Histamine modulates in vitro IgG production by pokeweed mitogen-stimulated human mononuclear cells

The specificity of the receptor for IgA (RFcα) on human peripheral blood monocytes and polymorphonuclear (PMN) cells was evaluated by the ability of various human IgA preparations to inhibit rosette formation between these cells and IgA-sensitized ox erythrocytes. RFc?α on PMNS and monocytes were blocked by both monomer and dimer IgA preparations indicating that multivalent expression of Fc regions does not play a major role in receptor binding and that neither secretory component nor J chain significantly influences the binding of RFcα to IgA. Immunoglobulins of both the IgA1 and IgA2 subclasses inhibited IgA rosette formation and were in fact quite similar in their efficiency of blocking of RFcα. An IgA paraprotein without a Cα₃ domain was an even better inhibitor of IgA rosette formation than the IgA1 or IgA2 immunoglobulins. This implicated the Cα₂ domain as the site on IgA which interacts with RFcα. Furthermore, that this Cα₃-deficient IgA, which exists as a half molecule, was very efficient at blocking RFcα also demonstrated that multivalent Fc expression is not important to binding of RFcα and moreover that the site on IgA which interacts with RFcα is not dependent on H-chain pairing. RFcα on both PMN cells and monocytes were susceptible to proteolysis by pronase at concentrations which did not affect the receptor for IgG on these cells. Within 18 hr after removal of RFcα these cells had resynthesized and displayed this receptor. Although it is unclear whether IgA alone can mediate the effector functions of PMNs and monocytes in mucosal areas, the present studies define more clearly the specificity and regenerative capacity of RFcα and provide a basis for understanding the role of receptors for IgA and the cells with which they are associated in immune defense especially on the mucosal surfaces.     

Suppression of pokeweed mitogen-stimulated immunoglobulin production in patients with rheumatoid arthritis after treatment with total lymphoid irradiation

Patients with intractable rheumatoid arthritis (RA) were treated with total lymphoid irradiation (TLI, 2000 rad). We previously reported long-lasting clinical improvement in this group associated with a persistent decrease in circulating Leu-3 (helper subset) T cells and marked impairment of in vitro lymphocyte function. In the present experiments, we studied the mechanisms underlying the decrease in pokeweed mitogen stimulated immunoglobulin (Ig) secretion observed after TLI. Peripheral blood mononuclear cells (PBL) from TLI-treated patients produced 10-fold less Ig (both IgM and IgG) in response to pokeweed mitogen than before radiotherapy. This decrease in Ig production was associated with the presence of suppressor cells in co-culture studies. By using responder cells obtained from normal individuals (allogeneic system), PBL from eight of 12 patients after TLI suppressed Ig synthesis by more than 50%. In contrast, PBL from the same patients before TLI failed to suppress Ig synthesis. Suppression by post-TLI PBL was also demonstrated in an autologous system by using responder cells cryopreserved before TLI. Again, only cells obtained after TLI were suppressive in four of seven patients. PBL with suppressive activity contained suppressor T cells, and the latter cells bore the Leu-2 surface antigen. In 50% of the patients studied, suppressor cells were also found in the non-T fraction and were adherent to plastic. Interestingly, the Leu-2+ cells from TLI-treated patients were no more potent on a cell per cell basis than purified Leu-2+ cells obtained before TLI. Additional experiments suggested that the suppression mediated by T cells after TLI is related to the increased ratio of Leu-2 to Leu-3 cells observed after radiotherapy.     

Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies. These three types of antibodies were then used in a solid phase radioimmunosorbent test to quantitate the amount of "IgE" synthesized by peripheral blood mononuclear cells from nonatopic and atopic donors cultured for 7 days in the presence and absence of PWM. ANTI-PS antibodies detected a PWM-induced "IgE formation" in cell culture supernatants of both non-atopic and atopic donors. (ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of pokeweed mitogen inhibition of rhIL-4-induced human IgE synthesis.

Pokeweed mitogen (PWM) suppressed rhIL-4-induced IgE synthesis in a concentration-dependent manner. When rhIL-4 was present from Day 0, PWM added to cultures on Day 0 or 3 inhibited MNC IgE synthesis but not when it was added on Day 6 or later. The concentration of interferon-gamma (IFN-gamma) in MNC culture supernatants varied directly with the quantity of PWM added. Conversely, rhIL-4-stimulated MNC culture IgE concentrations varied inversely with the dose of PWM added and the IFN-gamma concentrations induced. The addition of a rabbit polyclonal neutralizing anti-human IFN-gamma antibody to rhIL-4 plus PWM-stimulated cultures partially or completely reversed PWM-induced inhibition of rhIL-4-induced IgE synthesis. PWM failed to inhibit rhIL-4-induced IgE synthesis by isolated B cells cocultured with monocytes and T cells from a clone unable to produce IFN-gamma message or protein. These findings are consistent with the postulate that PWM inhibits rhIL-4-induced IgE synthesis by inducing the production of IFN-gamma.     

Emperipolesis of lymphoid cells by human follicular dendritic cells in vitro.

