Role of adenosine deaminase in lymphocyte proliferation.

Activity of adenosine deaminase (ADA), an enzyme known to be deficient in some patients with severe combined immunodeficiency, increased three-fold within a 24-hour exposure of human peripheral blood lymphocytes to phytohaemagglutinin (PHA) in culture. This increase took place before the onset of DNA synthesis. Increased levels of ADA activity were also observed in lymphocytes incubated with pokeweed mitogen (PWM) for 60 hr. DNA synthesis induced by PHA, PWM or mixed lymphocyte cultures (MLC) was strongly inhibited by adenosine at concentrations of 10(-4) M or higher when human peripheral blood lymphocytes were cultured in a medium supplemented with horse serum, which lacks ADA. 10(-6)-10(-8) M coformycin, a potent inhibitor of ADA, inhibited PHA-, PWM- and MLC-induced DNA synthesis to a variable extent, whereas thymidine incorporation induced by Salmonella lipopolysaccharide (LPS) in mouse spleen cell cultures was strongly inhibited (by 75% or more) by 10(-6) M coformycin. Combination of 10(-7)-10(-8) M coformycin and 10(-4)-10(-5) M adenosine synergistically inhibited mitogen- or MLC-induced DNA synthesis in human and mouse lymphocyte cultures. These results, together with observations on children with ADA deficiency, provide evidence that adenosine deaminase is highly important for lymphocyte proliferation. Human peripheral blood lymphocytes incubated with PHA, 10(-5) M adenosine and 10(-7) M coformycin showed some cytotoxicity whereas the rate of 51Cr release from normal lymphocytes was not modified by the drugs. These findings suggest that in vivo clones of lymphocytes responding to specific antigens might be eliminated by coformycin, which may prove to be useful as a specific immunosuppressive agent.     

T-lymphocyte dependency of B-lymphocyte blastogenic response to phytomitogens.

Human peripheral blood T and B lymphocytes were separated by a method based on the stable rosette formation of T lymphocytes with neuraminidase-treated sheep erythrocytes, followed by centrifugation over a Ficoll-Hypaque gradient. Monocytes were isolated from the T-depleted B lymphocyte preparation by allowing the monocytes to ingest iron particles and by subsequent centrifugation over a Ficoll-Hypaque gradient. The T lymphocytes responded extremely well to PHA and very well to PWM, while the B lymphocytes were unresponsive to either PHA or PWM. However, when the B lymphocytes were cultured together with irradiated autologous or allogeneic T lymphocytes (1:1, 1:2 or 1:4 ratio), both PHA and PWM became mitogenic to B lymphocytes. Irradiated T lymphocytes alone did not respond to either PHA or PWM, indicating that the 3H-thymidine incorporation seen in the mixed-cell culture was due to the activation of unirradiated B lymphocytes. The B lymphocytes failed to respond to these phytomitogens in the presence of lower concentrations of irradiated T lymphocytes. The monocytes were found to be incapable of helping the B lymphocytes to respond to PHA or PWM.     

Combined Effects of Phytohemagglutinin and Staphylococcal Enterotoxin B on Deoxyribonucleic Acid Synthesis During Blast Transformation in Human Lymphocytes

