Anti-pokeweed mitogen antiserum inhibits and enhances blastogenesis of mononuclear cells induced by pokeweed mitogen

The interaction between pokeweed mitogen (PWM) and peripheral blood mononuclear cells (PBMC) was investigated using rabbit anti-PWM antiserum (anti-PWM) and¹²⁵ I-PWM. Incubation of PBMC with PWM in the presence of anti-PWM resulted in an inhibition of the mitogenic effect of PWM. Anti-PWM predominantly blocked the interaction of PWM with monocytes, which is essential for optimal stimulation of lymphoid cells with PWM.Addition of anti-PWM to PBMC at several time-points after incubation with PWM showed inhibition of mitogenic activity when anti-PWM was added within 8 hours. However, enhancement of PWM-induced blast cell formation was found when anti-PWM was added after 48 hours. Further analysis revealed that the inhibition of PWM stimulation was mediated by the F(ab)₂ part of anti-PWM IgG. On the other hand F(ab)₂-anti-PWM was not able to enhance the effect of PWM.Incubation of PBMC with¹²⁵I-PWM and anti-PWM simultaneously, decreased the binding of PWM to both lymphocytes and monocytes. In contrast, addition of anti-PWM 48 hours after the incubation of PBMC with PWM resulted in an increased binding of PWM to monocytes.These results show that anti-PWM can moculate the lymphocyte reaction to PWM and suggest two possible mechanisms by which PWM can stimulate PBMC, both of which are dependent on the interaction of PWM with monocytes.     

Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen. Identification of proliferating cells by monoclonal antibodies

Using a two-colour immunofluorescence technique, we have investigated the mitogenic effects of phytohaemagglutinin-M (PHA-M) and of pokeweed nitrogen (PWM) on human lymphocyte subsets. These were identified by CD1, CD3, CD4, CD8, and CD16 monoclonal antibodies, and proliferation was demonstrated by a polyclonal anti-transferrin antibody. Evidence has been obtained for the generation of a population expressing both the CD4 and CD8 antigens simultaneously, in short-term cultures of peripheral blood mononuclear cells in the presence of PWM and of PHA-M.     

Prostatic infiltration in chronic lymphatic leukaemia.

Forty six men with chronic lymphatic leukaemia (CLL) were studied for up to seven years. Six patients required surgery for prostatic outlet obstruction. Histological examination of the prostatic chippings showed variable degrees of infiltration with small mature lymphocytes in all six patients, suggestive of a leukaemic origin for the cells. Patients with chronic lymphatic leukaemia who undergo prostatectomy may have a higher incidence of leukaemic infiltration than has been previously recognised.     

Influence of donor characteristics on responsiveness of mononuclear cells to pokeweed mitogen.

The variability in response of peripheral blood mononuclear cells (PBMC) from 95 healthy donors to pokeweed mitogen (PWM) was investigated. Age, sex, and blood group of the donors, the numbers of PBMC recovered from 500 ml blood, the percentage of monocytes therein, the presence of antilymphocyte antibodies (L-Cyt), or of anti-cytomegalovirus (CMV) antibodies, as well as HLA-B8 and HLA-DR4 were evaluated for their relation with the level of blast cell formation, proliferative response, and immunoglobulin G (IgG) synthesis, induced by PWM. The results showed that increasing age of the donors and the presence of anti-CMV antibodies are significantly associated with low proliferative responses of PBMC, whereas the HLA-B8 antigen and female donor sex were found to be associated with high blast cell formation after PWM stimulation. High percentages of monocytes in isolated PBMC at the onset of stimulation were inversely associated with IgG production, whereas the HLA-DR4 antigen was associated with high levels of IgG production, induced by PWM. The observed relations indicate that for comparison of responses of unseparated PBMC to PWM between patients and healthy controls, age, sex, and laboratory data, including the HLA background of donors, may have to be taken into account.     

Cell numbers in human lymphocyte cultures stimulated with pokeweed mitogen.

