Immunity during pregnancy: lymphocyte subpopulations and mitogen responsiveness

Many hypotheses have been proposed to explain the alteration of maternal immune status that allows the fetus to escape rejection. Published data using monoclonal antibodies have stated that there are small variable reductions in circulating T-lymphocytes and little or no change in helper-to-suppressor ratios. Specific decreased levels of helper T-cells have been claimed by other workers. Our laboratory has previously reported alterations in tritiated thymidine uptake (3H-TdR) and HLA antibodies during pregnancy. The present study evaluates total T-cells, lymphocyte T-cell subsets, helper-to-suppressor ratios of T-cells, B-cells, and lymphocyte blast transformation (LBT) throughout pregnancy. These lymphocyte measurements were compared to hormonal changes occurring during pregnancy to determine whether or not hormonal levels have a significant correlation on the maternal immune response during gestation. Data from 100 women revealed no significant alteration of total T-cells or T-cell subsets during pregnancy or after parturition, as measured by monoclonal antibodies. Helper-to-suppressor ratios were within normal limits. B-cells showed a significant decrease (P less than 0.001) during the postpartum period. There was decreased lymphocyte responsiveness to mitogenic stimulation by phytohemagglutinin-P (PHA-P), concanavalin-A (CON-A), and pokeweed mitogen (PWM) in the first, second and third trimesters (P less than 0.01). The mechanisms of fetal protection from maternal immune recognition remain obscure.     

Circulating lymphocyte subpopulations in Hashimoto thyroiditis

Peripheral blood and T and B lymphocytes and [¹²⁵I]thyroglobulin-binding lymphocytes were investigated in twenty-two euthyroid Hashimoto thyroiditis patients and in twenty-two age- and sex-matched normal subjects. Although the total lymphocyte count in Hashimoto patients (mean±SEM = 1226±187/mm³) was lower than in normal subjects (1603±156/mm³) this difference was not statistically significant. There was, however, a statistically significant reduction in the proportion of circulating T lymphocytes in the Hashimoto patients (mean±SEM = 57·4±2·5%) as assessed by the sheep red-cell rosette method when compared with the normal controls (mean±SEM = 66·7±1·8%). The proportion of B lymphocytes in the peripheral blood as assessed by indirect immunofluorescence, was not significantly different being 21·6±2·1% in the Hashimoto patients and 20·2±1·1% in normal subjects.

[¹²⁵I]thyroglobulin-binding lymphocytes, as assessed by autoradiography were present in the circulation of nineteen Hashimoto patients with a mean frequency of 8·37±1·15/10⁴ lymphocytes and in thirteen normal subjects with a mean of 8·84±0·93/10⁴ lymphocytes. There was no difference in the degree of [¹²⁵I]thyroglobulin binding between the two groups as determined by grain count analysis. There was no apparent correlation between age or thyroglobulin antibody titres and the frequency of [¹²⁵I]thyroglobulin-binding lymphocytes. Thyroglobulin-binding lymphocytes were increased 100-fold in a Hashimoto thyroid biopsy in comparison to the patient's peripheral blood.

     

Immunoglobulin turnover in B lymphocyte subpopulations

“In vitro” turnover of turnover of leucine-labeled and of radioiodinated IgM has been studied with cells from various lymphoid organs of nude mice, i.e. lymph nodes, thoracic duct, spleen and bone marrow, as well as with subpopulations of B cells fromspleen and bone marrow separated by free flow electrophoresis. Three types of IgM-producing lymphocytes could be distinguished by their turnover rates of IgM, by the size of the released IgM and by the capacity of the IgM molecules to be labeled by the lactoperoxidase-catalyzed radioiodination reaction and/or by incorporation of radioactive leucine. Type I cells release 7—8 S IgM rapidly (t_1/2 = 1—3 h); the released IgM is leucine-labeled and radioiodinated. Type II cells release 7—8S IgM slowly (t_1/2 = 10—30); the released IgM is leucine labeled and radioiodinated. Type III cells release 19 S IgM rapidly (t_1/2 = 2—4 h);the released IgM is leucine labeled, but not radioiodinated. Lymph nodesand thoracic duct contain predominantly type II cells, bone marrow contains type I and II cells, spleen contains type I, II and III cells. It is suggested that type III cells areIg-secreting, plaque-forming plasma cells, type II cells are small, resting “memory” B cells, and type I cells may be newly formed antigen-inexperienced B cells.     

