ALBERT

Description: 1. A method has been presented by which granulocytes can be labeled in vivo with diisopropylfluorophosphate containing radioactive phosphorus. The leukocytes are isolated from blood by dextran sedimentation of erythrocytes and are then treated with gramicidin and lysolecithin to remove remaining red cells. Platelets are removed by differential centrifugation. The isolated leukocytes are placed between two squares of scintillating plastic and counted with a scintillation counter. 2. Leukocytes essentially free of erythrocytes and platelets can be obtained by the method outlined. The efficiency of the plastic scintillation counting method for radioactive phosphorus is about 74 per cent and leukocyte samples obtained from 20 ml. samples of normal blood can be counted with a reproducibility of ±10 per cent. 3. The administration of 2 mg. of diisopropylfluorophosphate either intramuscularly or intravenously is without significant toxic side effects. 4. No evidence has been obtained that the label damages the leukocytes. 5. No evidence has been obtained that the label elutes from leukocytes under the conditions of these studies. 6. Diisopropylfluorophosphate labels granulocytes for a brief period of time following injection. The label is not reutilized after death of the cells.

Description: In these studies, the DF32P leukokinetic technique has been modified so that neutrophil kinetics may be studied in the nonsteady state. In order to achieve this modification, an acid citrate solution was used in preparing leukocytes for radioactive assay. This results in a suspension free of cell agglutination so that a specific activity related only to neutrophils is obtained. This technique has been successfully employed in the study of the early phase of cortisol-induced granulocytosis, allowing measurement of cell flow rate in and out of the circulating granulocyte pool. This method is also useful in the study of subjects with pronounced neutropenia.

Description: Present knowledge concerning the kinetics of granulopoiesis has been reviewed and quantitative data concerning granulokinetics in normal human subjects are presented. A. When granulocytes are labeled in vitro and returned to the circulation of the donor, the distribution of the cells in the circulation and the rate of disappearance of the cells from the circulation can be measured. 1. The total blood granulocyte pool (TBGP) consists of two compartments which are in equilibrium with each other. These pools have been designated the circulating granulocyte pool (CGP) and the marginal granulocyte pool (MGP). The size of the pools has been measured in 109 normal male subjects. The mean values, expressed as numbers of cells x 107 per Kg. of body weight were as follows: TBGP, 70; CGP, 31; and MGP, 39. The mean ratio of the CGP to the TBGP was 0.44. 2. The labeled granulocytes leave the TBGP in an exponential fashion with a mean half-time disappearance (T½) of 6.7 hours as determined in 56 normal male subjects. No evidence has been obtained for a return of granulocytes to the blood. 3. The mean value for the granulocyte turnover rate (GTR) in 56 normal male subjects was 163 x 107 granulocytes per Kg. of body weight per day. Thus, the TBGP turns over 2.3 times per day and the turnover time for the TBGP is 10.4 hours. B. When granulocytes are labeled in vivo by the intravenous administration of DFP32, the rate of disappearance of granulocytes from the circulation and the time required for myelocytes to divide, mature and appear in the blood can be measured. In addition, the generation time of myelocytes can be approximated. From the time parameters and the GTR, the bone marrow pool sizes and turnover times can be calculated. These determinations and calculations have been made for a group of 21 normal male subjects. 1. The mean half-time disappearance (T½) of in vivo labeled granulocytes from the circulation was 7.2 hours. This value agrees well with the value of 6.7 hours obtained after the in vitro labeling of granulocytes. 2. The mean time required for myelocytes to divide, mature and appear in the blood was 11.4 days. 3. The mean generation time of myelocytes was estimated to be not more than 2.9 days. 4. The total granulocyte pool in the bone marrow (neutrophilic myelocytes, neutrophilic metamyelocytes and PMN neutrophils) was calculated to be 186 x 108 cells per Kg. of body weight with a mean turnover time of 11.4 days. The myelocyte pool was estimated to be 41 x 108 cells per Kg. with a turnover time of 2.5 days; the metamyelocyte pool consisted of about 76 x 108 cells per Kg. with a turnover time of 4.7 days; the average size of the mature marrow PMN neutrophil pool was 69 x 108 cells per Kg. of body weight with a turnover time of 4.2 days. C. A kinetic model for granulopoiesis, based on the studies with the DFP32 label, is presented. In this model, myelocytes are depicted as approaching a self-perpetuating population of cells. Some cells enter this population from populations which are less mature but this latter source of cells is small under conditions of normal steady state kinetics. One of the daughter cells of a myelocyte division remains in the myelocyte population to divide again. The other daughter cell enters the metamyelocyte population. The metamyelocyte and PMN neutrophil population is incapable of division and cells move through this population in sequential fashion in the process of maturation. The cells then enter the blood where they equilibrate rapidly between the two blood compartments. The cells are removed from the total granulocyte pool in a random fashion. There is no appreciable pool of granulocytes in the extramedullary tissues of normal subjects and granulocytes do not return from the tissues to the blood. The entire movement of granulocytes from marrow to tissues is uni-directional.

