Comparison of lymphocyte subpopulations in arterial and venous blood of the rat

Lymphocyte subpopulation as well as other hematological properties were compared between arterial and venous bloods of Wistar-Imamichi rats. Lymphocyte subsets were defined with four monoclonal antibodies which were specific to the respective cell surface glycoproteins. Using these monoclonal antibodies, subsets of B lymphocytes, T lymphocytes, helper T lymphocytes, and suppressor and cytotoxic T lymphocytes in the peripheral lymphocytes were identified. The blood samples were taken from aorta abdominalis and venae cava caudalis. The population of these subsets were enumerated by a laser flow-cytometry system. The result showed that there was no significant difference in hematological properties between the arterial and venous blood except in leukocyte count and hemoglobin concentration. The difference in leukocyte counts was thought to depend mainly on the fluctuation of the lymphocyte counts. However, no significant difference was recognized in the proportion of positive cells to each monoclonal antibody. It was concluded that the difference in leukocyte counts found between the arterial and venous bloods of the Wistar-Imamichi rat did not produce any effects on the proportion of the subpopulation in the peripheral lymphocytes, and the lymphocyte subpopulations in both arterial and venous bloods were substantially equivalent to each other.     

Effect of music-assisted imagery on neutrophils and lymphocytes

The purpose of this study was to determine the effects of cell-specific mental imagery on neutrophil and lymphocyte cell counts. Subjects (N = 30) were randomly assigned to one of two experimental groups that underwent a 6-week training program focusing on images of morphology, location, and movement of either neutrophils or lymphocytes. Music was used to enhance the imagery of the subjects. Peripheral white blood cell and differential counts were determined before and after the final 20-minute imagery session. Results indicated that neutrophils decreased significantly (p less than .04) in the neutrophil-change group while lymphocytes did not. The reverse occurred in the lymphocyte-change group, with only the lymphocytes decreasing significantly (p less than .03). The authors concluded that under the conditions of the present study, cell-specific imagery was associated with decreases in peripheral blood cell counts of lymphocytes and neutrophils.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA, the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively. The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Effect of Beta-Adrenergic Stimulation on the Circulatory Lymphocyte Count: Studies in Normals and in Patients with Chronic Airflow Obstruction

In eight non-allergic patients with chronic airflow obstruction (CAO) and eight age and sex matched, healthy control subjects the circadian variation in circulatory lymphocyte count was studied in relation to serum Cortisol and urinary epinephrine levels. In addition, we investigated the effect of the beta-adrenergic agent terbutaline on the lymphocyte count in two ways: as a long-term effect after 8 days of oral slow-release terbutaline with constant diurnal and nocturnal serum levels in patients, and as a short-term effect by a constant rate infusion of 0.2μg/min over 4hr in normals. Both patients and controls showed similar circadian patterns of urinary epinephrine excretion and lymphocyte counts. Patients with CAO, however, had significantly lower epinephrine levels and significantly higher lymphocyte counts at all hours of observation (every 4 hr from 0800 to 0800 hr the next day), as compared with normal controls. After 8 days of slow-release terbutaline the lymphocyte count in the patient group decreased to levels not significantly different from that of normals. The circadian rhythm of the lymphocytes, however, persisted under terbutaline therapy. No correlation existed between the lymphocyte count modulating factor, serum Cortisol and the lymphocyte count over 24 hr. On placebo infusion in the control persons an increase of lymphocytes over 4hr occurred, as a consequence of circadian rhythmicity. On terbutaline infusion a significant increase of lymphocytes after 1hr was follwed by a decrease to levels significantly below those on the placebo day. The same pattern was found in the leucocyte count. From this study it is concluded that beta-adrenergic stimulation corrects the relative lymphocytosis to counts comparable with normals. Other coinciding factors must regulate, however, the circadian rhythmicity.     