As determined by electronic cell counting, the cell numbers in pokeweed mitogen (PWM) stimulated cultures of normal lymphocytes decreased by about 13% during the first day and then remained almost constant up to day 8. In contrast, a progressive decrease of the cell count was observed in cultures of chronic lymphocytic leukaemia (CLL) lymphocytes reaching about 40% of the initial number on day 8. In PWM cultures of normal lymphocytes the transformed cells increased to about 20% of the cells present on day 4, whereas in cultures of CLL lymphocytes these cells reached only 11% on day 7.     

Variations in interferon production by lymphocytes from patients with chronic lymphatic leukaemia

In a group of patients with chronic lymphatic leukaemia leucocyte cultures showed a diminished capacity to synthesize interferon when compared with controls. The leucocyte culture with the lowest capacity to synthesize interferon came from the patients with the highest peripheral blood lymphocyte counts and vice versa. These results are presented as indirect evidence that T lymphocytes are more competent producers of interferon than B lymphocytes.     

Inactivation of the mitogenic property of pokeweed mitogen by erythrocytes

In whole blood culture, pokeweed mitogen (PWM) is unable to stimulate human lymphocytes. This is because the mitogenic property of PWM is inactivated by erythrocytes, presumably due to absorption. The inactivation was observed with as few as 5 x 10(6)/ml of human erythrocytes. Therefore, when PWM is used to study the functions of human lymphocytes, especially of suppressor T lymphocytes, erythrocytes should be removed from lymphocyte preparations for accurate analysis.     

Kinetics of B-lymphocytes stimulation by pokeweed Pa-1 mitogen and bacterial lipopolysaccharide.

The presence of two subpopulations in mouse B lymphocytes responding to pokeweed Pa-1 mitogen or bacterial lipopolysaccharide (LPS) in terms of DNA synthesis was demonstrated. The analysis of surface markers of these two subpopulations revealed that one of the subpopulations carried the complement receptor (CR+) and the other lacked the complement receptor (CR-). The kinetics of stimulation by Pa-1 and LPS differed between these two subpopulations. CR+ -B cells exhibited the first peak of DNA synthesis 27 h after the addition of the mitogen, whereas CR- -B cells showed the first maximal response after 39 h. When the responses of CR+ -B cells 27 h and of CR- -B cells 39 h after various pulsed exposures to the mitogen were compared with those after continuous exposures to the mitogen, two successive 3 h exposures (0-3 and 12-15 h for CR+ -B cells; 0-3 and 21-24 h for CR- -B cells) yielded the same level of response as continuous exposure for 27 h (CR+ -B cells) or 39 h (CR- -B cells). These results indicate that both B-cell subpopulations require two signals for maximal stimulation.     

In vitro activation of human T and B lymphocytes by pokeweed mitogen.

In previous in vitro studies the DNA synthesis in human blood lymphocytes induced by low concentration of PWM correlated with the percentage of bone marrow-derived (B) lymphocytes in cell suspensions. No correlation with lymphocyte subpopulations was noted when lymphocytes were activated by high concentrations of PWM. In this paper the hypothesis that low concentration of PWM mainly activates B lymphocytes was tested by measuring the [14C]thymidine incorporation into purified T or B lymphocytes from healthy donors. B lymphocytes were purified to 90--95% by buoyant density centrifugation of T lymphocytes rosetted with sheep red blood cells. T lymphocytes were enriched by passage of lymphocytes through an IgG-anti-IgG-coated column. Low concentration of PWM-stimulated B lymphocytes but not T lymphocytes, while high concentrations of the stimulant activated both cell types. It was also noted that the B- but not the T-lymphocyte fraction contained cells which synthesized DNA in the absence of PWM.     

Induction of protein disulphide-isomerase and immunoglobulins by pokeweed mitogen in human lymphocytes.