Three mitogenic agents, phytohemagglutinin (PHA), staphylococcal enterotoxin B (SEB), and concanavalin A (Con A) were tested for their effects on deoxyribonucleic acid (DNA) synthesis in the normal human lymphocyte. When optimal concentrations of PHA and SEB were combined, tritiated thymidine incorporation in lymphocytes derived from several donors was enhanced significantly. In the presence of graded concentrations of one of these mitogens added to fixed optimal concentrations of the other, this enhancement was shown to be additive. By contrast, when PHA or SEB were combined with Con A, the resulting thymidine incorporation was slightly lower than for either mitogen alone. An inhibition of further thymidine incorporation when puromycin was added to lymphocytes incubated with PHA and SEB suggested that the additive effect of these mitogens was due to increased enzyme synthesis. To define potential differences in mechanisms of action underlying the additive effect of SEB and PHA, the relative contribution of the de novo and salvage pathways for pyrimidine biosynthesis was tested with cytidine, a specific salvage pathway inhibitor. Cytidine (10(-3) M) inhibited synthesis through the salvage pathway, but did not significantly alter induction of carbamyl phosphate synthetase II, the rate-limiting enzyme for the de novo pathway. An inhibition of DNA synthesis by millimolar cytidine concentrations in lymphocytes incubated with PHA or SEB, singly or in combination, suggested that pyrimidines for the observed enhancement of DNA synthesis were derived largely via the salvage pathway.     

Effect of thioproline in vitro on selected functions of peripheral blood lymphocytes from healthy persons and patients with lympho- proliferative syndromes]

Thioproline (thiazolidino-4-carboxylic acid) is one of promising antineoplastic preparations arousing interest since several years. In view of controversies with respect to the mechanism of thioproline action it was tried to study the effects of the preparation on certain functions of lymphocytes. The experimental model included cultures of T and B lymphocytes obtained from 20 healthy blood donors. The cultures were exposed to various concentrations of thioproline for determining the incorporation of 3H-thymidine into the DNA of these cells, their viability and intracellular concentrations of cAMP and cGMP. The lymphocytes from these healthy donors served also as a control for B-cells isolated from 24 patients with lymphoproliferative syndromes. It was found that at low concentrations thioproline added to the culture of T-cells from healthy donors caused a slight increase of 3H-thymidine incorporation, but at high concentrations a strong cytotoxic effect on T-cells was noted. B-cells isolated from the peripheral blood of healthy subjects and from the patients were highly sensitive to thioproline. These observations demonstrated a cytotoxic effect of thioproline on T and B lymphocytes.     

Mitogenic activity of extracellular cationic products produced by group A streptococci; analysis of the lymphocyte response.

The cationic fraction (isoelectric point greater than 8.5) of supernatant products of group A streptococcal cultures exerted a strong mitogenic effect on human peripheral lymphocytes at concentrations as low as 1 ng/well. Incorporation rates were highest at concentrations of 1-10 micrograms; rabbit peripheral lymphocytes also responded strongly, only a weak response was seen with mouse peripheral lymphocytes and rabbit thymocytes. Purified OKT4 positive (T helper) and OKT8 positive (T suppressor) lymphocyte subpopulations both responded, the former more strongly. Although accessory cells (monocytes) were not absolutely necessary, in their presence higher incorporation of 3H-thymidine was observed. Isolated B cells did not respond.     

Disturbed immunoregulatory properties of the neuropeptide substance P on lymphocyte proliferation in HIV infection.

The neuropeptide substance P (SP) is known to increase cell-mediated immune responses in animal models and healthy subjects. Several studies have suggested an involvement of neuropeptides in the immunopathogenesis of some diseases. The study of the immunomodulatory effects of neuropeptides, namely SP, may represent a model for the analysis of immunoregulatory defects in HIV infection at the level of the interaction between the immune and nervous systems, both of which are known to be affected by the virus. In the present study, we investigate the possibility of a disturbance in the immunomodulatory properties of SP in HIV infection by analysing the effects of SP (10(-10)-10(-6) M) on the lymphocyte proliferative responses to concanavalin A (Con A) and phytohaemagglutinin (PHA) assessed by 3H-thymidine incorporation in peripheral blood lymphocytes from 34 HIV-infected patients (16 asymptomatic (ASY)/persistent generalized lymphadenopathy (PGL); 18 ARC/AIDS) and in 37 healthy subjects. In ASY/PGL HIV-infected patients, SP 10(-7) M was identified as the concentration inducing the maximal increase in the lymphocyte responses to Con A and PHA, similarly to what was observed in healthy subjects. In ARC/AIDS patients, SP appeared to inhibit the mitogenic responses, particularly those induced by Con A, in contrast to the effects found either in healthy subjects or in ASY/PGL patients. These results suggest the existence of an alteration in the in vitro immunomodulatory properties of SP in ARC/AIDS patients compared with healthy subjects and ASY/PGL patients. In conclusion, the unexpected finding of an inhibitory effect of SP on lymphocyte proliferation from ARC/AIDS patients justifies further investigation of the neuropeptide-dependent immunoregulatory systems in HIV infection.     

