Pokeweed Mitogen-induced Immunoglobulin Production Research Articles

Immunological Responses Are Not Abnormal in Symptomatic Gulf War Veterans

The underlying etiology and pathogenesis of Gulf War veterans' illnesses continue to be under intense investigation. Reports have suggested the basis for these illnesses may be an altered immune system, but compelling evidence is lacking. We sought to determine whether in vitro immune responses were abnormal in symptomatic Gulf War veterans relative to matched controls. A randomized case-control study was conducted by blinded comparison of laboratory measures of in vitro immune responses in blood samples obtained from veterans in an outpatient facility of a Veterans Affairs medical center. Symptomatic Gulf War veterans with otherwise undefined illnesses (52 symptomatic subjects), asymptomatic Gulf War veterans (31 asymptomatic controls), and veterans who had applied for disability compensation and had not participated in the Gulf War (21 disability controls) represented the volunteer sample. In vitro cellular and humoral immune responses were measured to detect functional abnormalities in antigen presenting cells (autologous mixed leukocyte reactions and expression of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor-alpha); T cells (lymphocyte proliferation using the polyclonal T-cell activators phytohemagglutinin and Concanavalin A; primary immune responses in allogeneic mixed leukocyte reactions; secondary immune response using the recall antigens tetanus toxoid, Candida albicans, and anthrax vaccine; and soluble IL-2 receptor expression); type-1 T-helper cells (gamma interferon expression); type-2 T-helper cells (IL-4 and IL-10 expression); and B cells (polyclonal B-cell activator pokeweed mitogen-induced immunoglobulin production). In general, immune response measures did not differ significantly between groups. Heightened responses observed in the disability control group (sporadically greater responses to one mitogen and two antigens) and the Gulf War participation control group (greater recall responses to anthrax vaccine) did not suggest impaired immune cell function in symptomatic veterans when compared with controls. We conclude that in vitro immunological responses are not abnormal in symptomatic Gulf War veterans.

Human cytotoxic T cell responses to vaccinia virus vaccination

Recombinant vaccinia viruses have been proposed as live virus vectors in man to generate both specific humoral and cellular immune responses. The generation of such responses following revaccination was followed in five normal volunteers. All five showed elevated (35- to 6500-fold) vaccinia virus-specific IgG by 14 days post-vaccination, following uncomplicated vaccination by dermal scarification. Four of the five had elevated total serum immunoglobulins with increased spontaneous and pokeweed mitogen-induced immunoglobulin production. Major histocompatibility complex class I-restricted vaccinia virus-specific cytotoxic T lymphocytes (CTLs) were not detected either directly, from peripheral blood mononuclear cells, or following secondary in vitro stimulation, even by limiting dilution analysis. These studies would suggest that vaccination with recombinant vaccinia viruses may provide an insufficient stimulus to the generation of CTLs in vivo.

A multifunctional cytokine (IL-6/BSF-2) and its receptor.

Interleukin 6 (IL-6)/B cell stimulatory factor 2 is a multifunctional cytokine produced by both lymphoid and nonlymphoid cells. IL-6 regulates immune response, acute phase reaction, and hematopoiesis. It was found that IL-6 production by T cells is dependent on macrophages, and IL-6 is one of essential factors for pokeweed mitogen induced immunoglobulin production. Both high- and low-affinity IL-6 receptors were identified. The molecular cloning of IL-6 receptors demonstrated that this receptor is a member of the immunoglobulin superfamily. The deregulated production of IL-6 is suggested to be involved in the pathogenesis of autoimmune diseases and in the development of multiple myeloma.

In vitro effect of cyclosporin A on immunoglobulin production and concanavalin A induced suppression in primary biliary cirrhosis.

The in vitro effect of Cyclosporin A on the regulation of immunoglobulin production was investigated in 16 patients with primary biliary cirrhosis. A significant improvement in concanavalin A induced suppression of IgG and IgM producing cells was observed after prior incubation of mononuclear cells with 300 ng/ml Cyclosporin A for 30 minutes. No effect was seen on spontaneous or pokeweed mitogen induced immunoglobulin production, nor on con A induced suppression if Cyclosporin A was added after 24 hours. Incubation of mononuclear cells with a variable dose of Cyclosporin A showed an effect only at 250-500 ng/ml. Higher and lower doses had no effect. This dose dependent effect of Cyclosporin A is likely to be related to a differential inhibitory effect on T helper and T suppressor cells and may underlie the clinical benefit being observed in current clinical trials.

