Rat Peripheral Blood Lymphocytes Research Articles

Postnatal development of dopamine receptor expression in rat peripheral blood lymphocytes

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [ 3H]SCH 23390 and the dopamine D2-like receptor agonist [ 3H]7-OH-DPAT were used as radioligands. The affinity ( K d) of [ 3H]SCH 23390 or of [ 3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density ( B max) of [ 3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B max value of [ 3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.

Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes

We have previously shown that simultaneous exposure of rat lymphocytes to iron ions and 50 Hz magnetic field (MF) caused an increase in the number of cells with DNA strand breaks. Although the mechanism of MF-induced DNA damage is not known, we suppose that it involves free radicals. In the present study, to confirm our hypothesis, we have examined the effect of melatonin, an established free radicals scavenger, on DNA damage in rat peripheral blood lymphocytes exposed in vitro to iron ions and 50 Hz MF. The alkaline comet assay was chosen for the assessment of DNA damage. During pre-incubation, part of the cell samples were supplemented with melatonin (0.5 or 1.0 mM). The experiments were performed on the cell samples incubated for 3 h in Helmholtz coils at 7 mT 50 Hz MF. During MF exposure, some samples were treated with ferrous chloride (FeCl 2, 10 μg/ml), while the rest served as controls. A significant increase in the number of cells with DNA damage was found only after simultaneous exposure of lymphocytes to FeCl 2 and 7 mT 50 Hz MF, compared to the control samples or those incubated with FeCl 2 alone. However, when the cells were treated with melatonin and then exposed to iron ions and 50 Hz MF, the number of damaged cells was significantly reduced, and the effect depended on the concentration of melatonin. The reduction reached about 50% at 0.5 mM and about 100% at 1.0 mM. Our results indicate that melatonin provides protection against DNA damage in rat lymphocytes exposed in vitro to iron ions and 50 Hz MF (7 mT). Therefore, it can be suggested that free radicals may be involved in 50 Hz magnetic field and iron ions-induced DNA damage in rat blood lymphocytes. The future experimental studies, in vitro and in vivo, should provide an answer to the question concerning the role of melatonin in the free radical processes in the power frequency magnetic field.

Effects of amitriptyline on diurnal variations of oxidation-reduction enzyme activities in rat lymphocytes during experimental desynchronosis.

We studied the effects of amitriptyline on diurnal variations of oxidative-reduction enzyme activities (succinate dehydrogenase, lactate dehydrogenase, and NADPH diaphorase) in rat peripheral blood lymphocytes during experimental desynchronosis. Desynchronosis was induced by constant light exposure for 14 days and manifested in a morning shift of maximum lactate dehydrogenase activity and elevated morning NADPH diaphorase activity. Amitriptyline normalized morning lactate dehydrogenase activity and diurnal variations in NADPH diaphorase activity.

Phenytoin and electric shock-induced apoptosis in rat peripheral blood lymphocytes

The apoptotic index (AI) of peripheral blood lymphocytes (PBL) and plasma corticosterone (CS) levels were determined in Wistar rats treated with phenytoin (PHT) at therapeutic and toxic doses (100 or 200 mg/kg/day, respectively, over a period of 7 days) and stressed by bifrontal electric shock (60 Hz/40 mA/0.2 seg). The values of CS and AI were found to be significantly higher in rats submitted to electric shock (ES) and in rats treated with therapeutic and toxic doses of PHT plus ES, than in rats treated only with PHT ( P<0.001). The plasma concentrations of PHT were found to be significantly higher in rats treated with toxic doses than in those treated with therapeutic doses ( P<0.001), while the control group (without treatment) and vehicle group (propilenglycol–ethanol–water, 40:10:50), showed low levels of CS, and less than 1% of AI. The DNA analysis by electrophoresis in agarose in all the groups was positive, displaying the ladder pattern characteristic of apoptotic process (200 bp), except in the control groups (no treatment and vehicle treated). Our results demonstrate that chronic stress, caused by ES, produces an elevation of CS. The values of apoptosis were correlated with the CS levels, suggesting that the apoptotic inductor process is a consequence of an increase in the concentration of corticosterone in plasma, in response to the hypothalamic–pituitary–adrenals (HPA) axis activation, while phenytoin at therapeutic doses is only a moderate apoptosis inductor.

Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)(*).

CI-994 (acetyldinaline) is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 μM) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 μM and were statistically significant beginning at 10 μM. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 μM CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 μM CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 μM CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.

Transmission of HTLV-I to rats via peripheral blood mononuclear cells and serum from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) with amyotrophic lateral sclerosis (ALS)-like features.

