H. Cottier

Kinetics of megacaryocyte proliferation.

Conclusion The kinetics of the mega-caryocytic cell line of the rat bone marrow were studied using tritiated thymidine as a cell label. The changes in the percentage of labeled cells as a function of time after injection of the tracer were registered separately for arbitrarily chosen successive recognizable stages of megacaryocytic differentiation. Emphasis is put on the development of initially labeled cells into a stage of maturation corresponding to initially non-labeling cell forms. The following results were obtained: 1. The transit time for the most immature recognizable stage of megacaryocytic development to megacaryocytic disintegration is approximately 40 hours. 2. Evidence was obtained that the recognizable megacaryocytic elements originate from unrecognized precursors which continuously synthesize DNA for a period of at least 1 to 3 days prior to maturation into recognizable megacaryocytic precursors. 3. The immature megacaryocytic cells able to synthesize DNA take up more H3 thymidine than the rest of the bone marrow cells. This is consistent with polyploidy. 4. The process of nuclear lobulation is not accomplished by the end of DNA synthesis, thus being comparable to nuclear segmentation in neutrophilic granulocytes. This latest phase of maturation is relatively long (approximately 25–30 hours) as compared to the phase during which recognizable megacaryocytic precursors are able to synthesize DNA (less than 15 hours).

LYMPHOCYTE PRODUCTION MEASURED BY EXTRACORPOREAL IRRADIATION, CANNULATION, AND LABELING TECHNIQUES*

The lymphatic system has, truly an ancient history that is ably reviewed in Yoffey and Courtice.’ Apparently lymphatics were first observed around 300 B. C. In the 1600s, mesenteric lymphatic vessels and the thoracic duct were described. The classical injection of lymphatic vessels throughout the body by the Hunter Brothers in the 1700s are common knowledge. In 1800, Ludwig commenced cannulation of different lymphatic vessels. In 1851, lymphocytes were first seen within the lymph by Virchow.2 In 1875, Fleming observed mitoses in the germinal centers of lymph nodes. In the late 1800s, Ernest Starling pointed out that lyrnphatic vessels serve as a mechanism by which protein lost from the blood vessels can be returned to the blood. He thus firmly established one major function of the lymphatic system. In this century, a vast literature has developed on the lyrnphopoietic system that is too unwieldly to review in this brief paper. In Yoffey and Courtice’s book on lymphoid tissue, a large mass of the current literature is analyzed. A variation in the concentration of lymphocytes in afferent and efferent lymph has been presented by E h r i ~ h . ~ Following prolonged antigenic stimulus, the output of lymphocytes in the efferent lymphatic ducts of lymph nodes was greatly increased. There is also an increase in the lymphocytes in the lymph while passing through a lymph node. Drinker and Yoffey4 point out that this can not be explained by reabsorption of water in the node because the protein content of peripheal and central lymph is identical in amount. In fact, “if the blood capillaries of the lymph nodes are capable of large scale absorption of protein, they are not only unique members of the blood capillary system, but at the same time display a miraculous ability to absorb proteins so equally as to make afferent and efferent lymph precisely equal in protein content.” Much valuable information had been obtained by cannulation techniques. However, it was with the development of radioactive labels of DNA to tag the new cell production that knowledge on the magnitude of cell production and lifespan of cells began to grow. Again the literature is too large to review in detail. However, the classical studies of O ~ g o o d , ~ Otteson,6 and Hamilton’ are pertinent. Hamilton, utilizing C14-labeled adenine and guanine precursors of DNA, extended Otteson’s ideas on the possibility of two types of lymphocytes ( 1 ) one cell with a long lifespan and (2) another cell with a short lifespan. Alternatively, he could explain his data on the basis that there may be significant reutilization of the labeled DNA or its fragments. Hamilton also first pondered the possible significance of DNA reutilization in perpetuating immune responses. Gowans et ~ l . , * . ~ in a series of precise studies, has pointed out by utilizing classical cannulation techniques, radioactive labeling, and autoradiography that there is a significant recirculation of lymphocytes from blood to the lymphocytic organs and back to the blood again. His group has also confirmed the earlier

