Interaction of intact and irradiated macrophages with thymus lymphoid cells

The rat broncho-alveolar macrophages, subjected to gamma-irradiation, were incubated for 4 hours with irradiated (4 Gy) thymocytes. Following the total 24 hour incubation, some morphological features of macrophages were revealed in addition to their influence on survival, autologous rosetting and mitotic index of intact thymocytes. The increase in macrophage spreading was shown which was dose-dependent in the 1 to 4 Gy scale. Enhanced viability of thymocytes was revealed in the presence of macrophages irradiated at the dose of 1-2 Gy. Addition of 24 hour cultures of intact or irradiated macrophages elicited a significant decrease in rosette-forming capacity among thymocytes. Gamma-irradiation of 2 to 4 Gy inhibited the ability of macrophages to suppress the mitotic activity of thymic cells. A possibility of postradiational modification of some specific functions and properties of macrophages, including their thymotropic effects, is discussed.     

Mechanisms of T-lymphocyte stimulation of erythropoiesis under local body irradiation

Thymocytes transplanted into thymectomized mice with a locally irradiated lower limb in a dose of 7 Gy were accumulated in the depleted bone marrow to stimulate the processes of postradiation erythron regeneration. Cytosar treatment did not affect the regulatory function of thymocytes. At the same time thymocytes treated with actinomycin D possessed less powerful stimulating effect than intact ones.     

Modification of the regulatory effects of thymocytes on hematopoiesis in irradiated mice as affected by heterologous immunoglobulin G and low-dose ionizing radiation

The administration of heterologous immunoglobulin G (IgG) and/or exposure of mouse thymocyte donors to 1 and 2 Gy radiation were shown to change the regulatory effects of thymus lymphocytes on the recovery of haemopoiesis in syngeneic recipients irradiated with a median lethal dose of 6 Gy. Thymocytes of exposed (2 Gy) donors produced a stimulatory effect on the restoration of the myelokaryocytes number and increased the number of endogenous splenic colonies and bone marrow CFUs in animals exposed to a median lethal dose, whereas the administration of IgG to thymocyte donors given 2 Gy eliminated the stimulatory effect of thymocytes on the number of myelokaryocytes, and the amount of CFUs in irradiated recipients decreased.     

Immunochemical analysis of changes in the structure of the nuclear matrix in thymocytes of irradiated rats

It was shown that reactivity of the nuclear matrix of thymocytes for antibodies against chromatin of the control and irradiated thymocytes and PDN did not change immediately and increased markedly 2 h following irradiation of rats with a dose of 10 Gy. The method of immunoblotting failed to reveal any qualitative differences in the protein content of the thymocyte nuclear matrix of the control and exposed rats.     

Lipid peroxidation of rat spleen and thymus lymphocytes treated with x-rays (0-5-1.0 Gy)

It is established, that low doses of X-ray irradiation have affected activation of lipid peroxidation (LPO) in immunocompetent cells of the spleen and thymus. The amount of malonic dialdehyde (MDA) in lymphocytes of spleen and thymocytes increases 2 times twenty-four hours after animals' irradiation by X-rays in a dose of 0.5 Gy; when a dose grows to 1.0 Gy, the MDA content in the spleen lymphocytes increases from the 1st to the 6th days and in thymocytes from the 1st to the 3d days reaching its maximum at the 3d day. MDA accumulation in the immunocompetent cells of irradiated animals varies depending on the method of lipid peroxidation initiation.     

A study of DNA repair in the thymocytes of irradiated rats using a spectrofluorometric method

In studying DNA repair in thymocytes of irradiated rats it was shown that the increase in radiation dose from 2 to 20 Gy made DNA damages increase in number and caused changes in their spectrum and growth of irreparable damages. The one-hour study of DNA repair process exhibited its fast, median and slow phases.     

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.     

Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats

The pattern of DNA degradation in thymocytes of irradiated or hydrocortisone-treated rats has been studied by means of flow cytometry of the cells, treated with probes specifically bound to the AT or GC-pairs of DNA. It has been shown that the death of thymocytes is accompanied by a decrease in their DNA content. The main features of the occurrence and accumulation of cells with a DNA content less than the normal diploid level correspond with those of internucleosomal DNA fragmentation: such cells appear after a 1 hour lag-period, their accumulation is prevented by cycloheximide injection and is lower at 300 Gy than at doses of 10 to 30 Gy. At the same time, no increase in permeability of the cell membrane to ethidium bromide was observed up to the sixth hour after irradiation. Most of the thymocytes dying under the action of irradiation or hydrocortisone are in the G0 or G1 phases of the cell cycle. The method used allows detection of the cells with cleaved but not removed DNA.     

