Mitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice

Summary A murine osteosarcoma in which the number of tumor cells can be continually monitored by measuring the circulating plasma alkaline phosphatase levels was used to determine the effect of tumor burden on peripheral blood and splenic lymphocyte response to mitogens. In animals with tumors of different sizes, the pattern of response of the peripheral blood lymphocytes (PBL) to mitogens is different from that of splenic lymphocytes. PBL response to ConA, PHA, and LPS was initially depressed, but response to PHA and LPS recovered later, as the tumor burden exceeded 6%. However, the recovery of LPS response was not consistent, in that recovery was not seen when the tumor burden was 5%–6%. Response to ConA remained depressed. Splenic lymphocytes showed progressive decline of PHA response. Treatment of tumor-bearing mice with 2-mercaptoethanol (2-ME) restored the ConA response of PBL in 56% of mice. Treatment with 2-ME did not restore PBL response to PHA or LPS. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; peripheral blood lymphocytes; ConA, concanavalin A; PHA, phytohemagglutinin; LPS, lipopolysaccharide; 2-ME, 2-mercaptoethanol; FCS, fetal calf serum; AP, alkaline phosphatase; OS, osteosarcoma     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Lymphocyte reaction in schizophrenia patients to the phytomitogens, concanavalin and phytohemagglutinin]

The capability of the schizophrenic patients' lymphocytes and lymphocytes of healthy persons to respond to the stimulating action of T-mutagens -- concanavalin A and PHA -- was studied. The T-cell count was determined by the method of rosette formation; the influence of adhesive cells on the lymphocyte response to mitogens was ascertained. The response to both the mitogens in the patients' lymphocyte cultures was reduced as compared to control, and the T-cell count failed to differ from the normal. The removal of adhesive lymphocytes results in the disappearance of differences between the response of the patients' lymphocytes and normal lymphocytes to both the mitogens.     

Demonstration of significant differences in the proliferative capacity of lymphocytes from normal human subjects

Lymphocytes were obtained from six normal human subjects on three to five occasions. Lymphocyte response to pokeweed mitogen (PWM), phytohemagglutinin (PHA), and a pool of allogeneic lymphocytes was measured by incorporation of [³H]thymidine in cultures established in flat- and round-bottom multiwell plates. Unstimulated and mitogen-stimulated thymidine incorporation were not correlated. Therefore, incremental counts rather than stimulation indices were used to express data. Individual lymphocyte responses were more reproducible in round-bottom well plates. Using these data, correlation was found between the responses to PWM and PHA. Neither of these responses was correlated with the mixed lymphocyte reaction. There were no significant differences in the responses of these subjects to allogeneic lymphocytes. This response may, therefore, allow clear distinction between normal and abnormal lymphocyte reactivity. Among these normal subjects significant differences were found in their responses to both PWM and PHA. The biological import of these differences is not known.     

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A

Peripheral blood lymphocytes of the nurse shark (Ginglymostoma cirratum) respond to stimulation by concanavalin A (Con A) as evidenced by increased incorporation of tritiated thymidine. Separation by means of Ficoll-Isopaque yields two or more bands and a sediment, all of which contain lymphocytes responsive to Con A. Only the bottom cells react to phytohemagglutinin (PHA). This reaction cannot be detected in the unseparated lymphocyte population. Thus, only a unique subset of lymphocytes appears to be responsive to PHA and is probably blocked in its response by other cells. The findings suggest that differentiation toward Con A responsiveness may have preceded phylogenetically the responsiveness to PHA. Judging by the requirement for high concentrations of both mitogens the receptor sites on shark lymphocytes appear to be present in lower densities than on lymphocytes of higher vertebrates.     

Studies on rabbit lymphocytes in vitro : XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.     

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.     

Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Progressive loss in vitro immune response with tumor growth.

Peripheral blood lymphocytes from rats carrying a transplantable hepatoma were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (ConA) or dextran sulfate (DS) at various times after tumor cell inoculation or after its surgical removal. Mitogen-induced lymphocyte transformation, measured by tritiated thymidine incorporation, declined as the tumor size increased, especially when cells were cultured in autologous serum. The response to PHA and ConA declined prior to the response to DS. This inhibition could not be removed by extensive washing of the cells, alteration of serum concentration, time of incubation or mitogen dose. Culture for 24 hr prior to the addition of high doses of mitogen resulted in partial restoration of the PHA and ConA, but not DS, responses. Previously inhibited responses also returned when the tumor was surgically removed. Spleen cells from animals with large tumors were also inhibited.     