We tested the possibility that lymphocytes and serum obtained directly from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) induce infection in rats. Inbred Fischer F344 immunosuppressed rats were inoculated intravenously with 10x10(6) peripheral blood mononuclear cells (PBMC; 3 rats) and serum (3 rats) obtained from a HAM/TSP patient, who was seropositive and polymerase chain reaction (PCR)-positive for the HTLV-I proviral genome. Antibodies to HTLV-I appeared in the rat sera 2 months later; rat peripheral blood lymphocytes, spleen, salivary gland, and spinal cord were found to contain the proviral genome. Control rats inoculated with normal donor PBMC and serum tested negative for the HTLV-I antibodies and for the HTLV-I proviral genome by PCR. The positive control F344 rats inoculated with 5x10(6) cells of a SLB-1 HTLV-I cell line were found to be infected after 2 months. This study demonstrates for the first time that HTLV-I can be transmitted not only by human cellular components but also by human cell-free sera in a rat model.

Modulation of the immune response by progesterone-induced lymphocyte factors.

Rat spleen and peripheral blood lymphocytes express progesterone receptors whose concentration is increased greatly during the early phase of pregnancy. After stimulation of progesterone the expression of receptors was augmented 2-3 times. When cells were cultured in the presence of progesterone they released a soluble factor that inhibited cellular immunoreactions (MLR, CRC) and cellular proliferation as measured by thymidine incorporation by spleen-cell culture. This factor also inhibited the synthesis of anti-DNP antibodies by a mouse hybridoma and diminished the proportion of cells in phase S. However, the percentage of asymmetric molecules produced by the hybridoma remained unaltered. These results support the hypothesis that soluble factors released by rat lymphocytes modulate the immune response of the mother and participate in the mechanism that protects the fetus against antipaternal antibodies.

Cytochrome P450 1A1 in rat peripheral blood lymphocytes: inducibility in vivo and bioactivation of benzo[ a]pyrene in the Salmonella typhimurium mutagenicity assay in vitro

The presence and inducibility of CYP1A1 in freshly isolated peripheral blood lymphocytes was examined in untreated rats and in rats pretreated with agents known to induce the enzyme in other tissues, as well as dexamethasone [CAS #50-02-2], which is not commonly associated with CYP1A1 induction. CYP1A1 but not CYP1A2 was detected by Western blot analysis of lymphocytes from untreated rats and was induced in lymphocytes from rats treated with the known CYP1A inducers β-naphthoflavone [CAS #6051-87-2] or 3-methylcholanthrene [CAS #56-49-5] (7.3-fold), cigarette smoke (2.8-fold), and pyridine [CAS #108-86-1] (2.6-fold). CYP1A1 was also induced in lymphocytes from rats treated with the nonprototypic inducer dexamethasone (17.7-fold) or bromobenzene [CAS #108-86-1] (3.9-fold). Lymphocyte homogenate from rats treated with the inducers also catalyzed NADPH-dependent bioactivation of benzo[ a]pyrene [CAS #50-32-8] to mutagens. The benzo( a)pyrene mutagenicity was detected using Salmonella typhimurium TA100 in the Ames test, and correlated positively with lymphocyte CYP1A1 content. The data show that CYP1A1 is present in rat peripheral blood lymphocytes in vivo, and is inducible by prototypic, as well as nonprototypic, inducers of the enzyme.

Modulatory effects of LPS on 5-HT transporter in peripheral blood lymphocytes of rats

Modulatory effects of LPS on 5-HT transporter in peripheral blood lymphocytes of rats

Rat lymphocytes produce and secrete acetylcholine in dependence of differentiation and activation.

Previous data from this laboratory suggested for the first time that immune cells of the immune system of different species are capable to synthesize the neurotransmitter acetylcholine. In the present study we detected the RNA message for choline acetyltransferase in thymic, splenic and peripheral blood lymphocytes of rats using RT-PCR. Furthermore, using a sensitive radioimmunoassay, we measured acetylcholine in thymic, splenic and peripheral blood lymphocytes. T-cells were found to contain about three times the amount of acetylcholine as compared to B-cells, and CD4+ cells showed significantly higher levels as compared to CD8+ cells. Mitogenic stimulation with PHA increased the acetylcholine levels in lymphoid cells as well as the release into the supernatants.