Beta adrenergic receptors in lymphocyte subpopulations

To further evaluate the potential utility of lymphocyte beta adrenergic receptor assays in the study of receptor alterations in human disease, we studied highly purified populations of B and T lymphocytes in peripheral blood to see if differences existed in the concentration or affinity of beta adrenergic receptors and catecholamine-responsive cAMP levels. The mean number of receptors present in particulate fractions of B cells did not differ significantly from the number found in T cells. Similarly, no significant difference in the dissociation constant for (-)[3H]dihydroalprenolol was found. Cyclic adenosine monophosphate (cAMP) accumulation in whole lymphocytes as measured by radioimmunoassay was comparable, although a tendency toward lower basal and stimulated levels in the T cells was evident. The data suggest that differences observed in concentrations of beta adrenergic receptors or catecholamine-responsive cAMP accumulation in lymphocytes from patients with varying illnesses are not likely to be due to differences in the proportions of circulating B and T lymphocytes.     

Blood lymphocyte subpopulations in extrinsic and intrinsic asthmatics

Blood leukocyte numbers and proportions of T lymphocyte subsets were studied in extrinsic asthmatics (EA), intrinsic asthmatics (IA), IA systemically treated with corticosteroids (IA + C), and in age-matched control subjects. The EA and IA showed an increased number of eosinophils. During corticosteroid therapy of IA, the eosinophil number remained elevated, whereas there was a slight decrease in the number of circulating lymphocytes. The proportion of T cells of the suppressor/cytotoxic phenotype carrying the Leu-2a antigen was significantly lower in the IA than in all other groups. In the IA + C group, the proportions of Leu-3a/3b and Leu-2a positive T lymphocytes returned to normal, although the patients still exhibited asthmatic symptoms. These findings suggest that cellular immunologic factors might be involved in the pathogenesis of intrinsic asthma.     

The monitoring of lymphocyte subpopulations in non Hodgkin lymphoma patients during chemotherapy.

Numerous examinations of the changes in B, T, null subpopulations were systematically carried out in 20 non Hodgkin lymphoma patients 8, 24, 48 hours after the therapy onset and subsequently at intervals of a few days during and after each chemotherapy cycle (COP, Knospe). T cells were evaluated by examining the receptor for neuraminidase pretreated sheep erythrocytes (nE), B lymphocytes by detecting surface immunoglobulins (SmIg) with normal and Fc deprived anti Fab2 globulin, moreover receptors for mouse erythrocytes (Em) and for C3 (EAC) were examined. A characteristic rise of the dominating subpopulation was observed during the first phase of the drug application which, most probably, was due to an expulsion of the pathological cells from involved lymphatic organs to the blood. On the basis of our clinical observations a conclusion has been drown, that this type of monitoring possibly renders a maximal effective treatment, enables the control of the accompanying immunosuppression and offers an additional criterion of the remission.     

Immunoglobulin turnover in B lymphocyte subpopulations.

"In vitro" turnover of leucine-labeled and of radioiodinated IgM has been studied with cells from various lymphoid organs of nude mice, i.e. lymph nodes, thoracic duct, spleen and bone marrow, as well as with subpopulations of B cells from spleen and bone marrow separated by free flow electrophoresis. Three types of IgM-producing lymphocytes could be distinguished by their turnover rates of IgM, by the size of the released IgM and by the capacity of the IgM molecules to be labeled by the lactoperoxidase-catalyzed radioiodination reaction and/or by incorporation of radioactive leucine. Type I cells release 7-8 S IgM rapidly (t1/2 = 1-3h); the released IGM is leucine-labeled and radioiodinated. Type II cells release 7-8 S IgM slowly (t1/2 =10-30); the released IgM is leucine labeled and radioiodinated. Type III cells release 19 S IgM rapidly (t1/2 =2-4 h); the released IgM is leucine labeled, but not radioiodinated. Lymph nodes and thoracic duct contain predominantly type II cells, bone marrow contains type I and II cells, spleen contains type I,II and III cells. It is suggested that type III cells are Ig-secreting plaque-forming plasma cells, type II cells are small, resting "memory" B cells, and type I cells may be newly formed antigen-inexperienced B cells.     