Effect of Beta-Adrenergic Stimulation on the Circulatory Lymphocyte Count: Studies in Normals and in Patients with Chronic Airflow Obstruction

In eight non-allergic patients with chronic airflow obstruction (CAO) and eight age and sex matched, healthy control subjects the circadian variation in circulatory lymphocyte count was studied in relation to serum Cortisol and urinary epinephrine levels. In addition, we investigated the effect of the beta-adrenergic agent terbutaline on the lymphocyte count in two ways: as a long-term effect after 8 days of oral slow-release terbutaline with constant diurnal and nocturnal serum levels in patients, and as a short-term effect by a constant rate infusion of 0.2μg/min over 4hr in normals. Both patients and controls showed similar circadian patterns of urinary epinephrine excretion and lymphocyte counts. Patients with CAO, however, had significantly lower epinephrine levels and significantly higher lymphocyte counts at all hours of observation (every 4 hr from 0800 to 0800 hr the next day), as compared with normal controls. After 8 days of slow-release terbutaline the lymphocyte count in the patient group decreased to levels not significantly different from that of normals. The circadian rhythm of the lymphocytes, however, persisted under terbutaline therapy. No correlation existed between the lymphocyte count modulating factor, serum Cortisol and the lymphocyte count over 24 hr. On placebo infusion in the control persons an increase of lymphocytes over 4hr occurred, as a consequence of circadian rhythmicity. On terbutaline infusion a significant increase of lymphocytes after 1hr was follwed by a decrease to levels significantly below those on the placebo day. The same pattern was found in the leucocyte count. From this study it is concluded that beta-adrenergic stimulation corrects the relative lymphocytosis to counts comparable with normals. Other coinciding factors must regulate, however, the circadian rhythmicity.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA , the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively . The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Effect of music-assisted imagery on neutrophils and lymphocytes

The purpose of this study was to determine the effects of cell-specific mental imagery on neutrophil and lymphocyte cell counts. Subjects (N=30) were randomly assigned to one of two experimental groups that underwent a 6-week training program focusing on images of morphology, location, and movement of either neutrophils or lymphocytes. Music was used to enhance the imagery of the subjects. Peripheral white blood cell and differential counts were determined before and after the final 20-minute imagery session. Results indicated that neutrophils decreased significantly (p<.04) in the neutrophil-change group while lymphocytes did not. The reverse occurred in the lymphocyte-change group, with only the lymphocytes decreasing significantly (p<.03). The authors concluded that under the conditions of the present study, cell-specific imagery was associated with decreases in peripheral blood cell counts of lymphocytes and neutrophils.     

DNA Repair in Human Leukaemic Lymphocytes

CHRONIC lymphocytic leukaemia (CLL) is a common human leukaemia¹ in older people. Its gradual progressive clinical course is frequently associated with lymphocyte dysfunction². In this disease lymphocyte counts are elevated and lymph nodes and organs are infiltrated with small abnormal lymphocytes which have scanty blue cytoplasm and round or clefted nuclei with clumped chromatin.     

Patterns of peripheral cellular immune disorders in severe rheumatoid arthritis

The study is focused on the correlated peripheral cellular immune disorders registered in a group of 23 patients with severe, progressive rheumatoid arthritis, on methotrexate therapy. We investigated a panel of peripheral immune parameters: leukocyte counts, the proportions of lymphocyte populations (T, Thelper, Tcytotoxic/suppressor, B lymphocytes and NK cells) and the polyclonal activation of lymphocytes. Results show that leukocytosis is due to simultaneously elevated values of monocytes, granulocytes and, to a lesser extent, lymphocytes. The registered high values of the Th to Tc/s ratio are mainly attributed to the abnormal low proportions of the Tc/s subpopulation. Inverse correlations were emphasized between B, Tc/s lymphocytes and NK cells or granulocytes. The unbalance of the lymphocyte to monocyte ratio or of the Th to Tc/s ratio does not impair the polyclonal activation of lymphocytes. In conclusion, we have characterized different patterns of correlated cellular peripheral immune disorders in rheumatoid arthritis, associated to pathological processes in conjunction with the immunsuppressive and anti-inflammatory action of methotrexate that might be relevant for further investigation of disease and further therapy outcome. We emphasize the special relation between the adaptive and innate immune system at the level of cell counts and proportions. The correlations between the peripheral abnormalities in the rheumatoid arthritis group are better highlighted by analyzing subgroups of patients characterized by particular values of the investigated parameters.     