INCORPORATION OF TRITIATED NUCLEOSIDES AND AMINO ACIDS INTO LYMPHOID AND PLASMOCYTOID CELLS DURING SECONDARY RESPONSE TO TETANUS TOXOID IN MICE

It has become evident in recent years that almost all antibody-containing cells formed within four days after secondary stimulation with soluble antigen arise from precursor cells that synthesize DNA during this period.’ The kinetics and exact localization of this proliferative activity are not clearly ascertained.2 Renewed interest has focused on germinal-center cells as possible precursors of plasma c e k 3 There is lack of agreement as to the life cycle of germinal centers, their possible dependence on antigenic stimulation, their role in antibody synthesis, and their function in plasmocytopoiesis and/or lymphopoiesis.‘ The present report is concerned with the incorporation of tritiated nucleosides and amino acids into lymphoid and plasmocytoid cells of a regional lymph node during the secondary response to tetanus toxoid. The findings will be correlated with changes in cellular composition and histological appearance of the node. Emphasis is placed on growth and behavior of germinal centers in relation to the secondary tetanus antitoxin response. The following basic experimental conditions were established in order to evaluate cellular and histological changes during the secondary response to the specific antigen given at a time remote from primary stimulation: Fluid tetanus toxoid (without adjuvant) was used as the test antigen since nonimmunized mice do not have detectable specific antitoxin. Antitoxin titers may be determined very precisely based on the capacity of the serum to neutralize active tetanus toxin.5 A six-month time-interval between primary and secondary antigenic stimulation was sufficient to allow the primary response to subside. The site of the second injection of antigen differed from the site of primary immunization so as to avert local remnants from the primary response usually detectable in the regional lymph node. Secondary stimulation was given via injection into the footpad because, in nonstimulated mice, the popliteal lymph nodes as the first regional lymphatic “stations” contain very few germinal centers and plasma cells. I .

Evaluation of mitotic time in vivo, using tritiated thymidine as a cell marker: Successive labeling with time of separate mitotic phases

Abstract Following a single injection of tritiated thymidine in dogs, a reproducible type of curve was obtained for the time sequence of the progression of labeled cells in separate mitotic phases, as observed on serial bone marrow aspirations. The duration of mitosis in erythroblasts at an advanced stage of maturation toward erythrocytes was shown to be of the order of 30 to 40 min for at least 50 per cent of the cells undergoing division at that stage. Mitotic duration did not appear to vary markedly for the remaining 50 per cent. The variability in the duration of the premitotic synthetic gap appeared more pronounced than the variability in the duration of mitosis. The incorporated tritiated thymidine did not of itself cause any appreciable change in these durations. The values obtained were shown not to be influenced by morphological criteria used to register cells in mitosis.

Studies on lymphocytes: V. Short in vivo DNA synthesis and generation time of lymphoid cells in the calf thoracic duct after simulated or effective extracorporeal irradiation of circulating blood☆

Abstract The proliferative pattern of lymphoid cells in the thoracic duct of the calf was studied by the use of tritiated thymidine and autoradiographic evaluation of lymph samples taken at frequent time intervals. The fact that the thoracic duct contains a considerable number of cells in mitosis makes it possible to define more precisely the time parameters of their generation cycle in one and the same animal. A combined analysis of the mitotic labeling index (MLI) and the mean grain count per labeled mitotic figure (MGC/LMF), both as a function of time after a single i.v. injection of 3H-thymidine, revealed an in vivo G2 period of 30 to 40 min, a DNA synthesis time of 3 1 2 hr and a generation time of 5 1 2 to 6 hr for the majority of cells. The duration of these separate phases of the cell cycle was not changed to any appreciable degree by 48 hr of extracorporeal irradiation of the circulating blood (ECIB) prior to the study of lymphoid cell proliferation.