Thymocytes of irradiated rats studied using naphthalenesulfonic probes

The fluorescence intensity of 1.8-aniline naphthalene sulfonate (ANS) and 2.6-toluidine naphthalene sulfonate (TNS) introduced into irradiated rat thymocytes reached maximum 15 min and 4 h following irradiation (1-20 Gy). The parameters of 1.8-ANS binding to membranes did not contribute markedly to the effect observed.     

Modification of the interphase death of thymocytes by using colcemid

The pretreatment of thymocytes by colcemid (0.02 to 0.5 mu g/ml) induces a change in the plasma membrane state, registered by a pyrene fluorescent probe, and a decrease in the interphase cell death after 4 Gy X-irradiation. The authors discuss the role of the cell surface as a trigger initiating death program in irradiated lymphoid cells.     

The mechanism of radiation-induced interphase death of lymphoid cells: a new hypothesis

The interphase death of irradiated rat thymocytes depends on their concentration during postirradiation incubation. The kinetics of pycnosis and cell death determined with the trypan blue exclusion test in the samples with the highest cell concentration (1-2 x 10(7) cells/ml) is consistent with the data available in the literature, whereas the samples with the lowest concentration (2 x 10(5) cells/ml) undergo almost no pycnosis and death after irradiation with doses up to 50 Gy. On the basis of these results, we suggest a new mechanism of interphase death involving an interaction between irradiated thymocytes and the fraction of thymus cells possessing cytocidal activity. The observed correlation between the cytocidal activity and interphase death of thymocytes from animals of different ages favors our mechanism. It was found that the inhibitors which prevent the conjugation of killer cells and their targets do not influence interphase death, while the substances which block the secretion of cytotoxic factors or their action on the target membrane do protect from interphase death. Thus we suggest that the irradiation activates the killer cells to secrete some cytotoxic factors which induce pycnosis and interphase death of thymocytes.     

The effect of a low dose of ionizing radiation on the activity of cAMP-dependent phosphodiesterase and protein kinase of chicken thymus in ontogenesis

Dynamics of changes in activities of cAMP-dependent enzymes, phosphodiesterase and protein kinase, were studied in the thymus of intact and irradiated, prior to incubation, (0.05 Gy) chicken embryos and chicks. The changes observed were wave-like. In determining phosphodiesterase activity of irradiated chicken thymocytes during ontogenesis the values were obtained that correlated with the cAMP level and adenylate cyclase activity. It was also shown that the increase in the rate of cAMP-dependent phosphorylation under the effect of low-level radiation corresponded to the cyclic nucleotide content of a cell at different development stages.     

Role of a disorder in poly(ADP-ribosylation) in the activation of Ca/Mg-dependent endonuclease

The nuclei from the control and irradiated (3 h after irradiation at a dose of 10 Gy) thymocytes were preincubated with NAD in conditions optimal for poly (ADP) ribosylation. This was shown to decrease by 6-7- and 2-3 times, respectively, the rate of autolytic cleavage of DNA by Ca/Mg-dependent endonuclease. The inhibitors of poly (ADP-riboso)-polymerase, nicotine amide and thymidine, removed the effect of NAD. The data obtained prompt an assumption that the post-irradiation activation of Ca/Mg-nuclease in thymocytes is associated with the disturbance of its post-translation modification, poly(ADP)ribosylation.     

Postirradiation changes in the cGMP system of the mouse thymus and liver

Changes have been revealed in the function of cyclic GMP system of thymus and liver of irradiated (8 Gy) mice. In the thymus the cGMP level increased during the first 60 min following irradiation. In the liver the concentration of cGMP exhibited two peaks: 30 min and 24 hr after irradiation. The changes observed in the cGMP level are connected with the increased guanylate cyclase activity of thymocytes and liver of irradiated mice and, less likely, with changes in the activity of cGMP phosphodiesterase of these tissues.     

Study of changes in the surface of irradiated thymocytes using a method of separating cells in a 2-phase water-polymer system

The coefficient of cell distribution in a charged biphase water-polymeric system "polyethyleneglycol-dextran" decreased 60 min after irradiation of rat thymus lymphocytes with doses of 1 to 8 Gy. This dose-dependent effect was mainly related to radiosensitive thymocytes of a low fraction of density gradient; it disappeared with the uncharged system and was probably connected with the reduction in the negative electric charge of the irradiated cell surface.     