Characteristics of ribosomal RNA synthesis in the blood lymphocytes of chronic lympholeukemia patients

A study was made of the number of silver grains over the nucleoli and of the content of ribosomes in the lymphocyte cytoplasm for six healthy persons and for 20 patients with chronic lymphatic leukemia. Besides ratios of compact, nucleolonemic and ring types of nucleoli were evaluated in addition to counts of the specific radioactivity of mature 28S rRNA in lymphocytes. In the majority of cases examined, cells with 1 or 2 nucleoli of compact and nucleolonemic types were seen dominating. The number of silver grains over the nucleoli in the control healthy persons did not differ from that in patients who did not receive any treatment, which contrasted with high value grain counts in the treated patients. The lymphocyte ribosome contents varied within the normal and decreased values in both the patient groups. The specific radioactivity in 28S rRNA leukemic lymphocytes was significantly lower in groups of patients with low ribosome contents than in those with the normal ones. The data suggest that in the leukemic cells with a high or unaltered activity of ribosome cistrons and low ribosome levels rRNA processing is broken.     

Changes of the blood lymphocyte subpopulations and their functions following 131I treatment for nodular goitre and 32P treatment for polycythemia vera

The blood lymphocyte population was examined in 34 patients who were treated with 131I for toxic or atoxic nodular goitre. One to three doses of 300-550 MBq of 131I were administered at 1-week intervals. Lymphocyte counts were found to be significantly reduced at both 1 and 6 weeks after treatment. This decrease was accompanied by a changed composition of the lymphocyte subpopulations. The frequency of lymphocytes expressing membrane receptors for C'3 (EAC-rosette forming cells) was significantly reduced at 1 and 6 weeks following 131I administration. At 6 weeks there was a small but statistically significant increase of the frequency of T cells as identified by Leu 1 monoclonal antibodies. This was essentially due to an increased proportion of helper/inducer T cells as identified by Leu 3 monoclonals. 131I treatment also decreased the capacity of lymphocytes to secrete immunoglobulins (Ig) when stimulated with pokeweed mitogen (PWM). The greatest effect was observed for IgM. Secretion of IgG and IgA were less reduced. Mitogenic stimulations of lymphocytes with phytohemagglutinin (PHA) and concanavalin A were not significantly changed. It is concluded that these findings, with the exception of mitogen reactivity, are largely similar to those occurring following external radiation therapy for cancer. It is suggested that blood lymphocytes passing through the continuously irradiated gland are damaged mainly by beta-rays. The effect of 32P treatment on the blood lymphocyte population was examined in 16 patients with polycythemia vera. Before treatment the lymphocyte counts were within the normal range but the expression of certain membrane structures, as identified by monoclonal antibodies against total T cells (Leu 1 and 4), helper/inducer (Leu 3) and suppressor/cytotoxic T cells (Leu 2), were slightly decreased. Moreover, mitogenic responses of the lymphocytes to PHA and PWM-induced Ig secretion were impaired. Following a single oral dose of 32P (150-305 MBq), which normalized the production of erythrocytes and/or platelets, the blood lymphocyte counts were reduced by approximately 40 per cent 12 weeks after treatment. Examination of subsets demonstrated that the proportion of B-cells, as identified by B1 monoclonal antibodies, was decreased by the highest relative extent. On the other hand, lymphocytes expressing the above-mentioned T cell markers were somewhat increased. 32P treatment markedly increased PHA reactivity but it further reduced PWM-induced Ig secretion. The latter observation was in agreement with the finding that serum concentrations of Ig were reduced after treatment.     