beta2-Microglobulin production by highly purified human T and B lymphocytes in cell culture stimulated with various mitogens.

This study attempts to evaluate beta2-microglobulin production by highly purified (greater than 98%) peripheral and tonsil T and B lymphocytes cultured with various mitogens. beta2-Microglobulin was measured by the radioimmunoassay method. It was found that PHA and Con A markedly stimulated beta2-microglobulin production in cultures of T but not B lymphocytes. B lymphocytes were greatly activated, on the other hand, by Staphylococcus aureau Cowan I organisms cSpA), though the level of beta2-microglobulin production was less than that observed in PHA- and Con A-stimulated T lymphocytes. PWM only slightly increased beta2-microglobulin production of T lymphocytes, although the incorporation of [3H]-thymidine was highly enhanced. The highest level of beta2-microglobulin obtained with PHA or Con A was observed when the T/B lymphocyte ratio was between 90/10 and 80/20. These results lead to the conclusion that: (1) SpA is a specific mitogen for B lymphocytes, and its mitogenicity is independent of the presence of T lymphocytes, while PHA, Con A, and PWM are ineffective as stimulants of B lymphocytes; (2) the beta2-microglobulin producing ability of B lymphocytes is less than that of T lymphocytes, even when the lymphocytes are markedly activated; (3) the beta2-microglobulin production and DNA synthesis by T lymphocytes is markedly enhanced by the helper effect of B lymphocytes; (4) the level of beta2-microglobulin production reflects lymphocyte activation, especially in T lymphocytes stimulated with PHA or Con A.     

Mitogenic activity of 12-O-tetradecanoyl phorbol-13-acetate on peripheral blood lymphocytes from young and aged adults.

The effect of age on the proliferative response to 12-O-tetradecanoyl phorbol-13-acetate (TPA) was examined using peripheral blood lymphocytes from 185 adults. TPA-induced DNA synthesis measured by cellular 3H-thymidine incorporation was found, like the responses of cells activated by PHA and Con A, to markedly diminish with advancing age. The presence of indomethacin (1 microgram/ml) or Ro 20-5720 (10 micrograms/ml) in TPA activated cell cultures, unlike PHA stimulated cultures, did not result in augmentation of 3H-thymidine incorporation by cells from elderly individuals. These results demonstrate that prostaglandin synthesizing suppressor cells are not responsible for the age-related depression of cellular immune function observed in TPA activated cells and confirm the observation that decreased production and/or utilization of soluble mediators, such as IL-2, may account for the diminished mitogen responsiveness of lymphocytes from elderly individuals.     

The Effect of Levamisole on Peripheral T-Lymphocytes of Patients with Malignant Lymphomas

The effect of various concentrations (0.015-10 μg/ml) of Levamisole (LMS) on the peripheral lymphocytes of patients with malignant lymphomas (ML) and normal donors was investigated in vitro. The parameters studied include : E rosettes forming cells (total T lymphocytes), active E rosettes (early T lymphocytes) and DNA synthesis induced by pitogens PHA and Con A.

LMS improved significantly lymphocyte response both in patients with ML and normal donors when the cells were stimulated by Con A. In both groups no significant effect was observed on the response to PHA nor on the percentage of E-rosettes, whereas the mean number of active E rosettes was significantly increased on all concentrations of the drug.