Differential mobilization of leucocyte and lymphocyte subpopulations into the circulation during endurance exercise

Summary A total of 14 healthy subjects [means (SD): 27.6 (3.8) years; body mass 77.8 (6.6) kg; height 183 (6) cm] performed endurance exercise to exhaustion at 100% of the individual anaerobic threshold (Th_an) on a cycle ergometer (mean workload 207 (55) W; lactate concentrations 3.4 (1.2) mmol · l^–1; duration 83.8 (22.2) min, including 5 min at 50% of individual Th_an). Leucocyte subpopulations were measured by flow cytometry and catecholamines by radioimmunological methods. Blood samples were taken before and several times during exercise. Values were corrected for plasma volume changes and analysed using ANOVA for repeated measures. During the first 10 min of exercise, of all cell subpopulations the natural killer cells (CD3^–CD16/CD56+) increased the most (229%). Also CD3^÷CD16/CD56+ (84%), CD8^÷CD45RO^– (69%) cells, eosinophils (36%) and monocytes (62%) increased rapidly during thattime.CD3⁺, CD3⁺HLA-DR⁺, CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO^÷ and CD19⁺ cells either did not increase or increased only slightly during exercise. Adrenaline and noradrenaline increased nearly linearly by 36% and 77% respectively at 10 min exercise. The increase of natural killer cells and heart rates between rest and 10 min of exercise correlated significantly (r=0.576,P=0.031). We conclude that natural killer cells, cytotoxic, non-MHC-restricted T-cells, monocytes and eosinophils are mobilized rapidly during the first minutes of endurance exercise. Both catecholamines and increased blood flow are likely to contribute this effect.     

Correlation of plasma hormone levels and peripheral circulating lymphocyte subpopulations during human pregnancy

Using monoclonal antibodies (OKT3, OKT4, OKT8) peripheral blood lymphocyte subsets were determined in 40 normal primiparous pregnant women and compared with those of 31 nonpregnant controls. In pregnant women plasma concentrations of estradiol, progesterone, human placental lactogen (HPL), beta subunit of human chorionic gonadotropin (beta HCG), and alpha-fetoprotein were measured by means of radioimmunoassay. We studied if correlations between peripheral lymphocyte subsets and plasma hormone levels might exist. We observed in pregnant women from 10 to 40 wk of amenorrhea a decrease in the percentage of OKT3 and OKT8 cells, and during the course of pregnancy an increase in the percentage of OKT4 cells. This increase inversely correlated with plasma beta HCG levels and directly correlated with plasma HPL levels.     

Development of lymphocyte subpopulations in preterm infants

In preterm neonates the immune system is thought to be less developed at birth, but very little is known about the actual size of lymphocyte subpopulations, and even less about the maturation of these subpopulations during the first months after a premature birth. To evaluate the development of lymphocyte subpopulations in preterm infants during the first 3 months after birth, we performed a prospective longitudinal study in two hospitals in the Netherlands. Preterm neonates (n = 38) of all post-menstrual ages were included and blood samples were taken from cord blood, and at 1 week, 6 weeks, and 3 months. Lymphocyte subpopulations were measured by four-colour flow cytometry. The data were compared with follow-up data obtained in healthy term neonates (n = 8), and with single samples from school age children (n = 5) and adults (n = 5). Overall, we found a similar pattern of post-natal development of lymphocyte subpopulations in the term and preterm infants. Both B lymphocytes and helper and cytotoxic T lymphocytes mainly consist of naive cells at birth and during the 3 months of follow-up in all neonatal age groups. So, the preterm immune system seems to be able to generate an outburst of naive T and B lymphocytes from the thymus and bone marrow within the same time span after the start of post-natal antigenic stimulation from the environment as the term immune system, but, with lower post-menstrual age, the absolute counts of naive helper T lymphocytes are lower.     

Electroencephalogram activity,catecholamines, and lymphocyte subpopulations after resistance exercise and during regeneration