Immunotoxicity of environmentally relevant mixtures of polychlorinated aromatic hydrocarbons with methyl mercury on rat lymphocytes in vitro

AbstractThe immunosuppressive effects of methyl mercury (MH), polychlorinated biphenyls (PCBs), polychlorinated dibenzo‐p‐dioxins (PCDDs), and dibenzofurans (PCDFs) are well established at high exposure levels but unclear at low exposure levels. We exposed Fischer 344 rat splenocytes, thymocytes, and peripheral blood lymphocytes in vitro for 72 h to MHg (0.1, 2 μg/ml), PCDD/PCDF mixtures (1, 15 pg/ml) of three PCDDs (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, 1,2,3,7,8‐pentachlorodibenzo‐p‐dioxin, and 1,2,3,4,7,8‐hexachlorodibenzo‐p‐dioxin) and two PCDFs (2,3,7,8‐tetrachlorodibenzofuran and 1,2,3,7,8‐pentachlorodibenzo‐furan), three Aroclor® (1242, 1254, 1260), PCB mixtures (0.01, 0.5 μg/ml), or combinations of MHg/PCB/PCDD/PCDF mixtures Mitogenic responses of lymphocytes to concanavalin A, phytohemagglutinin, or lipopolysaccharide/dextran sulfate were determined by 3H‐thymidine uptake; cytotoxicity and intracellular Ca2+ were determined by flow cytometry. Methylmercury (2 μg/ml and PCB/ PCDD/PCDF mixtures with 2 μg/ml MHg decreased the viability of splenocytes to 57 and 40% at 4 and 24 h, respectively. Basal intracellular calcium ion levels were unaffected by the treatments. Methylmercury suppressed the responses of lymphocytes to T and B cell mitogens. All combinations of MHg/PCB/PCDD/PCDF mixtures decreased mitogenic responses to levels similar to those to MHg alone. In contrast, PCB and PCDD/PCDF mixtures did not suppress but augmented responses of splenocytes and peripheral blood lymphocytes to T cell mitogens. Overall, no interactive toxicity was observed with MHg/PCB/PCDD/PCDF mixtures on cytotoxicity and lymphocyte mitogenic responses. Therefore, MHg may pose a greater threat than organochlorines to the mammalian immune system.

Effect of garlic on micronuclei frequency in peripheral blood lymphocytes of rats with N-methyl-N'nitro-N-nitrosoguanidine-induced gastric carcinoma and precancerous lesions.

To investigate the effects of garlic on micronuclei frequency (MNF) in peripheral blood lymphocytes (PBLs) of Wistar rats with N-methyl-N'nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinoma (GC) and precancerous lesions (PLs). Wistar rats were exposed to MNNG at 1.25 mg/5 mL per day for 10 mo to induce GC and PLs (MNNG group, n = 30); rats not exposed to MNNG served as unmodeled controls (control group, n = 16). The MNNG rats were randomly divided into a prevention treatment group (n = 30; receiving 10 mL of a 10% garlic solution per day) and an untreated model control group (n = 20; receiving tap water). MNF in PBLs were detected at experiment months 10 and 16 mo by the microculture technique. The MNNG group had similar MNF levels at months 10 and 16. Compared to the control group, the MNNG, prevention and untreated model control groups had remarkably higher MNF levels (P < 0.01). The level of PLs was significantly lower in the prevention treatment group than in the untreated model control group (P < 0.01). The prevention group showed significantly lower MNF than the MNNG group (P < 0.01), and the MNF level was reduced in month 16 compared to month 10 (P < 0.01). However, the difference in MNF levels in groups given prevention or treatment was not significant. MNNG exposure exerted continuous mutagenicity and carcinogenicity properties on PBLs, and garlic exerted a remarkable anti-mutagenic and anti-carcinogenic effect. MNF in PBL may be a novel marker of early-stage GC.

Suppression of mitogen-induced IL-2R alpha expression in PBL by serum from microfilaraemic rats chronically infected with Brugia pahangi.

To study immunological responses in filarial infections, interleukin-2 receptor alpha (IL-2R alpha) expression in the peripheral blood lymphocytes (PBL) of Lewis rats infected with Brugia pahangi was observed by flow cytometry. During infection, no increase in IL-2R alpha was observed in either microfilaraemic or amicrofilaraemic rats. The PBL of rats were stimulated with phytohaemagglutinin (PHA) and the frequency of IL-2R alpha-positivity was examined. After microfilariae appeared, the increase in the rate of IL-2R alpha-positivity in microfilaraemic rats was less than in amicrofilaraemic rats. The anergy of IL-2R alpha expression in microfilaraemic rats was restored when their lymphocytes were cultured with normal rat serum. IL-2R alpha expression in normal uninfected rats decreased when cultured with serum from microfilaraemic rats. These results suggest that microfilariae-related factors in rat serum suppress PHA-induced IL-2R alpha expression in PBL and thus inhibit cell proliferation and host immunity.