While in the normal subjects a positive statistical correlation between active E-rosettes and Con A response was observed, in patients with ML an inverse correlation was found. This latter correlation was partially reversed by LMS.     

Degradation of Thymidine to Thymine by Rheumatoid Arthritis Synovial Tissue Eluate

A study was made of the effect of rheumatoid synovial tissue eluate on phytohaemagglutinin (PHA) stimulation of peripheral blood lymphocytes. It was found that rheumatoid synovial tissue eluate caused marked inhibition of 3H-thymidine incorporation by lymphocytes stimulated with PHA. Synovial tissue from traumatic joints had no effect. The inhibitor was a heat-sensitive and nondialysable substance. The inhibitory effect of PHA stimulation was diminished by thymine. In thin-layer chromatography it showed the capacity to convert thymidine to thymine. Because thymine is not incorporated by DNA-synthesizing cells, this report explains why negative results are obtained when lymphocytes are stimulated by rheumatoid synovial tissue using 3H-thymidine incorporation as an indicator of lymphocyte activation.     

Polyclonal human T lymphocyte activation results in the secondary functional activation of the human B lymphocyte.

The polyclonal T lymphocyte activators Con A and PHA were demonstrated to induce secretion of IgM and IgG as well as IgA antibodies in cultures of human peripheral blood lymphocytes. A similar response was seen in one-way mixed lymphocyte cultures and the kinetics, dose-response characteristics and optimal culture conditions are presented. The presence of functional T lymphocytes is a prerequisite for in vitro B lymphocyte activation in response to T lymphocyte mitogens. A narrow dose-response profile is a characteristic for both Con A and PHA and polyclonal B cell activation occurred at what have been previously regarded as suboptimal concentrations of these agents. The use of higher doses of these activators failed to generate immunoglobulin-secreting cells despite the presence of early and optimal levels of DNA synthesis in the cultures.     

Effects of biorhythm regulator melatonin on DNA synthesis in short-term cultures of human malignant tumors

The cytotoxic action of melatonin on DNA synthesis was studied in short-term human tumor tissue cultures. Twenty tumors (ovarian, renal, colorectal, gastric, skin, testicular, thyroid, adrenal gland, endometrial, cervical uterus ones, and melanoma) were isolated from patients at surgery. The effect of melatonin (concentrations of 5 x 10(-5) M to 5 x 10(-13) M on the level of 3H-thymidine incorporation into tumor tissue DNA (4-hour exposure) was evaluated. After exposure, 3H-thymidine was added to the medium for an hour. The quantity of DNA was determined in the hydrolysate by spectrometry; the level of 3H-thymidine incorporation into DNA was radiometrically evaluated by a scintillation counter. The quantity corresponding to 3H-thymidine incorporation per unit of DNA was determined. The control group and the melatonin-treated groups were compared by ANOVA. Melatonin inhibited DNA synthesis in 11 (55%) of the 20 tumors and was ineffective in 9 (45%) of the 20 ones. Melatonin-sensitive tumors were as follows: endometrial CA (33% vs 75% inhibition, p < 0.01), gastric CA (71% inhibition, p < 0.05), adrenal gland CA (38% inhibition, p < 0.01). Ovarian, cervical, skin CA and melanoma were unresponsive to melatonin. Renal cell CA, colonic and rectal CA were sensitive in some cases. In sensitive tumors, melatonin was effective even at concentrations of 5 x 10(-13) M to 5 x 10(-7) M. Thus, melatonin has an oncostatic effect on some human tumors in vitro occasionally at physiological concentrations.     

Activation of human peripheral blood lymphocytes by concanavalin A dependence of monocytes.