We examined the effect in ten male sports students of 30-min resistance exercise followed by either 45-min regeneration with massage treatment on a massage bench or supine rest serving as control, on plasma catecholamine concentration, number and distribution of circulating white blood cells and central activitity. Resistance exercise increased free plasma adrenaline (A) and noradrenaline (NA), whereas sulpho-conjugated catecholamine concentration remained unchanged as determined by high performance liquid chromatography. Exercise induced leucocytosis and lymphocytosis measured by flow cytometry was predominantly manifested by an increase in the number of lymphocytes, monocytes, CD3⁺ cells, CD8⁺ cells and CD3^– CD16/56⁺ cells. Computer-aided electroencephalography (EEG) revealed significant increases in absolute EEG band power. The increase was highest in alpha 2 with 51.6 (SD 40.2) % (P<0.01), followed by beta 1 with 33.3 (SD 21.0) % (P<0.01), alpha 1 with 31.9 (SD 25.2) % (P<0.01), beta 2 with 30.8 (SD 26.7) % (P<0.01), delta with 26.1 (SD 28.7) % (P<0.05), and theta with 19.8 (SD 16.5) % (P<0.01). All hormone and immunological variables returned to pre-exercise values 45 min after exercise with no differences between massage and control treatments. However, during regeneration differences in absolute EEG-band power were observed between massage and control treatments. In central (C_z, C₃, C₄) and fronto-lateral (F₃, F₄) electrode positions absolute beta 1 spectral power density was significantly lower during massage treatment than during control (Wilcoxon test:P<0.01). Overall, these data demonstrated that an influence of massage treatment on deactivation characteristics could be observed in EEG measurements but not in plasma catecholamine concentration or blood lymphocytes, indicating that computer-aided topographical EEG may be a useful technique for studying activation and regeneration characteristics.     

Mobilization of circulating leucocyte and lymphocyte subpopulations during and after short,anaerobic exercise

Summary A group of 11 healthy athletes [age, 27.4 (SD 6.7) years; body mass, 75.3 (SD 9.2) kg; height, 182 (SD 8) cm; maximal oxygen uptake, 58.0 (SD 9.9) ml · kg^–1 · min^–1] conducted maximal exercise of 60-s duration on a cycle ergometer [mean exercise intensity, 520 (SD 72) W; maximal lactate concentration, 12.26 (SD 1.35) mmol · l^–1]. Adrenaline and noradrenaline, and leucocyte subpopulations were measured flow cytometrically at rest, after 5-min warming up at 50% of each individual's anaerobic threshold (followed by 5-min rest), immediately after (0 min), 15 min, 30 min, and 1, 2, 4 and 24 h after exercise. Granulocytes showed two increases, the first at 15 min and, after return to pre-exercise values, the second more than 2 h after exercise. Eosinophils also increased at 15 min but decreased below pre-exercise values 2 h after exercise. Total lymphocytes and monocytes had their maximal increases at 0 min. Out of all lymphocyte subpopulations CD3^–CD16/CD56⁺- and CD8S⁺ CD45RO^–-cells increased most and had their maximal cell counts at 0 min. The CD3⁺-, CD4⁺CD45RO⁺-, CD8⁺ CD45RO⁺-, and CD19⁺- increased at 0 min, but had their maximum at 15 min. During the hours after exercise CD3^– CD16/CD56⁺-, CD3⁺CD16/CD56⁺-, CD8⁺CD45RO⁺- and CD8⁺ CD45RO^–-cells were responsible for the lymphocytopenia. The CD3⁺- and CD3^– CD16/CD56⁺-cells were lower 24h after exercise than before exercise. Adrenaline and noradrenaline increased during exercise. In conclusion, short anaerobic exercise led to a sequential mobilization of leucocyte subpopulations. The rapid increase of natural killer cells and monocytes may have been due to increased blood flow and catecholamine concentrations. We interpreted from these results that those cells forming the first line of defence can be mobilized faster and disappear out of circulation more rapidly than all other cell populations.     

Atopic asthma: T lymphocyte subpopulations

Atopy is associated with diminished cell-mediated immunity and increased amounts of IgE, both of which may be caused by imbalances of T lymphocyte subsets. We compared the composition of highly purified peripheral-blood T cells of fifteen atopic asthmatics with ten non-atopic control subjects. Each subject was examined on five separate occasions. Indirect immunofluorescence using monoclonal antibodies was used to define T cell subsets. We examined the proportion of T cells with T3 (most T cells), T4 (helper/inducer), T8 (suppressor/cytotoxic), M1 (natural killer), and Ia (activated T cells) surface antigens. Blood was obtained at the same time of day to eliminate the effects of circadian rhythm. Subjects were taking no medications. We found no difference between the groups of the percentage of T cells with T4, T8, M1, and Ia antigens, nor the ratio of T4+ (helper) to T8+ (suppressor) cells. T3 percent was slightly (94.3 vs 92.5%) higher in the atopic group. We conclude that atopic asthma is not associated with imbalances of peripheral-blood T cell subsets.     

