Leukocyte Labeling With 51Chromium. IV. The Kinetics of Chronic Lymphocytic Leukemic Lymphocytes

The kinetics of radiochromate-labeledautologous blood lymphocytes werestudied in ten cases of chronic lymphocytic leukemia (CLL). The labeledcells equilibrated with an enlarged recirculating lymphocyte pool (RLP) distributed between the blood, the spleen,and the bone marrow. The size of theRLP, estimated from dilution of thelabeled cells, was better correlatedwith the degree of splenomegaly andmarrow infiltration than was the bloodlymphocyte count. The marrow fraction of the RLP turned over moreslowly than the splenic pool. The half-life of the labeled cells in the RLPvaried in individual cases from one to11 days. Lymphocyte life span was notrelated either to the blood lymphocytecount or to the size of the RLP. Themean half-life of 3.8 days was significantly shorter than the normal lymphocyte half-life of 18 days. We conclude that CLL is characterized byoverproduction of a strain of abnormally short-lived lymphocytes.

Submitted on June 12, 1972 Revised on August 28, 1972 Accepted on August 30, 1972     

Leukocyte Labeling With 51Chromium. II. Leukocyte Kinetics in Chronic Myelocytic Leukemia

Sequential or simultaneous leukocytekinetic studies using radioactive diisopropylfluorophosphate and radiochromate (⁵¹Cr) yielded similar or identicalblood leukocyte disappearance curvesin seven patients with chronic myelocytic leukemia (CML). Body surface⁵¹Cr counting regularly showed a risein the spleen counting rate during thefirst hours after infusions of granulocytepopulations of mixed maturity. Epinephrine-induced leukocytosis was associated with a fall in the spleen countingrate, lesser decreases over the liver andmarrow, rises in the heart and lungcounting rates, and an unchanged bloodleukocyte disappearance curve. Thesechanges are consistent with the mobilization by epinephrine of a marginalgranulocyte pool (MGP), which is largely localized in the spleen and is in equilibrium with the circulating pool. Immature CML granulocyte fractions werecleared from the blood more rapidlythan mixed cell populations. The immature cells failed to equilibrate withthe splenic MGP, and instead accumulated in the marrow and later recirculated into the blood as mature cells.These findings indicate that the delayedand variably contoured blood granulocyte disappearance curves found inCML are composites resulting from therecirculation of immature granulocytesin the presence of an enlarged totalblood pool of mature cells.

Submitted on January 4, 1971 Revised on March 11, 1971 Accepted on March 26, 1971     

The Separation and 51Chromium Labeling of Human Lymphocytes With In Vivo Studies of Survival and Migration

This study looks at the application of⁵¹Cr labeling of lymphocytes as a methodof obtaining in vivo information aboutthe lymphocyte in human beings. Lymphocytes were separated from wholeblood by methods based on isopycnicand rate zonal centrifugation techniquesand the conditions for ⁵¹Cr uptake by theseparated lymphocytes standardized toenable a known amount of radioactivityto be injected into the subjects understudy. The uptake of the label into various sites in the body was studied by themeans of surface probes linked synchronously to a digital printout device and thesurvival in the circulation estimated byscintillation counting of blood samplestaken at various times after injection ofthe label. The in vivo studies of survivaland migration in 10 normal subjects showan initial rapid clearance of cells fromthe circulation associated with an uptakeof cells into spleen and liver sites, andto a lesser extent, into sites over bonemarrow and the abdomen. Survival ofthe circulating lymphocytes after thisperiod appears to be relatively short,with a half-life of 1.7 days. As the available evidence suggests, this short lifemay be due to the differential trappingof short-lived lymphocytes in the circulation at the expense of the long-livedlymphocytes. Kinetic interpretations ofthe data indicate an inverse exponentialuptake of cells into the sites studied, andthe decline over the organs appears tofollow the death rate of the cells in thebody as a whole. Comparisons with studies in patients having chronic lymphaticleukemia show a relative inability ofleukemic lymphocytes to leave the circulation and enter some sites in the body.These preliminary studies indicate thepotential of ⁵¹Cr labeling as a usefulclinical research tool in the study oflymphocytes in human beings.