The activation of human peripheral blood lymphocytes or isolated T lymphocytes by concanavalin A (Con A) is hightly potentiated by the presence of autologous, mitomycin C-treated monocytes. The optimal lymphocyte: monocyte ratio within a broad dose range is 1:1 when the incorporation of [14C]thymidine is expressed as total incorporation per culture tube and 1:10 when expressed per lymphocyte. A five-to-ten-fold increase of total DNA synthesis is noted in the presence of 10-90% monocytes. The data may help to explain the wide variations in Con A responsiveness of human peripheral lymphocytes which may be partly related to differences in purification which give rise to cell preparations containing varying amounts of monocytes.     

The significance of changes in blood lymphocyte populations following surgical operations.

After surgery blood lymphocyte levels fell to one-third of the pre-operative value. Since this depression was transient, and followed the peak of serum cortisol closely, it was probably due to a redistribution of lymphocytes from the blood to the tissues. The proportion of activated lymphocytes, as measured by the incorporation of 3H-thymidine in vitro without added mitogen, was substantially increased about 5-8 days after operation. The greatest number of S-phase lymphocytes was found after operation combined with blood transfusion but surgery alone and blood transfusion alone each produced significant increases. The proportions of B lymphocytes, T helper cells and T suppressor cells remained nearly constant despite the fluctuations in total lymphocyte counts. The response of lymphocytes in vitro to phytohaemagglutin (PHA) was doubled on average 7 days after surgery provided the test was performed in the patient's own serum. In pooled serum there was no consistent change in the response to PHA. It is argued that none of the observed changes necessarily signify that a phase of immunosuppression follows surgical operations.     

Alteration of lymphocyte reactivities by thyroid hormones

The effects of thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit peripheral blood lymphocytes to T-cell mitogens, PHA and Con A, and B cell specific goat anti-rabbit light chain antibodies (Anti-L) were investigated. It was observed that L-T4 potentiated the lymphocyte response to mitogens and Anti-L in a dose-dependent manner: 10(-9) M and 10(-8) M had no effect while 10(-7)-10(-5) M significantly enhanced the lymphocyte response. L-T3 (10(-11)-10(-8) M) had no effect on the lymphocyte response to PHA and Con A. At 10(-7) M, L-T3 inhibited the response to PHA but not Con A. L-T3 (10(-11)-10(-7) M) suppressed the lymphocyte response to Anti-L. The suppression was directly proportional to the L-T3 concentration. rT3 (10(-11)-10(-7) M) inhibited the proliferative response to PHA and Anti-L in a dose-related manner. Its effect on the lymphocyte response to Con A was stimulatory at 10(-11) M but inhibitory at higher concentrations (10(-8) and 10(-7) M). rT3 suppressed the enhancement by L-T4 of the lymphocyte response to the mitogens and Anti-L. The degree of suppression was proportional to its concentration. This data indicated that thyroid hormones can alter the reactivities of lymphocytes. The direction and magnitude of the alteration appear to depend on the concentration of a specific thyroid hormone encountered by the responding cells.     

Glucocorticosteroid enhancement of immunoglobulin synthesis by pokeweed mitogen-stimulated human lymphocytes.

We studied the role of the T lymphocyte in GCS enhancement of PWM-stimulated IgG synthesis by human peripheral blood mononuclear cells. Purified T or B lymphocyte subpopulations were pretreated with 10(-6) M prednisolone or recombined at various T:B ratios and 10(-6) M prednisolone was added. PWM-stimulated IgG synthesis was measured in the culture supernatants at 8 days by radioimmunoassay. Addition of prednisolone to cultures of autologous and allogeneic reconstituted mixtures of T and B lymphocytes resulted in enhancement of PWM-stimulated IgG synthesis. This effect was observed with constant and increasing numbers of lymphocytes in culture, independent of T:B ratio and occurred with purified B lymphocytes containing monocytes. Pretreatment of purified B lymphocytes containing monocytes but not purified T lymphocytes with prednisolone enhanced PWM-stimulated IgG synthesis in reconstituted mixtures of T and B lymphocytes. We propose that GCS enhancement of PWM-stimulated IgG synthesis by human mononuclear cells is independent of T lymphocyte regulation.     

