Murine Peripheral Blood Leukocytes Research Articles

Thymic microenvironment's impact on immunosenescence.

Age-related thymic involution is characterized by the loss of T cell development and the supporting epithelial network, which are replaced by adipose tissue. We previously showed that aging functionally impairs lymphohematopoietic progenitor cells, including thymic early T cell progenitors (ETPs), contributing to thymic involution. Considering that the thymic microenvironment is essential for thymocyte incubation, we aimed to investigate its role in age-related thymic involution and the mechanisms underlying these changes. The challenge in studying these processes led us to transplant T cell-depleted fetal thymus tissue into the kidney capsule of aged mice. This model allowed us to identify the mechanisms driving age-related changes in the thymic microenvironment and to assess whether these changes could be reversed. Flow cytometry was used to detect naïve T cells (CD62L+CD44-), including CD4 CD8 double-negative, double-positive, and single-positive T cells. Real-time PCR was used to detect and quantify signal-joint T cell receptor excision circles. We rearranged δRec-ΨJα in murine peripheral blood leukocytes to evaluate the thymic output of newly developed naïve T cells in the mice and gene expression in the thymus. Age-related thymic involution decreased naïve T cells and increased memory T cells, while fetal thymus transplantation improved thymic output and T cell production and reversed the impairment of thymopoiesis due to thymic involution in aged mice. Furthermore, the expression of key cytokines was restored and ETPs in the aged mice showed normal thymic T cell development. Our study suggests that degenerative changes in the thymic microenvironment are the primary cause of thymic dysfunction, leading to immunosenescence associated with age-related thymic involution.

Kinetics of the in vivo genotoxic and radioprotective effects of methyl gallate and epigallocatechin gallate.

The aim of this study was to compare the kinetics of the in vivo action of equimolar doses of methyl gallate (MG) and epigallocatechin gallate (EGCG) on their capacity to induce DNA damage and to protect against DNA damage induced by 60 Co gamma rays. DNA-damaged cells were determined by single-cell gel electrophoresis (comets) in murine peripheral blood leukocytes. The maximum radioprotective effects of MG and EGCG (approximately 70%) occurred at 15 min after administration when their effect was determined 2 min following irradiation. MG and EGCG have similar radioprotective indexes, which due to their fast response indicate that they are involved in free radical scavenging. Due to the similar radioprotective activities of MG and EGCG, the in vivo radioprotective effects of these agents do not seem to be dependent on the number of hydroxyl groups present in their structures but instead on the presence of the galloyl radical. EGCG induces an early, significant, and persistent increase in the number of DNA-damaged cells and a later and more important increase in the number of damaged cells, suggesting that it has two mechanisms by which it can induce DNA damage. MG at the same molar dose as EGCG caused a significant and persistent increase in DNA damaged cells but to a much lesser extent to that induce by EGCG, suggesting that the galloyl radical is not involved in the mechanism of DNA breaks induction.

There Is (Scientific) Strength in Numbers: A Comprehensive Quantitation of Fc Gamma Receptor Numbers on Human and Murine Peripheral Blood Leukocytes.

Antibodies are essential mediators of immunological defense mechanisms, are clinically used as therapeutic agents, but are also functionally involved in various immune-mediated disorders. Whereas IgG antibodies accomplish some of their biological tasks autonomously, many functions depend on their binding to activating and inhibitory Fcγ receptors (FcγR). From a qualitative point of view expression patterns of FcγR on immunologically relevant cell types are well-characterized both for mice and humans. Surprisingly, however, there is only quite limited information available on actual quantities of FcγR expressed by the different leukocyte populations. In this study we provide a comprehensive data set assessing quantitatively how many individual human and mouse FcγRs are expressed on B cells, NK cells, eosinophils, neutrophils, basophils and both classical, and non-classical monocytes under steady state conditions. Moreover, among human donors we found two groups with different expression levels of the inhibitory FcγRIIb on monocytes which appears to correlate with haplotypes of the activating FcγRIIIa.

Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy.

We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.

Ferulic Acid Inhibits Gamma Radiation-Induced DNA Strand Breaks and Enhances the Survival of Mice

Ferulic acid (FA) is a monophenolic phenylpropanoid occurring in plant products such as rice bran, green tea, and coffee beans. It has been shown to have significant antioxidant effects in many studies. In the present study, we show that intraperitoneal administration of FA at a dose of 50 mg/kg body weight 1 hour prior to or immediately after whole-body γ-irradiation of mice with 4 Gy results in considerable reduction in the micronuclei formation in peripheral blood reticulocytes. Administration of the same amount of FA immediately after 4 Gy γ-irradiation showed significant decrease in the amount of DNA strand breaks in murine peripheral blood leukocytes and bone marrow cells as examined by comet assay. Further, immunostaining of mouse splenic lymphocytes for phspho-γH2AX was carried out, and it was observed that FA inhibits the γH2AX foci formation. Finally, the survival of mice upon 6, 8, and 10 Gy γ-ray exposure was monitored. FA enhances the survival of mice by a factor of 2.5 at a dose of 6 Gy γ-radiation but not at higher doses. In conclusion, FA has protective potential in both pre- and postirradiation exposure scenarios and enhances the survival of mice possibly by decreasing DNA damage as examined by γH2AX foci, micronuclei formation, and comet assay.