The Pathogenesis of Chronic Lymphocytic Leukemia

The pathogenesis of chronic lymphocytic leukemia was examined in a series of 88 cases observed during a 15-year period. In untreated cases the trend of the absolute lymphocyte counts followed two main patterns. In the type I trend, the counts rose throughout the observation period; in the type II trend, the tendency to rise ceased and the counts stabilized above and below a mean value, the stationary trend being maintained for months or years. The type II trend was associated with relatively benign disease. The development of lymphocytosis was correlated with the progression of lymphadenopathy. It is suggested that lymphocytosis may result from the physiological process of recirculation and that the accumulation of lymphocytes may result from the proliferation of a single slightly abnormal cell-line. The abnormal cells might survive an unusually long time because they are unable to respond to stimuli which cause normal lymphocytes to transform.     

Activity of some lysosomal enzymes in peripheral blood lymphocytes of patients with lung cancer. A cytochemical study

In 33 patients with lung cancer (6 women and 27 men, aged at average 61.2 years) the activity and intracellular localization of acid phosphatase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in peripheral blood lymphocytes were determined by means of semiquantitative cytochemical methods. In comparison to the control group of healthy subjects, the patients with lung cancer showed increased counts of acid phosphatase-positive lymphocytes with granular-diffuse cytochemical reaction, increased counts of beta-glucuronidase-positive lymphocytes with solely granular type of reaction and increased numbers of N-acetyl-beta-glucosaminidase-positive cells showing the granular, granular-diffuse and diffuse type of reaction. The total count of beta-glucuronidase-positive and N-acetyl-beta-glucosaminidase-positive lymphocytes was significantly elevated in these patients. The authors discuss the significance of their observations for evaluating lymphocyte response in patients with lung cancer.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. VI. Roles of T and B lymphocytes in cluster formation.

The role of immune T and B lymphocytes in the in vitro production of antigen-specific clusters with macrophages pulsed with soluble protein antigen was studied by assaying the cluster-producing capability of lymphocyte populations deprived of either T or B lymphocytes. Populations deprived of B lymphocytes were able to produce clusters to the same extent as unfractionated lymphocyte populations, whereas populations deprived of T lymphocytes produced very few clusters. Staining of the cluster lymphocytes for membrane Ig using the immunoperoxidase technique showed that Ig-bearing cells were to a large extent excluded from the clusters. We suggest that each cluster is initiated by an antigen-committed T lymphocyte and that the assay for clusters may be used to enumerate the number of antigen-committed T lymphocytes in a given population.     

Asymptomatic Simian Immunodeficiency Virus Infection Decreases Blood CD4+ T Cells by Accumulating Recirculating Lymphocytes in the Lymphoid Tissues

Declining blood CD4⁺ T-cell counts mark the progress of simian immunodeficiency virus (SIV) disease in macaques and model the consequences of untreated human immunodeficiency virus infection in humans. However, blood lymphocytes are only a fraction of the recirculating lymphocyte pool, and their numbers are affected by cell synthesis, cell depletion, and distribution among blood and lymphoid tissue compartments. Asymptomatic, SIV-infected macaques maintained constant and nearly normal numbers of recirculating lymphocytes despite the decline in CD4⁺ T-cell counts. Substantial depletion was detected only when blood CD4⁺ T-cell counts fell below 300/μl. In asymptomatic animals, changes in CD4⁺ T-cell distribution were more important than lymphocyte depletion for controlling the blood cell levels.     

The pattern of lymphocytes in the thymus and spleen after labeling with 3H-thymidine and 3H-deoxycytidine.

Adult male untreated mice (NMRI) were investigated after radioactive labeling with 3H-thymidine and 3H-deoxycytidine to find out whether the lymphocytes in the cortex and medulla of the thymus as well as in the perifollicular and periarteriolar regions of the spleen show a labeling pattern which allows a classification into T- and B-lymphocytes. The percentages of radioactively labeled small lymphocytes and their mean grain counts were determined. The percentages of radioactively labeled small lymphocytes after 3H-TdR and 3H-CdR showed no significant differences in both splenic zones. The grain counts over the lymphocyte nuclei in the periarteriolar zone showed lower values after 3H-TdR than after 3H-CdR. The lymphocytes in the perifollicular zone were strongly labeled with 3H-TdR and weakly labeled with 3H-CdR. In the thymus medulla, lymphocytes were weakly labeled with 3H-thymidine and strongly labeled with 3H-CdR. In the cortex no significant differences were observed. 75 to 80% of the small lymphocytes in the peripheral blood were weakly and 20-25% strongly labeled after 3H-TdR. Therefore there are similarities in the radioactive labeling pattern of thymic medulla lymphocytes and that of small lymphocytes of the periarteriolar zone of the spleen by both DNA precursors. The small lymphocytes in the peripheral T-dependent tissue zones, for example in the spleen, as well as in the mixed lymphocyte population of the peripheral blood can be differentiated from the B-lymphocytes through the difference in the amount of incorporation of 3H-thymidine and 3H-deoxycytidine.     