Submitted on November 30, 1970 Revised on March 29, 1971 Accepted on April 20, 1971     

Leukocyte Kinetics in Hematologic Disorders Studied by DNA-Phosphorus Labeling

Leukocyte physiology in the normal and in hematopoietic disease statesin humans has been studied by DNA-P labeling with inorganic P³². In thenormal there is a post-mitotic granulocyte reservoir in marrow about 17times the size of the intravascular compartment. Progress through this reservoir is orderly and requires about 6 days. Some 1-2 x 10¹¹ cells dailyare released from it into the blood. In polycythemia vera there is an increasedproduction of granulocytes. DNA-P labeling in patients with chronic lymphocytic leukemia occurs at a very low level and is compatible with a veryslow rate of cell renewal. In one patient with chronic lymphocytic leukemia,no disturbances in kinetics caused by a dose of 20 µc. of P³²/Kg. weredetected during the time of the study. Although a progressively rising concentration of label in circulating leukocyte DNA was found in patients withchronic granulocytic leukemia, without the lag suggesting a distinct marrow phase, it is concluded that the blood and extravascular leukopoietic compartments cannot be a single compartment. An essentially normal curve isobtained after induction of a complete remission with busulfan.

Submitted on October 1, 1963 Accepted on December 3, 1963     

Leukocyte Kinetics Studied by Nitrogen Mustard Induced Alterations in DNA Phosphorus Labeling

The effect of a large dose of nitrogen mustard on total and differentialleukocyte counts in rabbits has been studied in conjunction with the effecton DNA labeling with inorganic radiophosphorus. Considered together, thefindings indicate that this dose of nitrogen mustard has a direct lethal effecton lymphocytes and effectively blocks P³² incorporation into leukocyte DNAfor several days by killing the elements capable of division. The post-mitoticgranulocyte reservoir seems to remain intact and is of sufficient size to replacethose lost from the circulation for about 56 hours. Granulocyte circulatinghalf-time under the conditions of these experiments falls between two andseven hours. The post-mitotic granulocyte reservoir is calculated to be 40 timesthe size of the circulating pool. During the period of rapid granulocyte recovery, leukocytes have been shown to exhibit a decreasing concentration ofDNA label per element and maintainence of the same level of label perelement depending on the availability of precursor P³². At present no conclusion about DNA labeling characteristics with inorganic P³² under steadystate conditions is warranted.

Submitted on January 22, 1962 Accepted on March 23, 1962     

Kinetics of Small Lymphocytes in Normal and Nude Mice after Splenectomy

Autoradiography and various quantitations on lymphoid tissues have been used to evaluate the kinetics of small lymphocytes in normal (+/nu or +/+) and congenitally athymic nude (nu/nu) NMRI mice 1 month after splenectomy or sham-splenectomy. The results indicate that splenectomy causes depressed thymic activity and diminished numbers of T lymphocytes in peripheral lymphoid tissues. The total number of cells in these tissues as well as the blast cell activity, were within normal limits. Bone marrow lymphocyte numbers and kinetics as well as blood lymphocyte levels in splenectomized and sham-splenectomized normal animals were comparable. Blood lymphocyte numbers were at normal levels in splenectomized nude mice, in spite of reduced numbers of bone marrow and thoracic duct lymphocytes. It is suggested that increased number of newly-formed lymphocytes, found in lymph nodes and blood of splenectomized mice, are released from the lympho-myeloid organs in compensation for the loss of long-lived, thymus-derived cells.     

Studies on the Fate of Lymphocytes. I. Labeling Small Thymic Lymphocytes with Tritiated Thymidine

A procedure is described whereby 0.5 ml. suspensions containing 3-5 x 10⁸small lymphocytes per ml., nearly 50 per cent of which were labeled with H³thymidine, have been prepared from rat thymus. Repeated doses of thymidinetotaling 7-8 µc/Gm. and from two to three days were required to producethis percentage, and it is suggested that longer times would not be muchmore effective. Preliminary results indicate that cells of such suspensions injected in irradiated hosts are widely distributed, do not appear to disintegraterapidly or to be massively ingested by the macrophages of the reticuloendothelial system, and are still present in good condition one and two daysafter injection.