The influence of autologous cell interactions on spontaneous and pokeweed mitogen-induced immunoglobulin production

Both helper- and suppressor-T-cell activities are generated in the autologous naked lymphocyte reaction and in pokeweed mitogen (PWM)-stimulated cultures. The addition of low numbers of irradiated non-T cells enhance while high numbers suppress spontaneous and PWM-stimulated IgG synthesis by autologous cells. Monocytes are the principal inducers of suppression and exert their influence within the first 24 hr of culture. Suppression in association with PWM stimulation is nonspecific in nature, T-cell mediated, partially radiosensitive, and resistant to hydrocortisone. Neither indomethacin nor dibutyryl cyclic AMP reverses monocyte-related suppression. These findings suggest that the outcome of in vitro Ig synthesis assays is critically dependent upon monocyte-T-cell interaction.

Augmentation of pokeweed mitogen-induced immunoglobulin production by cord T-cell supernatants.

Adult mononuclear cells were stimulated with pokeweed mitogen (PWM) in the presence of supernatants from cord T cells which had been previously stimulated with PWM for 24 h. Increased IgM, IgG and IgA production, as measured by ELISA, was observed with the addition of increasing concentrations of cord T-cell supernatants. The most significant increase in immunoglobulin production was observed with IgM (p less than 0.001). Cord T lymphocytes appear to be capable of producing soluble helper factors which augment immunoglobulin production by adult lymphocytes.

Inhibition by DL-α-difluoromethylornithine of the pokeweed mitogen-induced immunoglobulin production in cultured human lymphocytes

The effects of DL-α-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, on immunoglobulin production were studied in vitro using human peripheral blood lymphocytes stimulated with pokeweed mitogen. DFMeOrn inhibits in a concentation-dependent manner the usual pokeweed mitogen-induced increases of polyamine contents (putrescine, spermidine, spermine) and of [3H]thymidine incorporation. In parallel with the reduction of polyamine content and of thymidine incorporation, IgG and IgM productions are diminished, a 70% decrease being observed at 5 mM DFMeOrn concentration. Therefore, it appears that inhibition of polyamine biosynthesis may ultimately interfere with the cellulr immunologic response by blocking cell proliferation. These findings certainly deserve further consideration both under in vitro and in vivo conditions.

Characterization of a long-term T-helper-cell line which produces IL-2 and induces immunoglobulin secretion

Six OKT4 + human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.

Abnormalities of in vitro immunoglobulin synthesis by peripheral blood lymphocytes from untreated patients with Hodgkin's disease.

The immunoglobulin-synthesizing activities of peripheral blood mononuclear cells from 57 untreated patients with Hodgkin's disease and 47 normal subjects were compared. Cumulative amounts of IgM and IgG synthesized and secreted by unstimulated and pokeweed mitogen-stimulated cells over a 7-d period were determined in a solid-phase radioimmunoassay. Synthesis of IgM in unstimulated cultures and of both IgM and IgG in cultures stimulated with pokeweed mitogen was markedly reduced in patients with Hodgkin's disease, whereas the mean level of the spontaneous IgG synthesis was enhanced. The degree and frequency of in vitro abnormalities were not influenced by disease stage or histology. Depression of pokeweed mitogen-induced immunoglobulin synthesis did not correlate with excessive number of monocytes and it was unaffected by removal of phagocytic cells or addition to the cultures of monocytes from normal individuals. On the other hand, monocytes isolated from blood of patients with Hodgkin's disease were even more effective than normal monocytes in supporting pokeweed mitogen-induced immunoglobulin synthesis by normal phagocyte-depleted mononuclear cells. Synthesis of both IgM and IgG induced by pokeweed mitogen remained subnormal after addition to patient B cell cultures of autologous irradiated T cells or allogeneic normal T lymphocytes. T cells from patients with Hodgkin's disease appeared at least as effective as normal T cells in helping pokeweed mitogen-induced immunoglobulin production by normal B cells. However, when normal T cells were co-cultured with B cells from patients with Hodgkin's disease, spontaneous IgG synthesis declined, whereas the addition of patient T cells to normal B cells resulted in an increase of spontaneous IgG synthesis. In patients showing depression of pokeweed mitogen-induced immunoglobulin synthesis the lymphoproliferative response and immunoglobulin synthesis stimulated by Staphylococcus aureus bacteria of the Cowan first strain, a T cell independent B cell mitogen, were also markedly reduced. These studies demonstrate impairment of immunoglobulin synthesis by cultured lymphocytes from untreated patients with Hodgkin's disease after stimulation with polyclonal B cell activators and suggest that the in vitro abnormalities may be, at least in part, the result of a preexisting in vivo activation of lymphocytes in Hodgkin's disease patients.