Investigations of genotoxic activity of the antimicrobial/antiviral agent FS-1 in rodents by means of the micronucleus and the comet assays

Background: FS-1 is a complex of iodine and synthesized polysaccharides and it is very effective against a number of microbe and virus strains. The aim of the study was to evaluate possible genotoxic properties of FS-1. Methods: The compound was studied in rat and mouse bone marrow micronucleus (MN) assay and the comet assay in murine peripheral blood leukocytes, hepatocytes, and kidney cells. Two treatment protocols were applied, namely acute and subacute ones. In the first protocol, the compound was administered orally once and in subacute treatment two times, 24 h apart. The animals were sacrificed 24 h after the last treatment, and appropriate cells were used to assess DNA damage and MN induction. Results: In none of the tests (MN and comet assays) significant increase compared with respective negative controls was observed. Conclusion: The fact that the compound neither induces DNA damage in various organs of mice nor is effective in the induction of MN in bone marrow cells of rats and mice is important for future genotoxicity studies of FS-1, which can be used in clinical medicine after additional testing of safety for humans.

Roles of SIRT1 in the Acute and Restorative Phases following Induction of Inflammation

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that in vivo endotoxin triggers a rapid and transient decline in ATP concentration in human peripheral blood leukocytes and murine peripheral blood leukocytes and liver, which is associated with a brief increase in expression of the autophagy indicator LC3-II. In both of these tissues, the ATP concentration reaches a nadir, and autophagy is induced between 2 and 4 h post-endotoxin infusion, and homeostasis is restored within 12 h. Mouse liver SIRT1 and AMP-activated protein kinase (AMPK) protein expression levels decline precipitously within 10 min and remain below detection levels for up to 12 h post-endotoxin administration. In marked contrast, the expression of HIF-1α is induced within 90 min and remains elevated for up to 12 h. The ATP recovery is delayed, and the increases in both HIF-1α expression and autophagy are prolonged in endotoxin-challenged SIRT1 liver knock-out mice. Resveratrol prevents the decline in ATP concentration and SIRT1 expression, as well as the increase in HIF-1α expression and autophagy in liver of endotoxin-challenged wild type mice but not in SIRT1 liver knock-out mice. These results provide novel insight into the state of both cellular bioenergetics and metabolic networks during the acute phase of systemic inflammation and suggest a role for SIRT1 in acute metabolic decline, as well as the restoration of metabolic homeostasis during an inflammatory challenge.

Neutrophil Influx and Chemokine Production during the Early Phases of the Antitumor Response to the Vascular Disrupting Agent DMXAA (ASA404)

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G+CD11b+F4/80−. cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b+ cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor á, interleukin 6 (IL-6), and macrophage inflammatory 1α (MIP-1α). CD49b+ natural killer cells produced IP-10, whereas CD45R+ B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1α (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Prolonged Reduction of Leukocyte Membrane-Associated Dectin-1 Levels following β-Glucan Administration

Dectin-1 is the primary pattern recognition receptor for fungal glucans. Dectin-1 mediates the internalization and biological response to glucans. We examined the effect of i.v. or i.p. glucan phosphate (GP) administration on Dectin-1 membrane expression in murine peripheral blood leukocytes, splenocytes, bone marrow, and peritoneal cells from 3 h to 10 days after injection. Circulating leukocytes were also examined for uptake and internalization of glucans from the blood. Fluorescent-labeled GP was taken up from the systemic circulation by circulating peripheral leukocytes, splenocytes, and peritoneal cells. Following internalization, glucan colocalized with Dectin-1 in an intracellular vesicle. A single parenteral injection of GP resulted in a significant reduction (approximately 33-85%) in peripheral leukocyte membrane-associated Dectin-1 positivity that lasted for up to 7 days. The loss of leukocyte membrane-associated Dectin-1 after GP administration was primarily due to decreased levels of Dectin-1 on neutrophil and monocyte membranes with no significant changes in the percentage of neutrophils or monocytes circulating in the blood. Administration of control carbohydrate polymers, i.e., mannan or pullulan, which are not ligands for Dectin-1, did not decrease Dectin-1 leukocyte positivity, indicating that the effect on Dectin-1 is specific to glucans. In fact, mannan administration increased leukocyte Dectin-1 positivity, thus demonstrating a differential effect on leukocyte Dectin-1, compared with GP. We conclude that systemic administration of GP has a specific and prolonged effect on loss of leukocyte membrane Dectin-1 positivity. These data may have important implications for developing dosing regimens for immunomodulatory carbohydrates.