Submitted on June 29, 1961 Accepted on August 16, 1961     

51Cr Labelling of Normal Human T and B Lymphocytes for Kinetic Studies in Vivo

Lymphocyte traffic between blood and tissues was assessed by ⁵¹Cr labelling of lymphocytes and subsequent autologous reinfusion in 10 normal elderly persons. The technique for isolation and platelet depletion of lymphocyte suspensions is described. By the labelling procedure used about 70 μCi ⁵¹Cr may be incubated in about 100 million lymphocytes. This permits measurement of lymphocyte-bound radioactivity on the T and B fractions separately. The blood disappearance curves for labelled lymphocytes indicate the existence of exchangeable pools in the tissues of T as well as of B lymphocytes, that of the T lymphocytes being apparently larger. A characteristic finding in the blood disappearance curves for total lymphocytes is an increase in lymphocyte-bound radioactivity in the blood 4–6 h after reinfusion, designated reappearance. The disappearance curve of the B lymphocytes shows reappearance 4–10 h after reinfusion, whereas that of the T lymphocytes falls exponentially without any recordable reappearance. On the basis of the disappearance curves and a knowledge of the topographic distribution of T and B lymphocytes in the lymphoid tissues, a model of T and B lymphocyte traffic in the lymph nodes is discussed. This model operates with T and B lymphocyte passage by way of postcapillary venules and describes the migration in and around the germinal centres. The T lymphocytes in the periphery of the germinal centres are assumed to derive mainly from the afferent lymph, whereas the B lymphocytes in the centres are exchanged with lymphocytes in the blood in an exchangeable pool. The functional implications are discussed.     

The Effect of Endotoxin on Circulating Lymphocytes in Normal Man

S ummary . The effect of endotoxin administration on peripheral blood lymphocytes was studied in normal man. A partially purified bacterial endotoxin from Pseudomonas aeruginosa was given intravenously to 10 healthy volunteers. Endotoxin administration resulted in a substantial lymphocytopenia with reduction of both thymus-dependent T and bone marrow-derived B lymphocytes. Absolute numbers of circulating B lymphocytes decreased to a greater extent than T lymphocytes. T-lymphocyte functional characteristics, including mitogenic response to phyto-haernagglutinin and responder cell activity in mixed lymphocyte culture, were unaffected by endotoxin administration. Response to concanavalin A was consistently reduced. Stimulation of allogeneic lymphocytes in mixed lymphocyte culture, a function of B lymphocytes and monocytes, was also decreased. When endotoxin was used as an in vitro mitogen, a significant increase in DNA synthesis was observed only in lymphocytes from individuals previously given endotoxin and not in normal control lymphocytes. These studies indicate that experimental endotoxaemia is associated with a reduction in absolute numbers of circulating T and B lymphocytes, a decrease in B-lymphocyte bur not T-lymphocyte functional activity, and redistribution of a concanavalin A-responsive subpopulation of T lymphocytes. The data suggest that endotoxin has important effects on lymphocyte distribution and function in man.     

Respiration and Glycolysis of Normal Human Lymphocytes

The metabolism of intact, normal, human lymphocytes in vitro was studiedfrom a total of 80 subjects. Corrected for the metabolism of contaminating redblood cells, the glucose uptake, lactic acid production, and oxygen consumption were 62, 95, and 117 µmoles per 10¹⁰ lymphocytes per hour, respectively,provided the cells were incubated at concentrations greater than 40 x 10⁶lymphocytes per ml. At lower lymphocyte concentrations the oxygen consumption per lymphocyte rose steeply with decreasing cell concentration (crowdingeffect). A similar but weaker crowding effect was noted for the lactic acidproduction, but not for the utilization of glucose.

The oxygen uptake was lower with 20 per cent than with 100 per centoxygen as gas phase. Small Pasteur and Crabtree effects were demonstrated.The oxygen consumption and lactic acid production proceeded linear withtime, while the glucose utilization was higher during the first 30 minutes ofincubation than later on.

It is concluded that lymphocytes have a low aerobic glycolysis accountingfor 75 per cent of the glucose utilization. The respiration is severely inhibitedat high cell concentrations and it is suggested that this is caused by an insufficient availability of oxygen to the cells.

Submitted on September 24, 1965 Accepted on November 24, 1965     

Chromium51 Uptake as A Function of Platelet Age

This investigation was supported by the U. S. Atomic Energy Commission Contract AT (30–1) 3590. It is known that chromate uptake by platelets is mediated by a bimodal transport mechanism with a diffusional and a saturable transport component. These models of uptake were studied in rat platelet cohorts of different age, obtained during the recovery phase of thrombocytopenia induced by specific hetero-antibody. At low concentrations of chromate in the medium the saturable transport mechanism prevailed and was found to be markedly dependent on platelet age. Platelets one day or less in age accumulated more than twice the amount of randomly-aged platelets. At high concentrations of chromate at which diffusional transport predominates, no difference in total chromate accumulation was noted in these two platelet populations. The inhomogeneous distribution of ⁵¹Cr in a platelet population may have important bearing on the interpretation of platelet survival curves since by the generally employed labelling method, chromate concentrations are utilized well within the range in which saturable transport prevails and younger platelets are more heavily labelled than older ones.