Description: The effect of a single injection of vinblastine sulfate was studied in 50 mongrel dogs. Nine of 34 dogs given 0.2 mg./Kg. of VLB died with gastrointestinal toxicity and the mortality rate increased as the dosage of VLB was increased. The morphologic pattern of leukocyte suppression and recovery in the bone marrow and blood was studied in detail in surviving animals. The cells of the bone marrow were markedly affected by VLB. Within 4 hours there was an increase in the number of cells in metaphase and, by day 1, virtually all proliferating leukocytes and erythrocytes had disappeared. An orderly repopulation of the bone marrow followed. The neutrophils, eosinophils, lymphocytes and monocytes of the blood were all markedly altered in concentration after VLB. Each type of cell first decreased to abnormally small numbers and then increased to abnormally large numbers in the blood. The curve of disappearance from and reappearance in the blood differed for each cell type. The changes in blood neutrophil number and morphology were correlated with changes in the blood neutrophil precursor cells of the marrow. The following conclusions were reached concerning the neutrophils and the assumptions implicit to these conclusions were detailed. 1. In the dog, the marrow contains enough post-mitotic granulocytes to replace those lost from the blood for at least 3 to 4 days. 2. The release of mature neutrophils from the bone marrow is a function of the rate at which blood neutrophils are lost and proceeds normally even when the marrow granulocyte reserve is partially depleted.

Description: These studies were designed to determine whether colony stimulating factor (CSF) and diffusible granulocytopoietic stimulator (DGS) represented a single substance which stimulates the production of granulocytes in vitro and in vivo. The effect of endogenous CSF upon DGS activity was studied after injection of 40 µg endotoxin. This resuited in both DGS activity and high serum CSF levels, but the time of appearance and duration of the two activities followed different patterns. In particular, CSF peaked and was declining 24 hr following endotoxin at which time DGS was not yet detectable. Then, as serum CSF fell, the DGS activity rose. These data support the concept that the DGS activity detectable with short term Millipore chamber cultures is not due to CSF and suggests the effects of CSF in vitro and DGS in vivo are due to different factors.

Description: Since lithium causes granulocytosis in some patients, its effect upon granulocyte production was investigated using mouse marrow in the agar culture system. When lithium was added to semisolid cultures of mouse marrow, there was no stimulation of colony formation in the absence of colony-stimulating activity (CSA). In addition, lithium did not potentiate the action of already formed CSA. However, lithium did stimulate the production of CSA by lung tissue. Lithium enhancement of CSA production was blocked by puromycin, indicating that lithium action required active new protein synthesis. It was concluded that lithium promoted enhanced granulocyte production in vitro by stimulating the synthesis of CSA.

Description: A technic for the in vitro labeling of human platelets with DFP32 is presented, critically evaluated, and compared to in vivo methods employing DFP32 and to in vitro methods using Cr51. The initial recovery of platelets labeled in vitro with DFP32 averaged 79 per cent, but the survival curve was characterized by an irreversible initial loss of platelet radioactivity. Experiments in which platelets were simultaneously labeled in vitro with both DFP32 and Cr51 suggest that this is not due to elution of DFP32. The survival curve of platelets labeled in vivo with DFP32 shows an initial transient reduction in platelet radioactivity. It is suggested that both of these aberrations in initial survival are the result of platelet injury by DFP32. Significant "tailing" was observed in the survival curves obtained with DFP32, and possible explanations of this phenomenon are discussed. DFP32-labeled platelets circulating after 5 hours apparently survive normally and disappear from the circulation as a rectilinear function over the next 6-8 days. Although both in vitro and in vivo labeling methods employing DFP32 provide a meaningful approximation of platelet lifespan, the initial and terminal aberrations of the survival curves greatly complicate further interpretation. Dextran had no detectable effect on platelet survival, and epinephrine, Mecholyl, and cutaneous vasodilatation did not alter the platelet count or the specific activity of circulating labeling platelets in human subjects. The problem of initial platelet survival and the question of an extravascular or marginal platelet pool is discussed in the light of these data.

Description: Since lithium causes granulocytosis in some patients, its effect upon granulocyte production was investigated using mouse marrow in the agar culture system. When lithium was added to semisolid cultures of mouse marrow, there was no stimulation of colony formation in the absence of colony-stimulating activity (CSA). In addition, lithium did not potentiate the action of already formed CSA. However, lithium did stimulate the production of CSA by lung tissue. Lithium enhancement of CSA production was blocked by puromycin, indicating that lithium action required active new protein synthesis. It was concluded that lithium promoted enhanced granulocyte production in vitro by stimulating the synthesis of CSA.

Description: To test the hypothesis that there is normally a 14-21 day cycle in blood neutrophil concentration, the blood of 13 normal male volunteers was sampled daily for 42 consecutive days. No evidence for cyclic variation in neutrophil concentration was found. The effect of deleting four samples per week and interpolating values linearly or parabolically was tested by power spectrum analysis, since this type of analysis was used in the studies in which cycling was found. The interpolation of missing values was found to introduce apparent cycling, and the results of the analysis of daily neutrophil counts was no different than that obtained with a series of random numbers.