Kinetic of Genotoxic Expression in the Pharmacodynamics of Busulfan

Busulfan (BUS) is a highly toxic antineoplastic bifunctional-alkylating agent and has a narrow therapeutic window. Our previous study revealed a narrow dose-range of BUS, which causes a sudden dose-dependent transition from early- to late-expressing micronucleus induction and from a non-cytotoxic to a cytotoxic effect. In the present study, the kinetics of DNA-damaged cell induction by BUS and its dose-effect relationship were established. This was achieved by using the kinetics of DNA-damaged cell induction, determined by the comet assay in murine peripheral blood leukocytes of mice, after the intraperitoneal exposure to 16, 30, 45, 60 or 80 micromol/kg of BUS. Doses of 15 or 30 micromol/kg of BUS were able to increase DNA-damaged cell frequency, but doses of 45 micromol/kg body weight or higher caused a sudden drop in this frequency. This suggests that higher doses cause lesions that inhibit the expression of damage as comets, i.e., DNA-protein or interstrand crosslinks. The latter could be explained by sudden monoadduct-to-crosslink transformation due to a DNA conformational change induced by monoadduct accumulation that facilitates crosslink formation. This narrow dose-dependent transition could contribute to the narrow therapeutic window of BUS.

Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions

The effect of ferulic acid was studied on gamma-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body gamma-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of ferulic acid 1 h prior to 4 Gy gamma-radiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensuration with DNA repair. Administration of 50 mg/kg body weight of ferulic acid after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in ferulic acid treated animals.

SOCS3 Plays an important negative regulatory role in myeloid colony formation

The family of SOCS proteins are cytokine-responsive genes, which function as part of a negative feedback loop. In vitro, several growth factors including EPO, TPO, GM-CSF, IL3 and G-CSF have been shown to rapidly and transiently induce SOCS3 expression, and cycloheximide results in super-induction of SOCS3 message. We found that SOCS 3 basal expression was undetectable in human bone marrow and other tissues surveyed, but low levels of SOCS mRNA were detected in both human and murine peripheral blood leukocytes. Culture of BM with G-CSF for 3 to 7 days resulted in increased SOCS 3 mRNA, whereas SOCS 1 expression was not induced. In vivo expression of SOCS 3 was studied in mice treated twice daily with 50 ug/kg G-CSF for 4 days. SOCS 3 mRNA expression was rapidly induced in bone marrow and PBLs, but by 24 hours decayed to background levels in both tissues. Repeated daily injections of G-CSF yielded the same rapid and transient pattern, with some increase in maximal mRNA levels over the 4-day period. To determine if SOCS 3 regulates myeloid response to G-CSF, we tested anti-sense oiigonucleotides (ASO) on CFU-G formation in human bone marrow. SOCS 3 ASO increased the number of CFU-G colonies in the presence of sub-optimal or maximal G-CSF by 3-10-fold. The sensitivity to G-CSF was also increased up to ten-fold. The responses to SOCS3 ASO appear to be stage-specific, because the CFU-GEMM response was unchanged. Treatment with SOCS1, SOCS2 or CIS ASO had no significant effect on CFU-G assays. These results indicate that SOCS3 has an important regulatory role in regulation of normal myelopoiesis.

Repair kinetics of gamma-ray induced DNA damage determined by the single cell gel electrophoresis assay in murine leukocytes in vivo

The repair kinetics of the gamma rays induced DNA damage was determined in murine peripheral blood leukocytes in vivo by the comet assay. Mice were exposed to 1.0 Gy of gamma rays in a 137Cs source and samples of peripheral blood were taken from their tails at different times. The repair was evaluated per mice in separate experiments by measuring the proportion of cells with tail (comets) in each sample. An average of nearly 80% of comets was obtained at the initial time after the exposure; 2 min later the frequency decreased to 45% and continued diminishing to 22% at 15 min. This evidences the presence of a rapid repair mechanism. For a period of 25 to 40 min after exposure there was a slight but consistent increase of comets from 22 to 38% followed by a second reduction, which could be due to a late repair process that causes strand breaks and then joined them. In summary our results indicated that this system seems to be appropriate for the study of the repair capacity of cells following exposure to ionizing radiation.

Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation.

1. Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N-terminus peptide, cell-associated LC1-like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane. 2. Treatment of mice with dexamethasone (Dex; 0.5-5 micrograms per mouse corresponding to approximately 0.015-1.5 mg kg-1) increased LC1 levels in neutrophils and monocytes. The 2-3 fold increase in LC1 levels was time-dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg-1 orally) decreased LC1-like immunoreactivity in all three types of circulating leukocytes by > or = 50%. 3. Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (> or = 50%) in LC1 levels compared with circulating neutrophils. A high LC1-like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane-associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane-associated LC1 to a significant extent, i.e. up to 70%. 4. In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.

Thymocyte-stimulating factor from peripheral blood cells

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.