Competence of B cells for T-cell help in pokeweed mitogen-induced immunoglobulin production

Pokeweed mitogen (PWM)-induced polyclonal immunoglobulin (Ig) production of B cells is highly reproducible and the response has been shown to be dependent on T cells. We investigated whether or not human helper T cells are able to collaborate with B cells in PWM-induced polyclonal Ig production across allogeneic barriers, using T and B cells obtained from healthy individuals and a(hypo)-gammaglobulinemia patients. In results, it has become clear that the B-cell competence is required for T-cell help among healthy individuals and between healthy and a(hypo)-gammaglobulinemic individuals. These phenomena were associated with suppressor or killer T cells rather than HLA-A, -B, -C, and -DR antigens. They also imply that in order to investigate the T-cell helper activities of a(hypo)-gammaglobulinemia patients their T cells should be tested in cocultures not only with B cells of the third party individuals but also with B cells of patient family members.

A human T cell-derived soluble factor able to suppress pokeweed mitogen-induced immunoglobulin production.

Human peripheral blood T cells or Fc gamma receptor-positive T cells activated with the mitogen concanavalin A produce a soluble suppressor factor that inhibits polyclonal B cell immunoglobulin production. The factor suppresses pokeweed mitogen- (PWM) induced plaque-forming cells (PFC) by a noncytotoxic effect. It does not significantly affect lymphocyte proliferation, and exerts its effect independently of the presence of T cells. It is active in suppressing PFC when added from day 0 to day 3 of PWM cultures. Suppression is blocked by the addition of the monosaccharide L-rhamnose. The factor is stable at pH 2.0 and stable at 56 degrees C, but is inactivated by 2-mercaptoethanol. The approximate m.w. by gel filtration is 20,000 to 25,000. The above characteristics distinguish this suppressor factor from soluble immune suppressor supernatant, lymphotoxin, and gamma-interferon. They do not distinguish the suppressor factor from migration inhibition factor, which is also present in the supernatants.

Functions of T cells and B cells of a (hypo)-gammaglobulinemia patients in pokeweed mitogen induced immunoglobulin production system

Functions of T cells and B cells of a (hypo)-gammaglobulinemia patients were investigated, using pokeweed mitogen (PWM) induced immunoglobulin production system. As the B cell competence is required for T cell help among allogeneic individuals, T cells or B cells were tested in co-cultures not only with B cells or T cells of the third party individuals but also with B cells or T cells of patient family members. Detections of immunoglobulin secreting cells were performed by plaque assay using protein A coated sheep red blood cells. In three of four cases with common variable immunodeficiencies, B cell functions were disturbed, in another case T cell helper or suppressor function was disturbed and B cell function was slightly disturbed. In two cases with Bloom's syndrome and one case with 18 ring chromosome, B cell functions were disturbed.

696 IDENTIFICATION AND CHARACTERIZATION OF HUMAN SUPPRESSOR LYMPHOCYTES

The capacity of human peripheral blood lymphocytes (PBL) to suppress in vitro immune responses was investigated.PBL were incubated with Concanavalin A (Con A) (50 ug/ml) for 48 hours, washed in Hank's Balanced Salt Solution containing α-methyl-D-mannoside (5 mg/ml), and subsequently added at a 1:1 ratio to cultures of normal untreated lymphocytes. Con A treated cells suppressed the proliferative response of untreated lymphocytes to mitogens (phytohemagglutinin, Con A), antigens (tetanus toxoid) and alloantigens (one-way mixed lymphocyte culture), as measured by 3H-thymidine incorporation into DNA. Con A treated cells also suppressed pokeweed mitogen induced immunoglobulin production in vitro by normal lymphocytes, as measured by radio-immunoassay.Con A treated cells lost their suppressor activity following treatment with mitomycin C (50 ug/ml) or X-irradiation (> 500 RADS). It was shown that the Con A activated suppressor cell was a T cell which was nylon nonadherent and which bore surface receptors for histamine. Treatment of PBL or of T cells with antisera to B cell alloantigens (Ia like antisera) either before or after activation with Con A did not interfere with the suppressor activity of these cells.Assay of suppressor activity of Con A treated PBL could be helpful in the study of immunodeficiency diseases, graft-versushost disease, autoimmune diseases and atopic states.