE main peaks of the mean power density are different for MT in the steady and swollen state. The coherence of the ML and RE signals in mitochondria is registered. In steady state mitochondria the ATP quantity was more than in swollen organelles by 5.2×10 −8 M/10 6 cells. Our experimental results indicate that there is a correlation between a relaxation of tense energy and BMCHE process in an MT membrane. The phenomenological consideration of BMCHE mechanisms in the MT membrane energy transformation which is utilized for various processes of a living cell functioning is given. A model of BMCHE synthesis of ATP is suggested.

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.

Effects of glucocorticoids on lymphocyte metabolism

The immunosuppressive effect of glucocorticoids has been widely reported; however, the mechanism of action of these hormones on the immune system has not been fully established. In the present study, the effect of glucocorticoids on glucose, glutamine, and pyruvate metabolism in lymph node lymphocytes was investigated. Addition of dexamethasone to the incubation medium did not alter glucose and glutamine metabolism but inhibited pyruvate utilization by 40%. This latter effect took 1 h to occur and remained for up to 6 h, even after removal of dexamethasone from the culture medium. Measurements of the activity of pyruvate dehydrogenase in lymphocytes and the rate of [1-14C]-pyruvate conversion into 14CO2 in incubated lymphocyte mitochondria demonstrated that glucocorticoids decrease pyruvate utilization by inhibiting the activity of this key regulatory enzyme. The effect of such an inhibition of pyruvate utilization on the function of cells of the immune system remains to be clarified.

The effect of adenine nucleotides on the rate and fate of glutamine utilization by incubated mitochondria isolated from rat mesenteric lymph nodes.

The rate of glutamine utilization by isolated lymphocyte mitochondria is 21.4 nmol/min per mg protein, of which 72% is converted to glutamate. Addition of ATP, ADP or AMP increased the rate of glutamine utilization by 60%. Evidence is presented that this is due to a stimulation of oxoglutarate dehydrogenase activity by ADP: this may account for the stimulation of glutamine utilization by concanavalin A in lymphocyte.

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.

Observations on the Origin of Lymphocyte-like Cells in Mouse Bone Marrow

APPRECIABLE numbers of lymphocyte-like cells are found in the bone marrow of mice1,2 and other mammals3,4. Their origin has intrigued investigators for many years3. Yoffey3,4 considers that these cells have a haematogeneous origin, while Osmond and Everett5 find no evidence for the influx into the bone marrow of large numbers of blood lymphocytes. Evidence is accumulating that mitochon-drial content is a useful criterion for characterization of lymphocytes6–9 and that electron microscopy may help to elucidate the origin of some lymphocyte-like cells8. The present investigation was undertaken to compare the number of mitochondria in lymphocytes from blood and lymphocyte-like cells in bone marrow and to examine the ultrastructure of the latter.

Fine Structural Localization of Adenosine Triphosphatase in Human Platelets and Other Blood Cells

Abstract Adenosine triphosphatase activity has been localized in human blood cells by combined histochemical and electron microscopic technics. Deposits of lead phosphate, indicative of enzymic activity, were observed on the surface membranes of erythrocytes, lymphocytes and platelets. Intracellular ATPase activity was found in lymphocyte mitochondria and nucleoli. Blood platelets contained organelles in their hyaloplasm with dense accumulations of the enzyme reaction product. The distribution of lead phosphate in platelet organelles suggested that they were mitochondria, but some platelet granules may also contain ATPase activity.