Characteristics of T-Suppressors concentrated by elution from an allogeneic cell monolayer

Quantitative enrichment of the immune lymphocyte fraction with H-2 antigen-specified suppressor cells was obtained by their elution from the relevant allogeneic monolayer of target cells. This was accompanied by 2-3-fold gain in the content of T lymphocytes and DNA synthetizing cells, as well as in the total (3)H-thymidine incorporation resulting from an increase in the percentage of medium and large lymphocytes in the population and also in the proportion of DNA synthesizing small and medium lymphocytes. Complete abolition of the suppressor effect by treatment with anti-Thy-1 and anti-T antibodies rather than with anti-B serum, and the resistance of this effect to carrageenan and carbonyl iron indicate the T cell nature of the eluted suppressor cells.     

LYMPHOCYTE POPULATIONS IN MOUSE BONE MARROW: QUANTITATIVE KINETIC STUDIES IN YOUNG, PUBERTAL AND ADULT C3H MICE

Continuous ³H-thymidine infusion was used to characterize two kinetic subpopula-tions of small lymphocytes in mouse bone marrow during normal growth and development. Young (4 wk), pubertal (8 wk) and mature (16 wk) C3H mice were infused subcutaneously with ³H-thymidine for periods up to 10 days. Femoral marrow was then examined in radioautographic smears. During the first 3 days the proportion of marrow small lymphocytes labelled by ³H-thymidine showed a rapid exponential increase to 93%, 81% and 72% in 4 wk, 8 wk and 16 wk mice respectively. The rate of appearance of labelled small lymphocytes then declined markedly but remained higher in younger than in older animals. The labelling curves were found to represent the summation of two exponential curves from which the proportions and renewal rate of corresponding cell populations were calculated. Most marrow small lymphocytes comprised a rapidly renewing population but in mice of increasing age the relative incidence of these cells fell (93-3% at 4 wk; 88-0% at 8 wk; 78-5% at 16 wk) and their half-renewal time (T_½) lengthened (14 hr at 4 wk; 18 hr at 8 wk; 24 hr at 16 wk). The remaining small lymphocytes were slowly renewing with mean T_½ of 4, 7 and 14 days in 4, 8 and 16 wk mice, respectively. Some heavily labelled small lymphocytes persisted in the marrow up to 10 wk after fourteen daily ³H-thymidine injections in 10–12 wk mice. The numbers of rapidly renewing cells decreased from 604 times 10³ to 228 times 10³ per mm³ of marrow from 4 wk to 16 wk, respectively, while slowly renewing cells increased from 44 times 10³ to 61 times 10³ per mm³. The total number of nucleated marrow cells per femur increased from 4 wk to 16 wk but the rapidly renewing small lymphocytes per femur fell in numbers by 36% and in renewal rate by 63%. The results demonstrate a selective change in bone marrow small lymphocytes with age; rapidly renewing cells decline in number and renewal rate while the number of slowly renewing cells increases. The concept of bone marrow as a primary lymphoid organ is discussed.     

‘3H-testosterone distribution and binding in RAT thymus cells in vivo’

Summary Autoradiography and biochemical investigations showed that [³H]-testosterone where injected intraperitoneally into male white rats was incorporated rapidly into thymus lymphocytes. Thymic cortex contained more silver grains than medulla, and larger lymphocytes were more labelled than medium or small lymphocytes.Cytosol fraction of thymus cells labelledin vivo with [³H]-testosterone, contained the largest quantity of labelled hormone. A 4S cytosol fraction binds [³H]-testosterone. This could be separated by Sephadex chromatography or by linear sucrose gradient centrifugation. Nuclear extract contained also a small quantity of the labelled hormone.     

Lymphocyte subpopulation of human peripheral blood responds to the low doses of ionizing radiation,interleukin-2 and to both factors

Increasing of 3H-thymidine incorporation in lymphocytes of human peripheral blood which depends non-linearly on X-ray dose (3 cGy max) and interleukin-2 (IL-2) concentration (17.5 Units/ml) is shown. However addition of IL-2 (17.5 U/ml) into the medium of cells after irradiation (3 cGy) decreases almost to the control the effects induced by independently shown actions. Lymphocytes subpopulation responsible for the described phenomena are isolated during the fractionation of lymphocytes in the density gradient and pH (V-fraction BSA). Cell fraction less than 1-2% from the isolated lymphocytes is characterized by increasing of spontaneous corporation of 3H-thymidine, large sizes (d > 8 mkm), decreasing repair after UV-irradiation. It is believed that low dose irradiation and IL-2 activate this cell subpopulation of "last reaction", and higher doses of these factors and this both actions stopping 3H-thymidine incorporation initiate apoptosis. The relation of this sell subpopulation and before proposed ontogenetical reserve cells is discussed.     

Are all lymphoid blasts in the cell cycle?

