Technical Aspects of the Rosette Technique for Detecting Human Circulating B and T Lymphocytes Normal Values and Some Remarks on Null Lymphocytes

The effect of variations in several single steps in the rosette technique for detecting human circulating B, T and O cells is investigated in order to describe the optimal production of B and T rosettes. Variations in pH, duration of incubation, temperature, EAC cell production, age of EAC cells and definition of a rosette are investigated. Using the optimal rosette technique, normal values for 15 healthy men and 15 healthy women (age range 18–65 years) are found to be: B lymphocytes 27.5 % or 477/μ1, T lymphocytes 60.6 % or 1049/μl, O lymphocytes 11.9 % or 206/μl. It is concluded that variations in the analytical procedure may greatly affect the outcome. The existence of O cells is briefly discussed.     

The Effect of Total Body Irradiation on the Labeling of Circulating Mouse Lymphocytes with Tritiated Thymidine

Tritiated thymidine was administered to C3H mice at frequent intervals byintraperitoneal injection over a 2-week period. About one-third of the circulating small lymphocytes were labeled by this procedure. The animals werethen exposed to 208 rads of external irradiation which resulted in a fivefolddrop in the total circulating lymphocyte count. The labeled newly formedlymphocytes and the unlabeled older lymphocytes proved to be equally sensitive to radiation. The rate of labeling of peripheral blood lymphocytes wasunaffected by a single dose of radiation or by a subsequent dose delivered 4days later. The decline in the curve of labeling, following the discontinuationof labeling with tritiated thymidine, was the same for irradiated and unirradiated mice. These experiments suggest that the output of new lymphocytesis directly related to the level of circulating small lymphocytes and may possibly depend on transformation of small lymphocytes to large proliferatingcells.

Submitted on June 30, 1964 Accepted on September 20, 1964     

The Effect of Methotrexate on Transformation and Mitosis of Normal Human Blood Lymphocytes In Vitro

The effect of methotrexate (MTX) on transformation and mitosis of normal lymphocyte cultures was investigated. Mitosiswas 1000-fold more sensitive to MTX thanwas transformation; mitosis could be prevented without any observable effect ontransformation. The antimitotic effect ofMTX depended on its concentration, duration of contact with lymphocytes, as wellas on the developmental stage of thelymphocytes at the time of exposure. Incorporation of labeled precursors into protein, DNA, and RNA proceeded at normalrates unaffected by the presence of MTX.Transformation was partially inhibited byhigh concentrations of MTX (above 75µg/ml). Folinic acid abolished the antimitotic effect of MTX, with the number ofcells undergoing division directly proportional to the quantity of folinic acid added.

Submitted on May 11, 1973 Revised on August 22, 1973 Accepted on August 30, 1973     

Observations on Cryopreservation of Lymphocytes in Chronic Lymphocytic Leukaemia and Normal Human Lymphocytes

A set of conditions is defined for freeze-storage of lymphocytes circulating in chronic lymphocytic leukaemia (CLL-lymphocytes) and in healthy individuals (normal lymphocytes). Isolated cells are frozen in culture medium containing 15 % serum and 8.3 % (v/v) dimethyl sulphoxide (DMSO) at a post-freezing cooling rate in the range 1.8-–3.4° per min to ?40° or below followed by immersion in liquid nitrogen, and thawed rapidly (ca. 400° per min) at 37°. This procedure induced consistently low mortalities (2–7%) in normal and CLL populations, uninfluenced by freeze-storage lasting up to 57 weeks, and was accompanied by large-scale destruction (67–100%, average 95 %) of residual isolated red cells. The lymphocytes classified as surviving cells by cytologic analysis could display partial loss of initial capacity for respiration but capacity for survival in short-term culture was retained. Brief reference is made to unchanged ultrastructural appearance of surviving cells. No pronounced differences were observed in the capacity of normal populations to transform under the influence of phytohaemagglutinin (PHA) as a result of freezing and thawing, but tests for altered proliferative capacity of transformed cells were hampered by culture conditions proving sub-optimal. Ultrasensitivity of CLL-lymphocytes in vitro to cytocidal action of colchicine — a property currently utilized for measurement of abnormal cell content of CLL-lymphocyte populations — was closely reproduced following freezing and thawing in experiments with 9 patients.     

The Proliferative States of Circulating Granulopoietic Stem Cells in Man

The fraction of granulocyte-macrophage colony-forming cells (CFC) in DNA synthetic phase in blood from 25 normal adults and those in blood and bone marrow from 8 haematologically normal subjects were evaluated by in vitro culture of cells with and without prior exposure to ³H-thymidine (12.5 μCi) for 1 h at 37° C. The exposure of blood cells from normal adults to ³H-thymidine resulted in 26 ± 10 % reduction in colony formation and in 14 ± 10 % reduction in cluster formation. There was no difference in the magnitude of reduction in colony formation following exposure to ³H-thymidine by cells in blood and those in bone marrow in 6 of the 8 haematologically normal subjects. These findings indicated that about one fourth of the circulating CFC in normal adults are in proliferative state and that significant difference in proliferative states beween CFC in blood and those in bone marrow probably does not exist in the majority of haematologically normal subjects.     