The Protein disulphide-isomerase (PDI, EC 5.3.4.1, Thiol-proteindisulphide oxidoreductase, EC 1.8.4.2) is thought to regulate the sulfhydryl status of cells and to catalyze thiol/disulphide exchange reactions involved in the post-translational processing of disulphide containing secretory proteins. The aim of the present investigations was to study the possible function of this enzyme in differentiation of B lymphocytes and immunoglobulin synthesis. Non-adherent human mononuclear cells or purified T cells were cultured in presence and absence of Pokeweed mitogen over 3, 5 and 7 days. Monoclonal antibodies and a rabbit polyclonal antiserum specific for human liver PDI were produced to determine the concentration of PDI by an ELISA technique and cytoplasmic immunofluorescence. After PWM stimulation, both, the cellular content of PDI as well as that of immunoglobulin, particularly IgM, have been found to be induced in a time dependent manner with a 2-3 fold increase in comparison to unstimulated cells. The specific induction of PDI in human B lymphocytes was also confirmed in Western blotting. Our findings suggest that PDI plays a critical role in the final stages of B cell differentiation and immunoglobulin synthesis by activated B cells and plasma cells, respectively.     

Mouse rosette positive B cells fail to synthesize immunoglobulin following incubation with pokeweed mitogen

Mouse erythrocytes form spontaneous rosettes with a population of B lymphocytes from normal individuals and from patients with B cell chronic lymphocytic leukaemia (CLL). Since lymphocytes from patients with CLL respond poorly to pokeweed mitogen (PWM), we have compared mouse rosette positive (MR+), mouse rosette negative (MR-), and unfractionated B lymphocytes from normal individuals in their response to PWM. Mononuclear cells were fractionated into B, MR+ and MR- cell populations and then combined in 1:1 proportions with mitomycin-C treated T cells in culture media. Lymphocyte co-cultures were incubated for up to 10 days in the presence of PWM. Supernatant immunoglobulin (SIg) levels, percentage intracytoplasmic immunoglobulin (ICIg), and proliferative responses were determined. MR+ cells alone failed to produce significant levels of SIg (P less than 0.025) or percentages of ICIg positive cells This decreased synthesis of immunoglobulin by MR+ cells occurred in the presence of adequate T cells, macrophages and a satisfactory proliferative response.     

Community-acquired adenovirus pneumonia in a patient with chronic lymphatic leukaemia

Described here is a severe case of community-acquired adenovirus pneumonia that occurred in a previously healthy 54-year-old male who was later determined to have stage A chronic lymphatic leukemia. The clinical presentation was consistent with that of atypical pneumonia. Testing with PCR revealed adenovirus in a bronchoalveolar lavage sample, while all other tests to determine a bacterial or virological etiology were negative. Further examination of the patient revealed the previously undiagnosed chronic lymphatic leukemia. Following treatment with human immunoglobulin and oxygen therapy with continuous positive airway pressure support the patient recovered from the pneumonia completely.     

Cytotoxicity of human lymphocytes induced by pokeweed mitogen or in mixed lymphocyte culture. Specificity and nature of effector cells

Purified human peripheral blood lymphocytes were activated in vitro with pokeweed mitogen (PWM) or with mitomycin-treated allogeneic lymphocytes (MLC). After incubation for several days, stimulation to DNA synthesis and cytotoxicity for phytohemagglutinin (PHA)-activated lymphoblasts or for Chang cells was tested. Lymphocytes activated with PWM for 3–6 days were cytotoxic for both autologous and allogeneic PHA-induced lymphoblasts. In addition, they destroyed human tissue culture cells (Chang cells) effectively. In contrast, lymphocytes activated in MLC were cytotoxic for lymphoblasts bearing stimulator cell antigens, but not for autologous PHA-lymphoblasts. Cytotoxicity for Chang cells was variable. Lymphocytes which proliferate and become cytotoxic after activation in MLC are of T cell origin, since their ability is not reduced by prior removal of non-T cells on columns charged with human IgG-anti-IgG complexes. On the other hand, this procedure reduced both DNA synthesis and cytotoxicity of PWM-activated lymphocytes. This suggests that cells equipped with surface immunoglobulin and/or Fc recptors were required for activation of these cultures, or alternatively, were among the cells which responded to PWM by DNA synthesis and cytotoxicity.     