Quantitative titration of anti-horseradish peroxidase antibody in mouse serum

Abstract Both precipitable and non-precipitable antibody horseradish peroxidase was measured quantitatively by an enzyme-binding test in which the enzymatic activity served as a marker of the antigen. Results obtained with this method are compared with those found with a conventional hemagglutination techniqe.

Specificity of tritiated thymidine as a precursor of DNA under conditions of prolonged administration.

Summary After 90 injections of thymidine-methyl-3H into adult mice at intervals of 8 hr, the radioactivities of several biochemical fractions (acid-soluble material, lipids, RNA, DNA, and protein) were determined in homogenates of various organs. A high specificity of incorporation of labeled thymidine into DNA was observed in tissues characterized by a high rate of cell proliferation, while this specificity was of a lower degree in tissues exhibiting a low rate of cell proliferation. With few exceptions, the amounts of radioactivity (per gram wet weight) in the fractions other than DNA were similar for all organs tested. Approximately 70% of label contained in the acid-soluble fraction were in tritiated water, whereas no free thymidine-3H could be detected 1 hr after the last injection of this precursor.

DISTRIBUTION OF TRANSFUSED TRITIATED CYTIDINE‐LABELED LEUKOCYTES AND RED CELLS IN THE BONE MARROW OF NORMAL AND IRRADIATED RATS*

In normal rats and after whole-body irradiation with 550-r x-rays, the fate in the bone marrow of labeled nucleated and red cells of transfused peripheral blood was observed autoradiographically. Labeled nucleated cells, most of which were lymphocyte-like cells, readily migrated into the marrow parenchyma in normal animals (2 cells/1000 parenchymal cells/hr). Following irradiation to at least 27 to 51 hr, this migration appeared to be relatively increased. Beyond this time, parenchymal areas were more difficult to be defined as such. Labeled red cells were rarely observed to enter parenchyma despite the presence of nonlabeled erythrocytes within the parenchymal structure after irradiation. The vascular bed following irradiation increased greatly as the parenchyma diminished to a minimum of less than 10% of control at 75 hr. Despite these changes in the architecture of the marrow, the average density of labeled cells per area of marrow corresponded to values expected on the assumption of a free-flowing circulation. The presently-used technique did not allow distinguishirg at all times between a free-flowing circulation through intact sinusoids or through areas in which the sinusoidal wall as such was destroyed. (auth)

Fate of Intraintestinal Thymocytes Labeled with 125lododeoxyuridine or Tritiated Thymidine

Summary A suspension of thymocytes labeled with 125IUdR or 3 HTdR was injected into the jejunum of mice. The bulk of the radioactivity disappeared within few hours from the intestine and was recovered principally in the urine. This indicated a very rapid breakdown of labeled thymic cells, reabsorption and subsequent elimination of the tracer in the kidney. In mice injected with cells labeled with 3 HTdR, the initial rapid loss of radioactivity was of shorter duration, and slower during the second phase, presumably due to more extensive reutilization and/or prolonged persistence of acid-soluble radioactivity. Pretreatment of the recipients with antibiotics did not significantly reduce the rate of radioactivity loss.

Stimulation of proliferation of murine lymphocytes by the calcium ionophore A 23187 in the absence of serum: The requirement for thiols

The proliferation of mouse spleen cells and T-lymphocytes, initiated by the calcium ionophore A 23187 was studied by a serum-free culture technique. In contrast to Con A, A 23187 was capable of stimulating cells only if 2-mercaptoethanol, cysteine and glutathione (reduced form), respectively, were present in the culture medium. In the absence of one of these compounds a stimulating activity of A 23187 was observed only with high concentrations of cells (i.e., 10(7)/ml). With glutathione present, the cells could be stimulated only at concentrations of A 23187 which were found to be suboptimal in cultures with 2-mercaptoethanol. Human serum, fetal calf serum and bovine serum albumin shifted the active and optimally stimulating concentrations of A 23187 to higher values. A similar effect was observed with sera- and Con A-treated cells. The effect of sera and albumin was paralleled by a protecting effect of cells against high concentrations of A 23187.