Population kinetic study on the origin of guinea pig monocyte heterogeneity

Population kinetic studies were performed on guinea pig peripheral blood monocyte fractions isolated by counter-flow centrifugation elutriation following a single in vivo pulse of tritiated thymidine. Labeled large monocytes (volume 317 micron3; relative distribution 49%; circulating half-life 5.7 hr; and production rate 17,000 cells/ml blood/hr) accumulated in the circulation more rapidly, had a faster turnover time, and were produced in greater numbers than small monocytes (volume 283 micron3; relative distribution 34%; circulating half-life 10.8 hr; and production rate of 6100 cells/ml blood/hr). The kinetic data do not support a maturational sequence of small into large monocytes. Intermediate monocytes (volume 300 micron3; relative distribution 11%; circulating half-life 18.2 hr) and very large monocytes (volume 354 micron3; relative distribution 6%; circulating half-life 36.5 hr) had production rates, respectively, of only 1200 and 320 cells/ml blood/hr. Maxima in the labeling index curve for small and large monocytes suggested a generation time of 24 hr while grain count analysis revealed that these two cell fractions were derived from a precursor population with similar numbers of reductive divisions. Grain count analysis of intermediate and very large monocytes revealed that these cells differed from both small and large monocytes. Our data support the concept that monocyte subsets exist in guinea pig peripheral blood with different kinetics of production and survival.     

Estimation of effective eosinopoiesis and bone marrow eosinophil reserve capacity in normal man.

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [3H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 x 10(9) cells/kg body weight per day (mean 0.22 x 10(9) cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 x 10(9) cells/kg body weight (mean 0.14 x 10(9) cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.     

Human bone marrow lymphocytes. III. Polyclonal activation of B lymphocytes in human bone marrow measured by a direct plaque-forming cell assay.

Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.     

ESTIMATION OF EFFECTIVE EOSINOPOIESIS AND BONE MARROW EOSINOPHIL RESERVE CAPACITY IN NORMAL MAN

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [³H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 × 10⁹ cells/kg body weight per day (mean 0.22 × 10⁹ cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 × 10⁹ cells/kg body weight (mean 0.14 × 10⁹ cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.     

Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes.

A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.     

Blast cell and granulocyte production in human leukemia: pathophysiological concepts based on computer simulation using discrete modeling techniques

The granulocyte production of two patients suffering from leukemia was studied extensively by means of the tritiated thymidine method of cellular kinetics. The data obtained (1-h labeling index, pattern of cell labeling, labeling intensity, as well as other conventional parameters of bone marrow and blood) were used to develop a computer model (GPSS-language) to fit the observations. From these models, it was concluded that patients with leukemia may have an abnormal granulopoiesis, characterized by a high degree of inefficiency (premature cell death, skipping of divisions with undisturbed maturation). However, the underlying mechanisms may be quite different. While it cannot be excluded that in acute myelocytic leukemia there is a stem and/or progenitor cell pool that is highly ineffective but still capable of feeding some cells into the granulocytic pathway, it is nevertheless possible, as shown in plasma cell leukemia, that the ineffective granulopoiesis may be the result of direct or indirect interaction between the "leukemic" and the "normal" cell clone.     

The effect of food and water deprivation on the peripheral blood parameters of the mouse

Various peripheral blood and bone marrow parameters were determined during food and water deprivation and during food deprivation alone in order to obtain base lines that may be used to make comparisons with similar data from irradiated mice. The peripheral blood parameters following food and water deprivation were similar to those following food deprivation alone. The mean survival time was about 5 days and the weight loss 40% of the control weight. There was an absolute decrease in the total circulating lymphocyte and platelet counts, while the total granulocyte count remained unchanged or increased. The blood volume decreased, while the hematocrit and specific gravity of the blood increased. The bone marrow parameters following food and water deprivation showed that erythropoiesis was more markedly depressed than myelopoiesis. The tritiated thymidine labeling index for granulopoietic cells and megakaryocytes decreased progressively during starvation. The variations in the white blood count and the bone marrow parameters are not comparable with those found in irradiated mice having the G.I. syndrome; the changes in mean survival time, weight loss, hematocrit, and blood volume are similar.     

Studies of cell separation: A comparison of the osmotic response of human lymphocytes and granulocyte—Monocyte progenitor cells

The feasibility of using hypo- or hypertonic stress to selectively destroy lymphocytes while sparing stem cells was investigated. Lymphocytes were isolated from peripheral blood and exposed to Hanks' balanced salt solutions ranging in concentration from 66 to 2700 mOsm. The Boylevan't Hoff plot of cell volume versus reciprocal osmolality was linear. Following osmotic stress, viabilities of the lymphocytes and the granulocyte-monocyte progenitor cells (CFUc) were determined. Lymphocyte viability was assessed by tritiated thymidine incorporation following mitogen stimulation. CFUc viability was measured with the soft agar colony assay. Both types of cells were found to possess high osmotic tolerances compared to other blood cells. While progenitor cells in general appeared to survive anisotonic exposure somewhat better than lymphocytes, significant statistical differences were not established for most situations. The highest degree of CFUc enrichment was twofold, but there was a concomitant 50% drop in CFUc survival. These results suggest that osmotic stress is not a useful procedure for the separation of peripheral blood lymphocytes and stem cells.     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.     