Isolated follicular dendritic cells (FDCs) showed true and pseudoemperipolesis of fresh tonsillar lymphocytes, even after long-term (50-day) cultivation. Emperipolesis by FDCs was not restricted by allotype specificity, nor was it inhibited by the addition of antibodies against MHC-I & II antigens. Follicular dendritic cells predominantly engulfed B-cells; monocytes and macrophages were not found between FDC cytoplasmic extensions. When highly purified T-cell populations were added to FDC cultures emperipolesis of T-cells occurred, particularly those of the CD4-positive phenotype. Mitoses appeared within 6 h in the emperipolesed lymphocytes and, after an additional 18 h, some lymphocytes exhibited apoptosis.     

In vitro synthesis of human IgE by neonatal lymphocytes: enhancing effect of interferon-gamma

The present study demonstrates that neonatal human lymphocytes that are incapable of producing IgG and IgA antibodies may differentiate into IgE-secreting cells under the influence of IL4. Indeed, the addition of recombinant IL4 to cultures of unfractionated umbilical cord blood mononuclear cells (CBMC) induces a dose-dependent synthesis of IgE but not of the other classes of Ig. Moreover, IgE-secreting B lymphoblastoid cell lines can be derived from neonatal lymphocytes costimulated with EBV and IL4. Comparison of the mechanisms regulating the in vitro IgE synthesis by adult and neonatal lymphocytes indicates that in most cases IFN-gamma markedly potentiates the IgE synthesis in CBMC cultures whereas it has a reverse effect on adult lymphocytes. These reciprocal effects of IFN-gamma are specifically blocked by a neutralizing mAb to IFN-gamma; they are dose-dependent and they are observed when IFN-gamma is added at the initiation of the culture or shortly thereafter. Moreover, in a small number of cases IFN-gamma may also potentiate IgE synthesis by adult lymphocytes. The potentiation or the suppression of IgE synthesis by IFN-gamma is not explained by a differential effect of IFN-gamma on the production of soluble CD23 (sCD23); indeed in both cases IFN-gamma slightly increases the IL4-induced production of sCD23. Moreover, the spontaneous and the IL4-induced production of sCD23 by CBMC is comparable to that of adult lymphocytes. The IgE response is dependent upon the expression of Fc epsilon RII (CD23) inasmuch as it is specifically blocked by anti-CD23 mAb.     

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human an rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.     

Morphological aspects of human lymphoid cells grown in vitro

Summary An established immunoglobulin-producing human cell line exhibits a variety of morphological entities described as reticular, lymphoid, and plasmacytoid cells. Transitional forms are frequent, and time lapse photomicrography experiments demonstrate morphological interconversion. The round forms and their morphological precursors synthesize immunoglobulin and constitute about 44% of the cell population. This cell line displays a variety of morphological and functional properties assigned to in vivo lymphoid cell series. It is proposed as an in vitro model for the study of lymphoid cells Supported by Grant CA06939-05 and by Special Research Career Development Fellowship Award 1F03 CA42078 from the United States Public Health Service.     

Morphological aspects of human lymphoid cells grown in vitro

Summary An established immunoglobulin-producing human cell line exhibits a variety of morphological entities described as reticular, lymphoid, and plasmacytoid cells. Transitional forms are frequent, and time lapse photomicrography experiments demonstrate morphological interconversion. The round forms and their morphological precursors synthesize immunoglubulin and constitute about 44% of the cell population. This cell line displays a variety of morphological and functional properties assigned to in vivo lymphoid cell series. It is proposed as an in vitro model for the study of lymphoid cells. Supported by Grant CA06939-05 and by Special Research Career Development Fellowship Award 1F03 CA42078 from the United States Public Health Service.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

Activation of lymphoid cells by BCG in vitro

Bacillus Calmette-Guerin (BCG) stimulated splenic and thymic lymphocytes in vitro as measured by uptake of ³H-thymidine. This activation of lymphocytes by BCG required the presence of a critical concentration of macrophages. Thymus cells containing no more than 0.25% macrophages were stimulated by BCG, but reduction of macrophages below this level by adherence to plastic abolished the response. Reconstitution with purified macrophages completely restored the response. A high concentration of adherent cells (“macrophages”) depressed the response of splenic lymphocytes, as judged by the improvement in DNA synthesis after reduction of the proportion of adherent cells in the spleen cell population.Bacillus Calmette-Guerin augmented the production of lymphocyte-activating factor (LAF) from purified splenic adherent cells, but the presence of lymphocytes made that augmentation considerably greater.These data reaffirm the bidirectional nature of the relationship between lymphocytes and macrophages. They further show that BCG can create highly activated populations of each type of cell, in part by enhancing their interaction.     

Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

Modulation of IgE synthesis by IgE-binding factors released by T cells of asthmatic patients with elevated serum IgE

The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc epsilon R). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fc epsilon R+ T cells but not of Fc gamma R+ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc epsilon R+ T cells. With regard to circulating Fc epsilon R+ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77 +/- 0.15%) than in normal subjects (0.17 +/- 0.07%) in spite of a very small proportion of T cells bearing Fc epsilon R.