Studies of the mixed lymphocyte reaction by the virus plaque assay.

The virus plaque assay has been developed as a tool for enumerating activated lymphocytes. In previous studies using mitogens it was found that the assay detects activated T- but not activated B-lymphocytes. In the present studies, the mixed lymphocyte reaction was studied by the virus plaque assay as well as by incorporation of thymidine and development of cytotoxic lymphocytes. In combinations differing at the entire major histocompatibility complex, approximately 1% of the cells were activated and treatment with anti-thy.1 serum totally abrogated the virus plaque forming cell (V-PFC) response. In studies on the A.TH-A.TL recombinants and the AQR-B10/ 6R recombinant strains, incompatibility at the H-2-K or H-2-D loci were found not to be capable of activating T-cells to produce virus plaques. In contrast, differences in I or I plus S regions caused a marked T-cell activation, 3–6% of the cells being V-PFC. There was a general parallelism between thymidine incorporation and the V-PFC, and it was not possible to dissociate the cell types carrying out each of these functions by kinetic studies. However, preinfection of responding cells with the virus used in these studies, vesicular stomatitis virus, caused a complete abrogation of thymidine incorporation, indicating that the activation of virus is an earler stage than DNA synthesis, and that viral activation will block further cell replicaton and is probably a lytic event.     

Age and the response of peripheral blood lymphocytes to mitogens]

Reactions of the peripheral blood lymphocytes of normal persons from 19 to 49 years of age to the following mitogens was studied; phytohemagglutinin (PHA), concanavalin A (ConA), rabbit serum against human thymocytes (ATS). A significant reduction of the lymphocytes proliferative response to ConA was reported in persons above 30 years old. There was also demonstrated a significant negative correlation between the proliferative response indices and the donor's age (in ConA-stimulation-for the whole group examined, and in ATS-stimulation-for persons aged from 30 to 49 years only. Analysis of the intensity of thymidine-(3)H incorporation showed that with the advance of age there was a fall of the percentage of cells with an intensively labelled nuclei and an accumulation of cells with weakly labeled nuclei, this phenomenon was observed both when the proliferative response was decreased and when no significant differences were reported in these indices.     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Cultured lymphocytes in chronic alcoholics

Lymphocytes were isolated from the peripheral blood of 80 chronic alcoholics without an evident liver damage and filled up with 4 different mitogens. The DNA synthesis was investigated by means of the 3H thymidine incorporation rate. They were referred to a group of 15 healthy test persons. An increased proliferation rate of B-lymphocytes could be demonstrated, whereas there was no evidence for a change in the stimulatibility of T-lymphocytes. It was only the direct impact of ethyl alcohol on lymphocytes that produced a significant decrease in the ability of T-cell reactivity, whereas a further increase of stimulatibility could be observed in B-cells. Our findings allow the conclusion to be drawn that an immediate toxic impact of ethanol on lymphocytes can be identified in chronic alcoholics without manifest liver damage.     

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Dose-dependent hyporreactivity to phytohemagglutinin in systemic lupus erythematosus

The response of peripheral blood lymphocytes to stimulation with optimal and suboptimal doses of PHA was measured in patients with active SLE before initiation of therapy. The [³H]thymidine uptake of SLE patient's lymphocytes was significantly lower than that of their matched controls when cells were stimulated with suboptimal PHA doses in the presence of autologous plasma. A moderate improvement in the PHA response was observed by culturing washed patient's lymphocytes in medium supplemented with pooled normal human plasma, but only in one case the response reverted to normal values. A significant inhibitory effect of SLE plasma on the response of normal donor's lymphocytes to stimulation with low PHA doses, which was independent from the presence of lymphocytotoxic antibodies and persisted after complement inactivation was observed in further experiments.The results indicate that depression of lymphocyte transformation could be demonstrated in patients with active SLE using suboptimal doses of PHA and suggest that this depression may be caused by both a defect in the responding lymphocyte populalation and the presence of inhibitory factor(s) in SLE plasma.