Studies on Adrenaline-induced Leucocytosis in Normal Man

S ummary . Adrenaline-induced peripheral blood leucocytosis has been studied in three healthy normal and three healthy splenectomized males. An indwelling venous cannula allowed very frequent samples to be obtained before and after intramuscular injection of 1 mg adrenaline. It is confirmed that within the first 30 min there is a predominant lymphocytosis. Thereafter there is a variable neutrophilia with relative lymphopenia. Evidence was found for more than one peak in the early lymphocytosis. We have failed to find the mechanisms which are involved in leucocyte mobilization. Contraction of the spleen appears to play no significant part. In two experiments, sampling of blood simultaneously from the left innominate and right brachial veins yielded no evidence for a major additional influx of lymphocytes into the circulation via the thoracic duct in response to adrenaline.     

Erythropoietin Excretion in Normal Man

Levels of erythropoietin have been consistently demonstrated in urine collections from healthy adult males using a modification of the polycythemic mouseassay. This method is stable and reproducible, and it provides assay animalswith low background erythropoiesis. Six hour collections of urine can be concentrated without apparent loss of activity and the entire daily excretion canbe used in the assay. Quantitation has been achieved by comparing theresponse to a concentrate of normal urine directly to a linear dose/responsecurve of erythropoietin standard B. Based on 6 hour urine collections, erythropoietin excretion in the subjects tested averaged 4.0 units of erythropoietinstandard B (range 1.2-9.5). In addition to wide unexplained variations inresults seen from day to day, the data indicate that a diurnal pattern ofexcretion of the hormone may exist.

Submitted on October 26, 1965 Accepted on January 1, 1966     

Surface Immunoglobulins of Circulating Lymphocytes in Mouse Plasmacytoma. II. The Influence of Plasmacytoma RNA on Surface Immunoglobulins of Lymphocytes

Circulating lymphocytes of plasmacytoma-carrying BALB/c mice werefound to lose their normal surface immunoglobulins; in their place surfacestructures characteristic of the specific plasmacytoma globulin were demonstrated by the immunocytoadhesiontechnique. These changes were experimentally reproduced by the incubation of normal BALB/c lymphocyteswith an RNA preparation obtained byhot phenol extraction from the excisedplasmacytomas. RNA treated byRNAse was inactive, while DNAse ortrypsin had no inactivating effect.Lymphocytes, killed with heat or KCN,underwent no alteration of surfacereceptors following incubation with thetumor RNA. Plasmacytoma RNA, injected intraperitoneally into normalmice, also altered the reactivity of circulating lymphocytes. These observations suggest the possibility that thiseffect contributes to the functionalimpairment of the immune system inthis disease.

Submitted on August 16, 1971 Accepted on October 18, 1971     

Comparison of Hematologic and Febrile Response to Endotoxin in Man

The infusion of endotoxin in man is followed by a fall in mononuclear cellcount and subsequent granulocytosis. Significant granulocytopenia was notobserved, nor were changes in plasma fibrinogen levels or platelet countsnoted. Although a correlation was demonstrated between dose of endotoxinadministered and hematologic response, there was no correlation between themagnitude of fever and hematologic changes. Hematologic responsivenesswas unchanged following the development of febrile tolerance to endotoxin.

Submitted on September 22, 1964 Accepted on November 11, 1964     

The Effect of Antigens on DNA-Synthesis in Cultured Lymphocytes

Lymphocytes were isolated from the peripheral blood of healthy blood donors and stimulated in vitro to DNA-synthesis by the addition of antigens. The addition of two or four antigens to the same culture of lymphocytic cells did not increase the synthesis of DNA above the level induced by a single antigen. Controls with phytohaemagglutinin showed that the in vitro conditions did not represent limiting factors for the synthesis of DNA. It appears that the response of isolated peripheral blood lymphocytes to one antigen is maximal and that further stimulation with more antigens can not produce the additive effect expected if there were separate clones of lymphocytes for each antigen.     

Analytical Review: The Kinetics of Granulopoiesis in Normal Man

Present knowledge concerning the kinetics of granulopoiesis has been reviewed and quantitative data concerning granulokinetics in normal humansubjects are presented.

A. When granulocytes are labeled in vitro and returned to the circulationof 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 compartmentswhich are in equilibrium with each other. These pools have been designatedthe 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 weightwere as follows: TBGP, 70; CGP, 31; and MGP, 39. The mean ratio of the CGPto the TBGP was 0.44.

2. The labeled granulocytes leave the TBGP in an exponential fashion witha mean half-time disappearance (T) of 6.7 hours as determined in 56 normalmale subjects. No evidence has been obtained for a return of granulocytes tothe blood.

3. The mean value for the granulocyte turnover rate (GTR) in 56 normalmale 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 theTBGP is 10.4 hours.