Generation of B suppressor cells by phytohaemagglutinin.

Activation by phytohaemagglutinin-P (PHA-P) of B cells from peripheral blood of healthy subjects generated B suppressor cells which inhibited allogeneic mixed lymphocyte reactions (MLR) and PHA-induced DNA synthesis. Their action was direct, that is, not mediated through the induction of T suppressor cells. The finding that B cells can be induced by PHA to become highly effective suppressor cells may have important clinical and experimental implications.     

Local immune response in experimental pyelonephritis in the rabbit. I. Morphological and functional features of the lymphocytic infiltrate.

The cellular activity of circulating lymphocytes and lymphocytes isolated from the infected kidney of animals with experimental haematogenous pyelonephritis was evaluated. The incorporation of [3H-methyl]thymidine into DNA by lymphocytes was studied with mitogens such as phytohaemagglutinin (PHA), pokeweek mitogen (PWM) and goat anti-rabbit IgG (GARIG). Lymphocytes from infected kidney had a high baseline DNA synthesis compared to circulating lymphocytes from days 5 to 27 of infection. Infected kidney lymphocytes failed to respond to PHA, PWM, or GARIG, whereas circulating lymphocytes did respond to these mitogens. Uropod-bearing lymphocytes, which were shown to be T lymphocytes, were present from days 5 to 77 of infection. B lymphocytes, as determined by surface immunofluorescent technique, were present by day 12, coincident with the onset of local synthesis of antibody. These studies reveal that in pyelonephritis, the cellular response goes through sequential changes and indicate a dynamic interrelationship between T and B lymphocytes at an infected site.     

胰岛素在T淋巴细胞激活中的作用

本实验研究胰岛素对T淋巴细胞激活的影响。实验得出:胰岛素不是淋巴细胞的丝裂原,但可促进PHA激活T淋巴细胞的作用,并在一定条件下,胰岛素可不依赖PHA的存在,独自对淋巴细胞产生效应。在T淋巴细胞激活过程中,胰岛素可促进细胞DNA合成,但不改变淋巴细胞DNA合成峰时。     

Neuropeptide regulation of human thymocyte, guinea pig T lymphocyte and rat B lymphocyte mitogenesis

The effect of 15 defined neuropeptides on the mitogenic activation of lymphocytes from human thymus, guinea pig lymph nodes and rat spleen was investigated. Lymphocytes were incubated in the absence or presence of polyclonal T and B cell activators together with increasing doses of the neuropeptides, and harvested at 48 h of culture after pulse-labeling with 3H-thymidine to assess the DNA synthesis. A dose-related stimulatory effect on the spontaneous 3H-thymidine incorporation of human thymocytes was obtained with methionine-enkephalin (met-enk), motilin and neurotensin. Vasoactive intestinal polypeptide (VIP) and peptide HI (PHI) were inhibitory. A similar responsiveness was observed in cultures of phytohemagglutinin P (PHA)-activated human thymocytes. The low level of basal DNA synthesis of guinea pig lymph node cells was stimulated by VIP and inhibited by neuropeptide Y (NPY) and PHI. PHA-activated lymph node T lymphocytes were stimulated by neurotensin, bombesin and motilin, whereas NPY inhibited the thymidine uptake. The low rate of spontaneous DNA synthesis of rat spleen cells was increased in the presence of VIP. Met-enk stimulated both basal and dextran sulfate-activated splenic B cell proliferation, whereas PHI was inhibitory in both cases. The following peptides were found to be inactive in all the above assays: substance P, cholecystokinin-octapeptide, somatostatin, galanin, oxytocin, pentagastrin and gastrin-releasing peptide 1-27 and 14-27. Although the responses were generally of low magnitude and observed at high peptide concentrations, present study contributes to the understanding of possible mechanisms involved in interactions between the nervous and the immune system.