Role of T cell subsets in the modulation of Mycobacterium avium growth within human monocytes

Mycobacterium avium frequently causes disseminated disease in patients with advanced AIDS with low CD4 counts. The effects of T lymphocyte on intracellular M. avium replication were examined. Plastic adherent monocytes and nonadherent lymphocytes were separated from peripheral blood mononuclear cells. After infection with M. avium, monocytes were cultured with or without autologous lymphocytes (1-10 cells/monocyte) for up to 7 days. Addition of lymphocytes to M. avium-infected monocytes significantly decreased intracellular M. avium growth after 7 days culture (n = 11, P < 0.01, paired t test) and increased IFN-gamma production compared to monocytes alone. Neutralizing IFN-gamma partially abrogated lymphocyte activity. CD4 depletion diminished anti-mycobactericidal effects and CD8(+) lymphocytes increased intracellular M. avium growth (P < 0.05, n = 5, t test). These data suggest that interactions between monocytes and nonadherent cell fractions such as CD4(+) T cells and NK cells are important in intracellular M. avium growth modulation in monocytes from healthy humans.     

Lymphocyte and granulocyte migration across the endothelial layer of bovine pulmonary artery intimal explants towards lymphocyte conditioned medium

An intravenous infusion of endotoxin into sheep results in accumulation of equal numbers of lymphocytes and granulocytes in the pulmonary microcirculation. The role of the sequestered lymphocytes in acute lung injury is not known. The present study examines whether lymphocyte migration through pulmonary endothelium contributes to endothelial damage and also examines the effect of lymphokines on granulocyte migration. Bovine pulmonary artery intimal explants were mounted in Boyden chambers and conditioned media, prepared from bovine peripheral blood lymphocytes, was used as the chemoattractant. The rate of 51Cr labelled bovine granulocyte lymphocyte migration into intimal explants was determined over a 3 hr incubation period. Permeability changes were assessed by adding trace amounts of 14C-sucrose and 3H-water to the upper well and following their rate of equilibration with the lower well. 6-Keto-PGF1 alpha was measured in the upper well. Lymphocyte conditioned media was found to be chemotactic for both lymphocytes and granulocytes (lymphocyte migration at 60 min: lymphocyte conditioned media = 18.5 +/- 2.3%, mean +/- s.e. RPMI control = 12.5 +/- 1.5; granulocyte migration at 120 min: conditioned media = 36.1 +/- 5.7, RPMI control = 18.2 +/- 3.0). Ultrastructural examination revealed leukocyte migration followed an orderly sequence during which the leukocytes maintained close contact with the adjacent endothelial cells. No structural evidence of endothelial cell damage was seen at any time examined. Granulocyte migration was associated with an increased rate of 14C-sucrose equilibration after 2 hr of incubation (lower well counts/upper well counts at 2 hr, RPMI control = 0.18 +/- 0.02; lymphocyte conditioned medium = 0.30 +/- 0.04) indicating alteration in the endothelial barrier function. Leukocyte migration, particularly lymphocyte migration, was accompanied by a marked increase in prostacyclin accumulation (3 hr: no leukocytes, 188 +/- 17 ng/ml; lymphocytes, 560 +/- 104). These in vitro findings suggest that lymphocytes and lymphokines may be involved in acute lung injury and also that permeability changes associated with granulocyte migration may depend on the chemoattractant.