The effect of a low dose of ionizing radiation and of gangliosides on the adenylate cyclase activity of the chicken thymus and thyroid in ontogeny

Adenylate cyclase (AC) activity was studied in thymus and thyroid gland of intact chick embryos and those irradiated with a dose of 0.029 Gy prior to incubation, and newly hatched chicks in the presence of total ganglioside fractions extracted from the same organs. Gangliosides were shown to increase the enzyme activity of thymocytes and thyrocytes during the postnatal development. It is suggested that small radiation doses potentiate the stimulatory effect of ganglioside fractions on AC.     

The microelectrophoresis of the DNA in individual intact and gamma-irradiated thymocytes

The in vitro gamma-irradiated mouse thymocytes were embedded in low melting agarose at 37 degrees C. After getting at 4 degrees C, the cells were lysed in neutral detergent solution containing proteinase K and ethidium bromide. Microscopic visualization of single lysed and stained cells showed the presence of the central "core" (nuclear matrix) surrounded with "halo" (relaxed nuclear DNA). During electrophoresis (2-5 V/sm, 5 min) this "halo" migrated towards the anode forming a "tail". The use of microdensitometric system provided measuring the size of the tail (L) and quantity of migrated DNA (S) for individual cells as well as obtaining the distribution of these parameters among the cells. The latter may be characteristic of heterogeneity of the cell population. It was shown that L and S increased linearly with the dose irradiation at least between 0.2 and and 5.0 Gy. In irradiated thymocyte (3 Gy) the DNA repair occurred within 10-20 min, but residual DNA damage could be observed even after 60 min of incubation. These damages may initiate the degradation of DNA in irradiated thymocytes that was observed after the repair of DNA.     

Inhibition of poly(ADP-ribose) polymerase as a possible reason for activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats

The molecular mechanism of activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats was studied. Thymocyte nuclei of control and irradiated rats were pre-incubated with NAD under conditions favourable for poly ADP-ribosylation. Pre-incubation results in a decrease in the rate of autolytic DNA digestion by Ca2+/Mg2+-dependent endonuclease of 6-7- and 2-3-fold for control and irradiated animals, respectively. The activity of Ca2+/Mg2+-nuclease extracted from the nuclei pre-incubated with NAD is also considerably decreased. The presence of nicotinamide and thymidine in the preincubation medium prevents the suppression of Ca2+/Mg2+-nuclease activity. In the experiments performed with isolated nuclei and permeabilized thymocytes the synthesis of poly(ADP-ribose) does not significantly change within 1 h after irradiation at a dose of 10 Gy, whereas 2 and 3 h after the exposure it decreases by 35-40 and 45-55 per cent, respectively. The activity of poly(ADP-ribose) glycohydrolase in this period is similar to that in the controls. The average size of the de novo synthesized chains of poly(ADP-ribose) increases from 11 to 17 ADP-ribose units by the second hour after irradiation. Inhibition of poly(ADP-ribose) polymerase in the postirradiation period preceded the internucleosomal fragmentation of chromatin. The results suggest that activation of Ca2+/Mg2+-nuclease in irradiated thymocytes is accounted for by the disturbance of its poly ADP-ribosylation.     

Cytofluorimetric study of ploidy in the preleukemic thymus in C57BL/Ka mice under fractionated irradiation

The ploidy of the thymus was studied in C57B1/Ka mice irradiated with 4 weekly X-Ray doses of 1.75 Gy. The determination of nuclear DNA content was performed by flow cytometry of intact thymocytes labeled with propidium iodide in presence of a mixture of chicken and rainbow trout red blood cells as internal reference standards. The method has been tested by detecting the sex difference in DNA content of G0/G1 of normal thymic mouse cells. The mean value was 2.9% higher in female mice. The thymus of almost 60% of irradiated male mice present a slight hypoploidy of 2.6% one month after the last irradiation.     

Postirradiation changes in the chromatin structure of thymocytes in irradiated rats

In this work the antibodies were obtained against chromatin isolated from thymocytes of intact and irradiated rats (2 h after exposing to 10 Gy) and against polydeoxyribonucleotides (PDN) extracted from thymus nuclei 6 h following irradiation. All the antibodies under study reacted more readily with the chromatin obtained from the thymus of exposed rats than with the control chromatin. The complexes of DNA with the most firmly bound non-histone proteins, obtained from the three objects under study, reacted with the antibodies with equal efficiency. Thus, a higher reactivity of PDN and chromatin from thymocytes of exposed rats was associated with the decondensation of the latter leading to an increase in availability of a part of antigenic determinants. Using the immunoblotting method we failed to discover any qualitative differences in the protein composition of the chromatin from control and exposed rats.