The frequency of 6-thioguanine-resistant human peripheral blood lymphocytes as determined by flow cytometry and by clonal propagation

Human peripheral blood lymphocytes were selected for 6-thioguanine resistance in short-term cultures. Resistant cells, defined as cells capable of incorporating tritiated thymidine under the selective conditions, were flow-cytometrically differentiated with respect to their DNA content. This was carried out by sorting at two stages of the cell cycle, before and after mid-S-stage, yielding frequencies of resistant cells in the range of 10(-4) and 10(-5), respectively. Observed frequencies for cells from the whole cell cycle spectrum and for cells cultured according to the long-term protocol, the clonal assay, were in the range of 10(-4) and 10(-5), respectively. Our interpretation of these results is that the over-representation of tritiated thymidine-labelled cells occurring before mid-S-stage after short-term culture reflects less resistant cells or phenocopies which are probably eliminated during long-term culture with the clonal assay, hence leading to a decreased frequency of 6-thioguanine-resistant cells. We conclude, therefore, that short-term culture in combination with flow cytometric sorting after mid-S-stage in the cell cycle can be used as an alternative to the clonal assay for the determination of fully 6-thioguanine-resistant human peripheral lymphocytes.     

THE EFFECTS OF TRITIATED THYMIDINE INCORPORATION ON SECONDARY ROOT PRODUCTION BY PISUM SATIVUM

Hummon , Margaret R. (Montana State U., Missoula.) The effects of tritiated thymidine incorporation on secondary root production by Pisum sativum. Amer. Jour. Bot. 49(10): 1038–1046. Illus. 1962.—Most studies of effects of radiation on plants have involved a general exposure of all the cells and tissues of an organ or entire plant. Tritiated thymidine offers a tool for selective irradiation of the nucleus with little effect on the cytoplasm of a cell. Furthermore, differential incorporation due to variation in the pattern of DNA synthesis permits selective irradiation of cells and tissues. In this study, developing primary roots of Pisum sativum were submerged for brief periods in a solution of tritiated thymidine. This resulted in an alteration of the lateral root pattern. In the area corresponding to the region of elongation during treatment, subsequent lateral root production was suppressed. This correlated with the portion of the root in which there was incorporation of tritiated thymidine into a high percentage of pericycle nuclei. Abnormal development of vascular tissues also occurred, with evidence of altered polarity in the xylem. Although incorporation also occurred in the apical meristem, the latter was not affected at this level of exposure. Thus, differential sensitivity as well as differential incorporation may have been involved in producing the temporary alteration of lateral root production.     

Immunological studies of aging. IX. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry

Lymphocytes from humans over the age of 65 incorporate approximately 50% less tritiated thymidine than do lymphocytes from young donors when cultured with phytohemagglutinin. Because lymphocytes from elderly humans are more sensitive to cell cycle arrest induced by tritiated thymidine, it was impossible to determine to what extent impaired thymidine incorporation reflected a defect in proliferation or the increased sensitivity to the radioactive isotope. Flow cytometry was used to measure the proliferative response of T cells from young and old donors in culture with PHA. It was found that 25 percent fewer lymphocytes from old as compared to young humans enter the G1 or complete the S phase of the cell cycle. However, the rate of progression through the cell cycle by activated cells from young and old humans is comparable. Thus, flow cytometry suggested that the difference in thymidine incorporation by lymphocytes from old and young donors is attributable equally to a proliferative defect and to cell cycle arrest induced by tritiated thymidine. This conclusion was supported by the fact that the relative impairment of thymidine incorporation by lymphocytes from old donors was only one-half as great when a 20-min instead of a 24-hr pulse of tritiated thymidine was used.     

Characteristics of in vitro colony formation by cells from haemopoietic tissues

Summary The characteristics of stimulation of colony formationin vitro from cells of mouse haemopoietic tissues has been briefly reviewed. Mouse kidney or embryo feeder cells, media conditioned by the cells from these tissues, normal or leukemic mouse sera, sera from leukemic or infectious mononucleosis patients, human urine and mouse embryo extracts are all sources of colony stimulating activity and their properties have been described. All sources of colony-stimulating activity produce clones of cells of the granulocyte series. In tritiated thymidine treated mice injection of preparations rich in colony-stimulating activity has been shown to produce a neutrophil leucocytosis and accelerate the rate of accumulation of labelled neutrophils in the blood. It is suggested that thein vitro assay can detect factors capable of stimulating granulocyte development.     

RELATIONSHIP OF BONE MARROW CELLULARITY AND PROLIFERATIVE ACTIVITY: A LOCAL REGULATORY MECHANISM

An analysis has been made of tritiated thymidine (³H-TdR) uptake and cellularity in normal and regenerating marrow. Comparison of right and left femoral marrow in ninety normal rabbits has revealed a significant negative correlation (−0.75) between thymidine uptake per presumptive proliferative cell and the concentration of such cells. Locally irradiated marrow follows the normal marrow regression down to about 40% relative cellularity and then shows an upward displacement. The regenerating mechanically depopulated marrow manifests a similar displacement over the entire cellularity range studied which suggests that recovery was not yet complete as in the case of some of the irradiated marrow samples. The negative regression of ³H-TdR uptake on cellularity is not a consequence of altered thymidine availability, but rather of-a changing DNA synthesis rate and/or a changing fraction of proliferative cells. Since ³H-TdR uptake per presumptive proliferative cell reflects cell production, it follows that cell production is geared to the cellularity of a discrete marrow area. This provides a local self-regulating mechanism.     

PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.     

DNA synthesis rate and unlabeled cells with S-phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.