Lectin-activated killer cells rapidly induced by pokeweed mitogen conjugated beads and their in vivo antitumor effects

Pokeweed mitogen (PWM) was found to induce rapidly killer cells in human peripheral blood mononuclear cells (PBMC). To avoid PWM contamination in infused lymphocytes, PBMC were stimulated with PWM-coated beads, CMC-1. One hour of stimulation with CMC-1 led to distinct cytotoxic activity in PBMC at 7 h, this reaching a peak at 23 h in the following in vitro culture. The cytotoxic activity and target cell spectrum of CMC-1-activated killer (PWM-AK) cells were similar to those of lymphokine-activated killer (LAK) cells, the precursor cells of PWM-AK cells, as are those of LAK cells which are also low-density lymphocytes. The in vivo antitumor effects of PWM-AK cells were examined in nude mice with peritoneal carcinomatosis generated by the human colon cancer cell line, RPMI 4788. The intraperitoneal (i.p.) injection of PWM-AK cells immediately after stimulation with CMC-1 significantly prolonged the survival of tumor-bearing mice, suggesting that these cells could be of value for clinical cancer therapy.     

Induction of IgM production by pokeweed mitogen and interleukin-2: different responses by human peripheral blood cells and tonsil cells

Stimulation of tonsils or peripheral blood lymphocytes with either pokeweed mitogen (PWM) or interleukin-2 (IL-2) results in significant IgM production. However, the combination of PWM and IL-2 shows a synergistic IgM synthesis in tonsil cells, whereas the secretion in peripheral blood cells is diminished compared to activation with either stimulus alone. The heavy-chain isotype distribution does not significantly change in the two cell types when stimulated with either PWM, IL-2, or the combination. The Ig synthesis by tonsil cells to PWM or IL-2 drastically increases in the presence of monocytes, but the response to PWM + IL-2 together is not affected. The reason for the synergistic IgM production to PWM + IL-2 in tonsil cells is the relative lack of monocytes and the low number of T8+-suppressor cells. The decreased IgM production in peripheral blood cells after stimulation with both PWM and IL-2 is due to the presence of T8+ cells. These data show that the monocyte-dependent IL-2 production is an essential step in PWM-induced Ig secretion; the subsequent induction of the T-cell-helper signal for B-cell differentiation is determined by the balance between the strength of the stimulus for T cells and the T4/T8 ratio. Hence, these parameters should be included when the B-cell function is studied in vitro with the use of PWM and IL-2.     

Interaction of pokeweed mitogen with monocytes in the activation of human lymphocytes

The present study examines the role of monocytes in the in-vitro activation of human T cells and B cells by pokeweed mitogen (PWM). The T cell-dependent PWM-induced B-cell activation process was found to be monocyte dependent. Fluorescence-activated cell sorter (FACS) analysis revealed that upon addition to peripheral blood mononuclear cells, fluoresceinated PWM, at concentrations that provided optimal B-cell and T-cell activation, bound predominantly to human monocytes. The binding of PWM to monocytes was reversible and could be displaced within the first few hours of binding by oligomers of N-acetylglucosamine (GlcNAc). As a functional correlate of the binding studies, it was shown that PWM-pulsed monocytes could induce B lymphocytes to become plaque-forming cells (PFC) and T lymphocytes to undergo proliferation. In contrast, markedly reduced PFC and blastogenic responses were observed when monocyte-depleted B lymphocytes and T lymphocytes were respectively pulsed with PWM and washed, followed by the addition of non-PWM-pulsed monocytes to the cultures. Thus, the initial event in the PWM-induced activation of human lymphocytes, for both in-vitro T-lymphocyte blastogenic responses and B-lymphocyte Ig secretion, appears to be binding of the mitogen to sugar residues on the surface membrane of the monocyte, followed by subsequent interaction with the appropriate lymphocytes. The process of PWM binding to monocytes did not appear to affect the baseline production of interleukin-1 (IL-1) by human monocytes, nor could soluble factors from PWM-pulsed monocytes substitute for intact cells in the initiation of the lymphocyte-activation process.     

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.