B. When granulocytes are labeled in vivo by the intravenous administrationof DFP³², the rate of disappearance of granulocytes from the circulation andthe time required for myelocytes to divide, mature and appear in the bloodcan be measured. In addition, the generation time of myelocytes can be approximated. From the time parameters and the GTR, the bone marrow poolsizes 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 (T) of in vivo labeled granulocytesfrom the circulation was 7.2 hours. This value agrees well with the valueof 6.7 hours obtained after the in vitro labeling of granulocytes.

2. The mean time required for myelocytes to divide, mature and appear inthe blood was 11.4 days.

3. The mean generation time of myelocytes was estimated to be not morethan 2.9 days.

4. The total granulocyte pool in the bone marrow (neutrophilic myelocytes,neutrophilic metamyelocytes and PMN neutrophils) was calculated to be186 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 turnovertime of 2.5 days; the metamyelocyte pool consisted of about 76 x 10⁸ cells perKg. with a turnover time of 4.7 days; the average size of the mature marrowPMN 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 DFP³²label, is presented. In this model, myelocytes are depicted as approaching aself-perpetuating population of cells. Some cells enter this population frompopulations which are less mature but this latter source of cells is small underconditions of normal steady state kinetics. One of the daughter cells of amyelocyte division remains in the myelocyte population to divide again. Theother daughter cell enters the metamyelocyte population. The metamyelocyteand PMN neutrophil population is incapable of division and cells move throughthis population in sequential fashion in the process of maturation. The cellsthen enter the blood where they equilibrate rapidly between the two bloodcompartments. The cells are removed from the total granulocyte pool in arandom fashion. There is no appreciable pool of granulocytes in the extramedullary tissues of normal subjects and granulocytes do not return from thetissues to the blood. The entire movement of granulocytes from marrow totissues is uni-directional.

Submitted on March 16, 1964 Accepted on April 9, 1964     

PHA Responsiveness and Subpopulations of Circulating Lymphocytes in Pernicious Anemia

Lymphocyte transformation responses tothe mitogen phytohemagglutinin (PHA)were measured in 20 patients with provenpernicious anemia (PA) and 20 matchedcontrols using ³H-thymidine label. The patients with PA showed significant depression of lymphocyte transformation to thethree doses of PHA employed, as judgedby beta counting; however, radioautographic examination of PHA-stimulatedcells indicated that the results were due toa failure of intranuclear incorporation of³H-thymidine by PA lymphocytes, ratherthan a failure of PHA to induce blastogenesis. The percentages and numbers ofT and B lymphocytes in peripheral bloodwere measured in 30 patients and controlsby rosette and immunofluorescence techniques, respectively. There was no significant difference in the B cell subpopulations between patients and controls; theT cell subpopulation was slightly lower inthe PA patients (mean 62.4%) than in thecontrols (mean 65.5%), but the differencewas not statistically significant. The depressed uptake of ³H-thymidine by stimulated lymphocytes in PA would seem toreflect a chemical defect rather thaninherent immunologic abnormality.

Submitted on November 13, 1973 Accepted on June 12, 1974     

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     

Hypoxanthine-Guanine Phosphoribosyltransferase Activity in Normal and Leukaemic Lymphocytes

S ummary . The levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were determined in lymphocytes from normal people and patients with chronic lymphocytic leukaemia (CLL). The HGPRT level in the total lymphocyte population from patients with CLL was lower than that from normal subjects. The HGPRT activity was higher in normal non-T cells than normal T cells. The enzyme activity in CLL B cells was lower than in CLL T cells. The HGPRT level was higher in CLL T cells than in normal T cells; these data suggest CLL T cells differ biochemically from their normal counterparts.     

The Effect of Mucaine on Gastrin Release in Man

Summary: The effect of Mucaine and Aludrox on basal and food stimulated immunoreactive gastrin has been assessed in normal control subjects and patients with duodenal or gastric ulcer. No differences in gastrin responses were observed either in the basal period or after the protein meal with the two antacids. As previously described, release of gastrin was greatest in gastric ulcer patients but in contrast to previous results, normal subjects seemed to show a greater response than duodenal ulcer patients but this was not statistically significant. Thus the combination of a local anaesthetic oxethazaine with aluminium hydroxide gel does not lead to diminished gastrin re/ease and is not the prime mechanism of action of this agent.     

The Effect of Mucaine on Gastrin Release in Man

Summary: The effect of Mucaine and Aludrox on basal and food stimulated immunoreactive gastrin has been assessed in normal control subjects and patients with duodenal or gastric ulcer. No differences in gastrin responses were observed either in the basal period or after the protein meal with the two antacids. As previously described, release of gastrin was greatest in gastric ulcer patients but in contrast to previous results, normal subjects seemed to show a greater response than duodenal ulcer patients but this was not statistically significant. Thus the combination of a local anaesthetic oxethazaine with aluminium hydroxide gel does not lead to diminished gastrin re/ease and is not the prime mechanism of action of this agent.