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with mediumsized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no G_o cells. Supported by the Deutsche Forschungsgemeinschaft DFG Grant SFB 112     

Are all lymphoid blasts in the cell cycle? DNA synthesis in the lymphoid tissues of the dog

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with medium-sized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no Go cells.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

Mitotic cycle kinetics of root meristems of isolated barley embryos and intact seedlings. Labelling of nuclei by3H- thymidine and its cytogenetic consequences

The duration of the mitotic cycle and its individual phases was estimated in root meristems of isolated barley embryos and intact barley seedlings by means of pulse labelling with³H-thymidine and construction of labelled mitoses curve. The duration of the whole mitotic cycle in the cell population of root meristems of isolated barley embryos cultivated in the aerated liquid complete medium is 12.2 h. The mitotic cycle time of root meristems of intact barley seedlings, oultived in Petri dishes on wet blotting paper is 9.2 h. Most of root meristem cells belong to the fraction of rapidly proliferating cells, but this fraction exerts a high degree of variability by itself. Pulse treatment by³H-thymidine in our experimental conditions (74 kBq ml^-1 - or 2 μCi ml^-1, exposure 0.5 h) did not induoe any chromosomal aberrations in unlabelled cells and only a very low frequency of chromosomal aberrations in labelled cells. Measuring the cell population kinetics by pulse labelling with³H-thymidine can be used simultaneously with the study of induction of ohromosomal aberrations by mutagens.     

Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.     

Proteases stimulate proliferation of human fibroblasts.

Incubation of primary human fibroblasts in serum-free medium with small concentrations of thrombin, trypsin or plasmin resulted in manyfold increase in total DNA synthesis and in the number of 3H-thymidine labelled cells. Rise in the frequency of mitoses indicates that the proteases stimulated also cell division. Because proteases induced only a fraction of cells to proliferate increase in the total number of cells remained moderate. Calf, horse and rabbit serum inhibited the growth stimulating effect of trypsin but chicken, dog and monkey serum were permissive. Specific inhibitors of proteases prevented the stimulation of cell proliferation suggesting strongly that proteases act in virtue of their enzymatic activity.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

The dynamics of proliferation and differentiation of osteogenic cells under supportive unloading

With the use of radioactive marker of DNA synthesis--3H-thymidine we have studied the dynamics, peculiarities of proliferation and differentiation of osteogenic cells under hind limb unloading of white rats ("tail suspension" method at an angle 35 degrees) during 28 days. The 3H-thymidine was administered at a single dose at the end of the experiment, the biosamples were taken from femoral bones in 1, 48, 96 hr. Light and electron-microscopic radioautography with 3H-thymidine (in 1 hour) have shown, that basic fraction of DNA synthesizing cells in the zones of adaptive remodelling of bone tissue is represented by little-differentiated perivascular cells (that include osteogenic cell precursors). A tendency for a decrease of a labelling index in the 3H-thymidine osteogenic cells on metaphyseal bone trabeculae under hind limb unloading has been established. The dynamics of labelled cells during various time intervals after 3H-thymidine injection testifies to a delay in the differentiation precursors in osteoblasts and their transformation to osteocytes in experiment animals. The obtained data have shown that a long-term supportive unloading leads to lowering the intensity of osteogenetic processes in long bones and reducing bone mass.     

Accumulation and lethal effect of tritium (tritiated water) inRhodopseudomonas spheroides

Nonsulfur purple photosynthetic bacteria, Rhodopseudomonas spheroides cells were cultured in medium containing tritiated water (THO) under the light-anaerobic and dark-aerobic conditions. The experimental R value defined as specific activity ratio of organic bound 3H to THO in medium was 0.49 and 0.48 for the light-anaerobically grown cells and the dark-aerobically grown cells, respectively. From the relation of R value to number of weight doubling of the cells (n), ratio of experimental R to theoretical R, i.e., (2n-1)/2n derived by assuming no isotope effect, was 0.51 and 0.49 on an average for the light-anaerobically grown cells and the dark-aerobically grown cells, respectively. 3H-incorporation from THO-medium into the light-anaerobic nongrowing cells was affected by the light intensity and suppressed by adding HgCl2, KCN, and 2,4-dinitrophenol as well as 3H-labelling in the dark-aerobic nongrowing cells was affected by oxygen tension and suppressed by adding these metabolic inhibitors. From the fractionation of the lyophilized cells by modified Schneider method, the distribution of exchangeable 3H in cold acid-soluble and ether-ethanol-soluble fractions and nonexchangeable 3H bound to small molecules and macromolecules was 7.4/25.3/67.3 in the growing cells cultured anaerobically in the THO-medium up to late exponential phase in the light. The distribution in the nongrowing cells incubated anaerobically with the THO-medium for 18 h in the light of 300 and 3,000 lux was 82.1/8.4/9.5 and 58.2/19.2/22.6, respectively. These distributions of 3H were changed with growth phase and/or incubation time. On the biological effect of 3H-THO for the cells stocked at -196 degrees C to accumulate 3H-decays, the dark-aerobic nongrowing cells labelled with THO were rather radiosensitive than the dark-aerobically and light-anaerobically grown cells cultured in the THO-medium. The killing efficiencies, i.e., the probability that a single disintegration would be lethal, ranged from 1/200 to 1/275 for the above three kinds of cells labelled with THO. The killing efficiencies for R. spheroides labelled with THO were similar to that for radiosensitive strain CB13 and wild strain Hfr of Escherichia coli labelled with 3H-thymidine and stored at -196 degrees C.     