Alterations in lymphocyte subpopulations in copper-deficient mice.

Analyses of cell surface determinants of splenocytes from copper-deficient C58 mice indicate alterations in lymphocyte subpopulation characteristics. Both the absolute number and the relative percentage of surface immunoglobulin-bearing (B) cells from copper-deficient mice were significantly greater than those from copper-supplemented controls. The relative percentage of Thy 1.2-positive (T) cells was decreased, and the decrease was most prominent within the Lyt 1-positive (helper) T-cell subset. The functional responsiveness of both B cells and T cells was decreased in copper deficiency.     

Changes in T and B lymphocyte subpopulations before, during and after chemotherapy for malignant melanoma

The behaviour of T and B lymphocyte subpopulations before, during and after chemotherapy with DTIC or CCNU was studied in 87 melanoma patients. The effect on immune status of DTIC administered as adjuvant therapy was also reported. The immunosuppressive effect was found to be higher in patients with normal pretreatment T-cell values than in patients with low pretreatment values, and it was higher in metastatic than in nonmetastatic patients. Where T and B lymphocyte subpopulations were assessed in 10 metastatic patients after each course of chemotherapy, a progressive reduction of T-lymphocytes was observed, indicating a cumulative effect of the drugs with advancing course. The reduction of T lymphocytes was mainly due to the decrease of OKT4+ cells. A normalization of T lymphocyte subpopulations was observed after the suspension of therapy. No significant decrease in the number of B cells was observed in the DTIC treated metastatic patients, whereas a significative reduction in the absolute number was found in those treated with CCNU.     

Hydration status and cardiovascular function: effects of hydration enhancement on cardiovascular function at rest and during psychological stress.

This study examined the effects of a 3-day enhanced hydration regimen on resting cardiac function and reactivity to acute stress. Healthy volunteers (14 male, 14 female) were assigned to one of two groups: Enhanced Hydration and Normal Hydration Group. Participants in the Enhanced Group were given six 1-l bottles of water and instructed to drink two bottles a day in addition to normal fluid intake for 3 days preceding their laboratory session; no extra water was given to the Normal Group. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), cardiac output (CO), stroke volume (SV), and total peripheral resistance (TPR) were recorded during a 10-min baseline, 6-min Paced Auditory Serial Addition Test (PASAT), 5-min recovery, 5-min intermediate baseline, and 3-min Cold Pressor Test. Repeated-measures ANOVA revealed a significant Hydration GroupxTask interaction for DBP during the cold pressor (p<0.01) with the Enhanced Group exhibiting greater DBP reactivity to cold stress relative to the Normal Group. Analysis revealed significant Hydration GroupxGender interactions for SV and TPR (p<0.05) at rest and during both the PASAT and Cold Pressor Test. Females in the Enhanced Group displayed higher SV and lower TPR relative to Enhanced Group males, whereas females in the Normal Group displayed lower SV and greater TPR relative to Normal Group males. These results suggest that 3-day hydration enhancement influences blood pressure reactivity in both men and women, and that long-term hydration enhancement is related to resting gender differences in cardiac function.     

Peripheral lymphocyte subpopulations in human falciparum malaria.

The concentration of circulating T, B, and 'null' lymphocytes was determined in thirty children and three adults with Plasmodium falciparum infections in West Africa. During infection, both percentage as well as concentration of T cells were decreased as compared to levels following treatment. The percentage but not concentration of B cells was increased. Both percentage and concentration of 'null' cells were increased in malaria. Patients with splenomegaly had the most severe alterations in T-cell number; no other historic or clinical parameter correlated with the degree or pattern or change in circulating lymphocyte subpopulations. These alterations were rapidly reversible after antimalarial treatment and presumably represent the sequestration of T cells in the spleen or other organs.     

T lymphocyte subpopulations in congenital cytomegalovirus infection.

T lymphocyte subpopulations in the peripheral blood of children with congenital cytomegalovirus infections and in controls were enumerated with monoclonal antibodies. Infants of less than 1 year of age with symptomatic infections showed significant increases in the proportion of suppressor cells and decreases in the ratio of helper to suppressor cells, whereas T lymphocyte populations in older symptomatic patients and asymptomatic infants and children did not differ from those in controls.