Thymosin α 1 interacts with the VIP receptor-effector system in rat and mouse immunocompetent cells

Thymic peptide thymosin α 1 (10−11 to 10 -6 M) is shown to interact with the VIP receptor-effector system in rat and mouse peritoneal macrophages, and both rat peripheral blood lymphocytes and spleen lymphocytes. In all models, thymosin α I inhibits 125I-VIP binding with a potency that is in a range 1000–1700 times lower than that of the native VIP. Interaction of thymosin α 1 with VIP receptors is compared with that of some structurally VIP-related peptides such as helodermin, PHI, secretin, and glucagon. The order of potency in inhibiting 125I-VIP binding was VIP > helodermin > PHI > secretin > thymosin α 1. Thymosin α 1 (1010 to 10−6 M) was weak in stimulating adenylyl cyclase activity. Its efficacy is in a range 900–1800 times lower than that of native VIP in all cell types studied. The analysis of the sequence of both complete and N-terminal portion of thymosin α 1 reveals close structural and physicochemical similarities with the members of the so-called VIP family of polypeptides. Taken together, experimental data support that thymosin α 1 must be included like the lowest partial agonist of the VIP family of polypeptides and it is a VIP receptor antagonist with weak intrinsic activity.

Beta-blockade enhances adrenergic immunosuppression in rats via inhibition of melatonin release

We have recently shown in rats that an in vivo treatment with catecholamines via α 2,-receptors leads to a pronounced suppression of T-and B-cell mitogen responses of peripheral blood lymphocytes (PBL), provided that a β-blocker is administered concomitantly. Since melatonin (MEL) reportedly has stress-protective effects on several immune functions, and since the release of MEL from the pineal gland is inhibited by β-blockade, we tested the effect of MEL substitution on T-and B-cell mitogen responses of PBL in rats treated with two s.c. implanted retard tablets containing noradrenaline (NA) and propranolol. It was found that an oral treatment with MEL (about 40 /j,g/animal) abolished the adrenergic immunosuppression. Furthermore, functional pinealectomy induced by constant light had a similar enhancing effect on the α 2adrenergic immunosuppression as observed with β-blockers, whereas PBL from animals kept at the regular light/dark interval were resistant to the treatment with the selective α 2agonist clonidine. It is concluded that endogenous MEL effectively protects rat PBL from adrenergic immunosuppression, and that β-blockers enhance the immunosuppressive property of os-adrenergic agents via blocking the night-time release of MEL.

Regulation of prolactin receptor mRNA expression in peripheral lymphocytes in rats in response to changes in serum concentrations of prolactin

In the present study we have evaluated the absolute number of the two forms of prolactin (PRL) receptor mRNA in rat peripheral blood lymphocytes and the modulation of receptor mRNA induced by changes in serum levels of endogenous PRL or by administration of ovine PRL. Lymphocytes expressed low levels of both forms of PRL receptor transcripts. Repeated treatments with ovine PRL significantly reduced levels of mRNA encoding the long form PRL receptor, whereas expression was markedly increased by repeated doses of bromocriptine. In contrast, the mRNA level of short form PRL receptor was unchanged by both treatments. The expression of long form transcripts was also markedly decreased in lymphocytes from pituitary-grafted rats. Therefore it appears that in rat peripheral lymphocytes PRL has a negative effect on the expression of its own receptor.

Regulation of prolactin receptor mRNA expression in peripheral lymphocytes in rats in response to changes in serum concentrations of prolactin.

In the present study we have evaluated the absolute number of the two forms of prolactin (PRL) receptor mRNA in rat peripheral blood lymphocytes and the modulation of receptor mRNA induced by changes in serum levels of endogenous PRL or by administration of ovine PRL. Lymphocytes expressed low levels of both forms of PRL receptor transcripts. Repeated treatments with ovine PRL significantly reduced levels of mRNA encoding the long form PRL receptor, whereas expression was markedly increased by repeated doses of bromocriptine. In contrast, the mRNA level of short form PRL receptor was unchanged by both treatments. The expression of long form transcripts was also markedly decreased in lymphocytes from pituitary-grafted rats. Therefore it appears that in rat peripheral lymphocytes PRL has a negative effect on the expression of its own receptor.

Frequency and phase correlation of rhythms of dihygroorotate dehydrogenase activity in rat peripheral blood lymphocytes and solar activity rhythms and wave periods of planets

The common variation pattern and the synchronous increases of enzymatic and solar activity are explained by the identical frequencies of the bio- and heliorhythms and by the strict phase coordination of the latter. It is shown to be possible to predict enzymatic activity according to the sum of the resonance heliorhythms. The wave genesis of the resonance between enzymatic and solar fluctuations is confirmed by the fact that the periods of the bio- and heliorhythms are the same as the wave periods of planets.