DNA Synthesis in Microspores in Anther Culture of Wheat (Triticum aestivum L.)

Anthers with mid-unlnucleate microspores were cultured on W5 medium supplemented with 0.5 mg/l kinetin, 2 mg/l 2,4-D and 9% or 3% sucrose. At a series of interval (0, 1, 1.5, 2, 14 days) after cultured, the anthers were labelled with 3H-thymidine (4 MCi/mi) for 24 h, fixed, and then performed autoradiography according to conventional method. Results show that after cultured for 24 h, 3H-thymidine was incorporated into some late-uninucleate microspores (see Plate I, 3), and after for 2.5 days, vegetative nuclei in pollen grains were la- belled (see Plate I, 4). Usually, vegetative nuclei were labelled frequently and generative ones were labelled rarely. Sometimes generative cell which could synthesis DNA might develop suspensor-like structure individually (see Plate I, 13). During early stage of development of a multicellular pollen grain, the DNA synthesis in the cells were synchronized. With pollen development, the synchronism of DNA synthesis was destroyed. When anthers cultured on medium with 3% sucrose, DNA in microspores could be synthesized normally, and the number of labelled microspores was more than that of anthers cultured on medium with 9% sucrose. However, on medium with 3% sucrose, the nuclei in microspores stopped dividing after one or two divisions and the cell wall of them could not be formed and multicellular pollen was not observed. It seems that the absence of multicellular pollen on medium with 3% sucrose was primarily due to the block of cell division and cell wall formation, not due to the interruption of DNA synthesis.     

Proteases as mitogens : The effect of trypsin and pronase on mouse and human lymphocytes

Treatment of mouse spleen lymphocytes with trypsin (from 0.1 to 1.0 μg/ml) was found to cause a significant stimulation of the incorporation of ³H-thymidine. When spleen cells from nude (congenitally athymic) mice were incubated with trypsin in the absence of serum for 3 days, very high levels of incorporation were noted (stimulation index of 10 to 20). Trypsin was without effect on thymic lymphocytes of the mouse but caused significant activation of human peripheral blood lymphocytes. The stimulatory effect of trypsin was a consequence of its enzymatic activity. Prolonged treatment with pronase also caused small but significant increases in the incorporation of labelled thymidine (stimulation index of 2 to 4) into the thymic and splenic lymphocytes of the mouse and into human lymphocytes. The evidence suggests that trypsin stimulates the B-derived lymphocytes.     

Fate of H2-activated T lymphocytes in syngeneic hosts: II. Residence in recirculating lymphocyte pool and capacity to migrate to allografts

T.TDL—a purified population of lymph-borne H2-activated T lymphocytes—were transferred to syngeneic mice to examine their capacity to remain in the recirculating lymphocyte pool (RLP). Experiments with cells labelled with ³H-thymidine (³HTdR) or ⁵¹Chromium showed that although a considerable proportion of T.TDL joined the RLP (i.e., were mobilizable through a thoracic duct fistula) for several days after transfer, most of the cells soon left the pool. This applied whether the cells were transferred to normal mice or to “B” mice. Normal thoracic duct lymphocytes, by contrast, joined the RLP for long periods post-transfer.Studies with ³HTdR-labelled T.TDL showed that a small number of heavily labelled cells remained in the RLP for at least 3 months. Experiments with the θ-antigen as a cell marker suggested that a further small proportion of cells underwent division at some stage after transfer and then rejoined the RLP in expanded numbers.T.TDL showed a tendency to home to specific allografts of either skin, tumor cells or lymphoid cells. Although homing was specific it was very limited in extent.     

Cholinergic differentiation in neurogenic basal forebrain cultures.

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Comparative study of the proliferative activity of blood vessel cells of various calibers

Proliferative activity of the main tissue components of aorta, inferior vena cava and small vessels of paravasal tissues has been studied in 7 white adult rats after repeated injections of 3H-thymidine (12 injections of 0.8 microCi/g with the interval of 8 h). The greatest number of labelled endotheliocytes is revealed in capillaries, small and medium veins (29.4 +/- 6.2%). Proliferative activity of smooth muscle cells in arterioles was by 2 times greater than in other vessels. There was no difference between the number of labelled fibroblasts in the media of all types of studied vessels.     

Tritiated thymidine autoradiographic study on the origin and renewal of argentaffin cells in the pyloric gland of hamsters

Summary The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with ³H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt.By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with ³H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus.The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the random loss system. The half time of turnover of this cell population was 15 days on average.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan