Pokeweed Mitogen Stimulation Research Articles

Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells.

Human T-cell blasts were generated by stimulation with mitogens and antigens. A proportion of these blasts expressed Ia antigens detectable by immunofluorescence with both allo- and hetero-antiserums. The maximal expression of Ia antigens was delayed and usually occurred after the peak of blastogenesis. Among the three mitogens used, pokeweed mitogen (PWM) was most effective in giving a high percentage and intense Ia staining of T-cell blasts. Phytohemagglutinin and concanavalin A blasts gave weaker and lower percentages of Ia staining. Activation by alloantigens and soluble antigens such as tetanus toxoid and purified protein derivative resulted in Ia expression on T cells comparable to PWM stimulation. Depletion of Ia+ cells from freshly isolated T cells with anti-Ia and complement decreased subsequent Ia expression, suggesting that a proportion of Ia+ blasts were derived from Ia-bearing peripheral blood T cells. When the specificities of the Ia antigens on T-cell blasts were examined with alloantiserums, it was evident that the T blasts expressed similar HLA-DR determinants to those on B cells from the same donor; occasional minor differences between stimulated T cells and autologous B-cell lines or fresh B cells were encountered.

Differential synthesis of IgM and IgG anti-tetanus toxoid antibody in vitro following in vivo booster immunization of humans

The in vitro syntheses of IgM and IgG anti-tetanus toxoid antibody by human peripheral blood leukocytes were compared prior to and at various intervals following in vivo booster immunization with soluble tetanus toxoid. Prior to booster immunization, the in vitro synthesis of IgG anti-tetanus toxoid antibody by combinations of B cells and irradiated T lymphocytes was negligible following pokeweed mitogen stimulation. Within 2 weeks after booster immunization, the quantity of IgG anti-tetanus toxoid antibody synthesized in vitro increased 5- to 20-fold. There was no comparable increase in total IgG synthesis. In contrast to the synthesis of IgG antibody, in vitro synthesis of IgM anti-tetanus toxoid antibody occurred prior to booster immunization and did not increase significantly following booster immunization. This dichotomy in anti-tetanus antibody production was further demonstrated in an individual with common variable hypogammaglobulinemia whose lymphocytes synthesized normal quantities of total IgG, IgM, and IgM anti-tetanus toxoid antibody in vitro, but failed to synthesize IgG anti-tetanus antibody following in vivo booster immunization.

Association of helper activity for B-lymphocyte differentiation with lymphocytes having both SRBC and complement receptors in a patient with lymphoproliferative disease

Approximately 96% of the lymphocytes of a patient with lymphocytic leukemia were found to possess surface membrane complement receptors, showed T-lymphocyte antigen, and formed spontaneous rosettes with sheep erythrocytes (SRBC). These cells showed the morphology of small- to medium-size lymphocytes and lymphoblasts. A lymphocyte transformation study showed that these T lymphocytes with complement receptors gave no response to phytohemagglutinin-P or concanavalin A and a low response to pokeweed mitogen (PWM) stimulation. No production of anti-SRBC antibody was detected with these lymphocytes as determined by a plaque assay after the lymphocytes had been stimulated by PWM. Using this plaque assay, these lymphocytes were cocultured with normal lymphocytes to test for their influence on normal antibody production. It was shown that the T lymphocytes with complement receptors were capable of providing helper activity for B-lymphocyte differentiation.

ANTITHYRDGLOBULIN (ATG) PRODUCTION BY PERIPHERAL BLOOD LEUKOCYTESIN VITRO

Antithyroglobulin is produced in vitro by pokeweed mitogen stimulation of peripheral blood lymphocytes from patients whose serum contains the autoantibody. Antithyroglobulin synthesis requires T lymphocyte help but is suppressed by larger numbers of T lymphocytes. T cells from both patients and normals perform both of these functions.

Monocyte Dependence of Pokeweed Mitogen-Induced Differentiation of Immunoglobulin-Secreting Cells from Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.

Differential Sensitivity of Lymphocyte Subpopulations to Suppression by Low Density Lipoprotein Inhibitor, an Immunoregulatory Human Serum Low Density Lipoprotein

Reports by a number of investigators have described the thymus-derived (T)-cell dependence of immunoglobulin synthesis by pokeweed mitogen (PWM) stimulated human peripheral blood bone marrow-derived (B) cells. Because of the cooperative nature of this in vitro system, it was chosen for examination of the differential effects of low density lipoprotein inhibitor (LDL-In) on B- and T-cell functions. Supernates from 7-d cultures that contained either peripheral blood mononuclear cells (PBM) or combinations of isolated lymphocyte populations were assayed for immunoglobulin (Ig)G by competitive inhibition radio-immunoassay. LDL-In suppression of whole PBM IgG synthesis occurred at 5-20 mug protein/ml and was independent of PWM concentration. Maximal suppression required preincubation of cells with LDL-In before stimulation. Suppression was also observed when B cells alone were exposed for 24 h to LDL-In before PWM stimulation; these suppressed B cells were not rescued by normal T cells. Exposure of T cells alone to low doses of LDL-In for 24 h augmented, but high doses suppressed, IgG synthesis, suggesting a differential effect on T-helper vs T-suppressor cell populations. Independent LDL-In exposure of T-helper or T-suppressor cell enriched populations, separated by rosetting with IgG- or IgM-coated ox erythrocytes, identified the T-suppressor cell populations as the most sensitive of the lymphocyte populations tested. The sensitivities of lymphocyte subpopulations to LDL-In, relative to PBM, were 2.8, 1.2, and 0.3 for the T-suppressor cells, B cells and T-helper cells, respectively. Thus, both B and T lymphocytes are sensitive to and can be regulated by LDL-In. In addition, the biologic activity observed when unseparated PBM are exposed to LDL-In appears to represent a composite of the sensitivity of each of the lymphocyte subpopulations.

Restricted classes of immunoglobulin produced by a lymphoid cell line from a patient with agammaglobulinemia.

Restricted expression of immunoglobulin by a long-term B lymphoid cell line derived from a patient with X-linked agammaglobulinemia is reproted. The patient had peripheral blood B lymphocytes with surface IgD and IgM. Culture of the B cells in vitro with mitogens did not stimulate immunoglobulin secretion, although pokeweed mitogen stimulation resulted in the development of cytoplasmic IgD and IgM. The lymphoid cell line established from these B lymphocytes primarily bore surface IgD, with a small population of cells also bearing surface IgM. These cells also had cytoplasmic immunoglobulin, primarily IgD. The cell line did not have a cytoplasmic pool of immunoglobulin for export and did not secrete immunoglobulin. This B lymphoid cell line is an in vitro analogue of the arrest in differentiation of the patient's B lymphocytes. The IgD-bearing phenotype may represent a normal transitional stage in the differentiation of B lymphocytes.

Patterns of Expression of Human “Ia-Like” Antigens during the Terminal Stages of B Cell Development

The "Ia-like" antigens characteristically present on the membrane of B lymphocytes were shown to be absent in most terminally differentiated Ig-producing plasma cells. This was most evident in analyses of myeloma plasma cells that uniformly lacked the Ia-antigens. Also, in B lymphoid cell lines, the lymphoblasts were uniformly Ia-positive but the plasma cells were negative. In contrast, however, the majority of plasma cells produced after pokeweed mitogen stimulation remained positive. Plasma cells in tonsil and in the tissue of peripheral blood of patients with Wladenström's macroglobulinemia were also sometimes Ia-positive although the majority were negative. Studies of membrane IgM and IgD indicated a similar loss in some instances. However, the SIg and Ia antigens were not invariably associated. These results lead to the conclusion that Ia-antigens are differentiation antigens for B cells and are usually lost by the end stage cells in this series.

702 EVIDENCE FOR EXCESSIVE T SUPPRESSOR CELL ACTIVITY IN X-LINKED IMMUNODEFICIENCY WITH HYPER-IgM

Increased T suppressor cell activity has been detected in patients with common variable hypogammaglobulinemia. In the present study, we used both a plaque-forming cell (PFC) assay that detects cells producing IgM antibody to sheep red blood cells (SRBC) and a double antibody radioimmunoassay to measure IgM synthesized by mononuclear blood cells in evaluating two patients with x-linked immunodeficiency with hyper IgM (HypM). Cultures from 18 normal individuals gave a range of 180-2,254 PFC/106 viable cells, and co-cultures of cells from pairs of normals averaged 83% of the PFC's predicted from individual culture data. The two HypM patients failed to produce PFC to SRBC, and co-cultures of HypM and normal mononuclear cells showed complete suppression of PFC activity by the normal cells. The HypM patients cells were capable of synthesizing IgM following pokeweed mitogen stimulation but suppressed IgM synthesis by normal cells in co-culture. In patient DM, the nature of the suppressor cell was investigated by co-culturing lymphocyte subpopulations and by adding hydrocortisone (HC). The following data were obtained: The findings indicate that patients with this defect have excessive numbers of hydrocortisone-sensitive T cells that can suppress IgM synthesis by normal B cells and suggest that these cells develop secondary to the patients' excessive IgM production.

Activation of human B lymphocytes: VI. immunoregulation of antibody production by mitogen-induced and naturally occurring suppressor cells in normal individuals

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response ( P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells ( P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10 6 cells) usually suppressed the PFC response of allogeneic cells ( P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10 6 cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.

B‐ AND T‐CELLS AND INTRACELLULAR Ig‐SYNTHESIS OF PERIPHERAL LYMPHOCYTES IN CHILDREN WITH ASTHMA AND/OR PREVIOUS ADENO‐TONSILLECTOMY

Studies of IgG, IgA and IgM in serum, the number of circulating T- and B-cells, and the number of Ig-containing blast cells after pokeweed mitogen stimulation of peripheral lymphocytes in vitro were performed in children who had been found previously to have low IgA levels in serum and saliva. The first group comprises 24 children with tonsillectomy performed approximately 3 1/2 years earlier, among whom several cases of atopic diseases had been found. In these children, low IgA levels were found continuously, while the numbers of B- and T-cells and the number of IgG, IgA, IgM and IgE-containing blast cells were within normal levels. The second group comprises 28 children with recurrent and chronic respiratory infections, which often preceded attacks of asthma. Very low IgA levels were found in these children associated with a decreased number of T-cells, increased numbers of lymphocytes with membrane bound IgA and IgE and an increased number of IgE-containing blast cells. The third group comprises 20 children with severe extrinsic asthma. In these children, slightly decreased levels of IgA and low levels of IgM were found. Furthermore, the number of circulating T-cells was reduced and, in addition, an increased number of lymphocytes with surface IgE was observed.

B‐ AND T‐CELLS AND Ig‐CONTAINING BLAST CELLS IN HEALTHY CHILDREN

Studies on rosette-forming cells and IgG, IgA, IgM and IgE-bearing lymphocytes were carried out in 70 healthy children aged 5 to 15 years. Furthermore, in 20 of these children, the percentages of IgG, IgA, IgM and IgE-containing blast cells after pokeweed mitogen stimulation of peripheral lymphocytes were evaluated. No differences in the percentages of rosette-forming cells, or in Ig-bearing and Ig-containing cells were found when three different age-groups were compared.

PULMONARY WASHINGS AND BLOOD IN PULMONARY ALVEOLAR PROTEINOSIS (PAP)

Pulmonary alveolar proteinosis is a diffuse pulmonary disease of obscure etiology characterized by abnormal accumulations of phospholipids in the lung. Despite severe pulmonary disabilities there is a striking absence of inflammatory cells and fibrosis in the lungs of these patients. We have examined the lung washings and blood from five patients with this disease in an attempt to discover whether there are any inhibitors of the normal inflammatory responses locally or systemically. The complete peripheral blood counts and numbers of T and B lymphocytes and monocytes were entirely normal in all patients. The cellular components of the lung washings were primarily mononuclear cells which exhibited histochemical and surface characteristics typical of alveolar macrophages (AM). Morphologically, these AM were many times larger than AM from control patients and were filled with amorphous material. The lung washings of the patients inhibited Con A and pokeweed mitogen stimulation of allogenic peripheral blood lymphocytes by >90%, but had no significant effects on PHA stimulation. Their sera inhibited stimulation by all three mitogens >95%. This inhibition was not due to cytotoxicity. When the lung washings were injected into normal rabbit peritoneal cavities, there was no inhibition of accumulation of either PMNs or peritoneal macrophages as stimulated by mineral oil. In addition, the lung washings stimulated RNA synthesis in vitro by rabbit AM.

Influence of Autologous Serum on in Vitro Reactivity of Peripheral Lymphocytes of Patients with Breast Cancer

The influence of autologous serum on DNA synthesis and phytohemagglutinin (PHA) or pokeweed mitogen (PWM) stimulation of peripheral blood lymphocytes from mammary tumor-bearing women has been studied. Blood samples were collected from patients at the 4 stages of the TNM classification who had not undergone any therapeutic treatment. Lymphocyte functions were significantly reduced only in patients with the largest tumors and in the latest stage of disease. Autologous cancer sera inhibited spontaneous DNA synthesis of lymphocytes and did not inhibit lymphocyte stimulation by mitogens. It is possible that unrecognized inhibiting and stimulating factors, possibly acting in synergism with mitogens, are present in mammary cancer sera.

Activation of Human B Lymphocytes

Abstract A recently described system for the induction and assay of plaque-forming cells (PFC) following polyclonal stimulation of human B lymphocytes was employed to delineate the cellular requirements for pokeweed mitogen (PWM)-induced activation of human tonsillar and peripheral blood B lymphocytes. It was found that the process was not dependent on the presence of monocytes, but was dependent on the presence of T cells. T cell depleted suspensions of tonsillar as well as peripheral blood lymphocytes had markedly diminished if not absent PFC responses following PWM stimulation. The PFC responses of T cell depleted cultures of tonsil and blood could be reconstituted by adding back T cells. T cell supernatants could also reconstitute the PFC response of T cell depleted tonsillar lymphocytes but not of T cell depleted blood lymphocytes, thus suggesting a more stringent T cell requirement of the PWM-induced PFC response of blood lymphocytes. Enrichment for T cells on the other hand enhanced PFC responses in both organs, particularly in those cultures which had low responses in unfractionated suspensions demonstrating the requirement of optimal T cell help for maximal PFC responses. This study demonstrates the feasibility and effectiveness of this PFC system in elucidating some of the complex regulatory processes associated with the activation of human B lymphocytes.

The immunosuppressive mechanism of azathioprine. I. In vitro effect on lymphocyte function in the baboon.

The effect of azathioprine on in vitro baboon lymphocyte function tests was evaluated using the mitogen stimulation test, the mixed lymphocyte culture test, the migration inhibition factor test, the cell-mediated lymphocytotoxicity test and the antibody dependent cell-mediated cytotoxicity test. It was found that azathioprine inhibited phytohemagglutinin and Concanavalin A stimulation at lower concentrations than those required to inhibit pokeweed mitogen stimulation. It inhibited the MLC reaction with as little as 0.2 mug/culture in the microculture system. Azathioprine had no effect on (a) the release of migration inhibition factor, (b) the cell-mediated lymphocytotoxicity assay if presensitized cells were used, and (c) the antibody-dependent cell-mediated cytotoxicity assay. However, azathioprine inhibited CML if it was added during in vitro sensitization and induction of killer cells. These in vitro results suggest that azathioprine inhibits those reactions which require cellular division.

In vitro studies of lymphocytes from children with kwashiorkor

In vitro lymphocyte functions for children with kwashiorkor have been studied and compared to controls. Peripheral blood lymphocyte counts are similar to controls. Atypical mononuclear cells have been observed frequently in the peripheral blood smear. Electron microscopic observation revealed an increased number of plasmacytoid cells and occasionally lymphoblasts among isolated lymphocytes. In vitro response to phytohemagglutinin was decreased significantly, whereas pokeweed mitogen stimulation demonstrated higher response in 60% of the kwashiorkor patients studied and [ 3H]thymidine incorporation comparable to controls in 40%. The absolute number of rosette forming lymphocytes (T and B cell rosettes) was reduced in the kwashiorkor patients. Lymphocyte depletion of the thymus-dependent areas and reduction in the number of primary follicles and germinal centers were the main results of the histopathological examination of the thymolymphatic tissues. These findings suggest an acquired defect in cell-mediated immunity in kwashiorkor children.

Leukemic reticuloendotheliosis: A study of the origin of the malignant cell

A highly pure preparation of neoplastic cells from the spleen of a patient with leukemic reticuloendotheliosis was studied for function, membrane characteristics and glucose metabolism. Glass adherence and phagocytosis of small particles (latex and carbon black) were demonstrated with phase contrast microscopy. Staphylocidal activity was similar to that of normal monocytes. Immunofluorescent assays revealed nonspecific uptake of antiserums to immunoglobulkis G (IgG), M (IgM), A (IgA) and kappa and lambda light chains. Rosette assays Indicated the presence of receptors for IgG on the surface of all cells but no receptors for complement (C3) or sheep red blood cells. Glucose metabolic studies revealed a pattern that differed from that of normal monocytes or lymphocytes with intermediate values for glycolysls, low hexose monophosphate shunt activity and high Krebs cycle activity. Increments in tritiated ( 3H)-thymldine uptake and glucose metabolism in response to phytohemagglutinin stimulation were minimal (5 per cent of normal lymphocyte values) and no response was noted with pokeweed mitogen stimulation. These findings suggest that the leukemic reticuloendotheliosis cell most closely resembles cells of the monocyte-histiocyte series.

Genetic control of the immune response: in vitro stimulation of lymphocytes by (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L.

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4 degrees C followed by washing and incubation at 37 degrees C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition.

In vitro lymphocyte response to phytohemagglutinin and pokeweed mitogen in Hodgkin's disease. An electron microscopic and functional study.

Lymphocytes from eight patients with Hodgkin's disease were incubated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) over 7 days. Thymidine incorporation into DNA and ultrastructural features of transformed cells were studied. Response to these mitogens was either normal or diminished and/or delayed. In seven patients lymphocyte response to PHA was paralleled by a corresponding response to PWM. PHA-transformed lymphocytes showed fine structural features similar to transformed normal cells. After PWM stimulation, blast cells and plasmacytoid cells in various stages of differentiation were observed. The number of transformed cells corresponded to the magnitude of thymidine incorporation and in cultures with normal PWM response, plasmacytoid cells occurred with almost normal frequency. If the specificity of the mitogens is as postulated, then patients with Hodgkin's disease do not have a selective loss of T lymphocytes. Furthermore, the findings suggest that in some patients there may be a functional impairment of both T and B lymphocytes.