Kinetics of immunoglobulin synthesis and secretion by resting and pokeweed mitogen-transformed human lymphocytes

The kinetics of immunoglobulin (Ig) synthesis and secretion were examined in human plasma cells, resting lymphocytes, and pokeweed mitogen (PWM)-transformed lymphocytes, in an attempt to define the Ig production properties of the PWM-transformed lymphocyte which is often used as a model for the plasma cell. By pulse labeling with [³H]leucine, the time course of intracellular Ig synthesis was shown to be similar for lymphocytes and plasma cells. However, plasma cells differed basically by exhibiting secretion of labeled Ig into the culture medium exceeding intracellular Ig levels after several hours of pulsing, whereas resting lymphocytes were nonsecretory. PWM-transformed peripheral blood lymphocytes were shown to have a spectrum of morphologic differentiation from numerous large lymphoblasts to relatively few plasmacytoid cells. In addition, they were found to have enhanced intracellular Ig synthesis (2.5-fold increase over baseline resting state) but no significant rise in secreted Ig, thus exhibiting enhanced Ig production but retaining a lymphocyte-like Ig kinetic profile. This suggests that the PWM-transformed peripheral blood lymphocyte represents only a partial differentiation toward the mature antibody-secreting plasma cell.     

Synthesis and secretion of alpha 1-microglobulin by human lymphocytes.

alpha 1-Microglobulin (alpha 1-m) was purified by column chromatography from a supernatant fluid of cultured T and B lymphocytes stimulated with mitogens. This protein had a molecular weight of 33,000 as determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, migrated in the alpha 1-region on immunoelectrophoresis and proved immunologically identical to alpha 1-m which had been purified from the urine of patients with renal tubular disorders. Using indirect immunofluorescence, alpha 1-m was detected on the surface of both T and B lymphocytes, displaying the same intensity on each cell type. These findings indicate that alpha 1-m is actively produced and secreted by T and B lymphocytes.     

In vitro synthesis of IgE by human lymphocytes. I. The spontaneous secretion of IgE by B lymphocytes from allergic individuals: a model to investigate the regulation of human IgE synthesis

In view of the controversial data in the literature regarding the in vitro IgE synthesis by human lymphocytes, the conditions for culture of lymphocytes and the methodology for measurement of the IgE produced are described in detail. In the absence of any added mitogen, enriched B cell preparations derived from 70% of allergic donors actively secreted 100 to 3200 pg/ml of IgE after culture for 7 days, at which time the cell viability was higher than 85%. In comparable B cell cultures derived from non-allergic donors, only trace amounts of de novo synthesized IgE were detected in 20% of the cases. All B cell cultures actively secreted IgG, IgA, IgM and there was no apparent relationship between the secretion of IgE and that of the other classes of Ig. By contrast, the synthesis of IgE by unfractionated peripheral blood mononuclear cells of allergic individuals, which were stimulated with pokeweed mitogen (PWM) under several experimental conditions, was not consistently reproducible, i.e. the spontaneous synthesis of IgE in such cultures was either suppressed or enhanced by PWM. The most important finding was that the secretion of IgE was selectively enhanced by supplementing the B cell cultures with cell-free supernatants (CFS) of cultures of neonatal lymphocytes which had been preincubated with 10 micrograms/ml IgE. It is, therefore, concluded that B cell cultures from allergic individuals constitute an appropriate model for investigations of the mechanisms underlying the regulation of human IgE synthesis.     

Transcription, translation and secretion of both IgA subclasses in polyclonally activated human lymphocytes

To analyze human IgA subclass-specific mRNA we have developed RNA probes for quantitation of alpha 1 and alpha 2 heavy chain constant region genes by solution hybridization. Under our assay conditions, we can detect as little as 3 pg of specific mRNA and there is less than 2% cross-reactivity between the two IgA subclasses. Using this method, the relative proportions of IgA1 and IgA2 mRNA in pokeweed mitogen (PWM)-stimulated cells were found to be 66% and 34%, respectively, while in the Epstein-Barr virus (EBV)-stimulated cells they were 77% and 23%, respectively. Cytoplasmic staining of the plasma cells and determination of IgA subclass distribution by flow cytometry revealed an almost even distribution of the two IgA subclasses as induced by both activators. The culture supernatant contained 72% IgA1 and 28% IgA2 after PWM stimulation, while EBV stimulation induced 85% IgA1 and 15% IgA2. This report thus describes a method for the quantitative analysis of IgA subclass-specific mRNA. Furthermore, we present evidence that in response to in vitro stimulation of peripheral blood lymphocytes by polyclonal activators the IgA-producing B cells not only synthesize both isotypes but also have the potential to secrete them.     

Suppression by Trypanosoma cruzi of T-cell receptor expression by activated human lymphocytes.

The immunosuppression that develops during Chagas' disease and African sleeping sickness is thought to facilitate survival of the causative agents in their mammalian hosts. Whereas a number of manifestations of immunosuppression manifested during the course of these diseases has been reported in patients and animals, the mechanisms by which they are induced remain obscure. An in vitro system in which phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBMC) were co-cultured with purified Trypanosoma cruzi or T. brucei rhodesiense was used in the present work to establish whether these organisms were able to alter the capacity of activated helper/inducer (CD4+) or cytotoxic/suppressor (CD8+) cells to express T-cell receptor (TcR). Suppressed interleukin-2 receptor (IL-2R), known to be caused by both the trypanosomes and supernatants containing their secretion products, was the independent parameter used to demonstrate the occurrence of immunosuppression in all experiments. We found marked reductions in the percentage of TcR+ cells in T. cruzi-containing cultures as early as 18 hr after PHA stimulation. This alteration was still readily demonstrable after 72 hr of culture, i.e. when last tested for. Suppressed TcR expression occurred concomitantly with reduced levels of CD4 or CD8 molecules on the surface of helper/inducer and cytotoxic/suppressor T lymphocytes, respectively, indicating that the parasite had induced more than one alteration in the same cells. These effects were reproduced when the trypanosomes were separated from the PBMC by a 0.45 micron pore size filter or when filtrates from T. cruzi suspensions substituted for the parasite in the cultures, indicating that TcR suppression was mediated by a parasite secretion product(s). Interestingly, neither T. b. rhodesiense nor filtrates of suspensions of this organism altered significantly the level of TcR expression in cultures in which suppressed IL-2R expression by activated human T cells took place. Thus despite sharing the ability to impair IL-2R expression, T. cruzi and T.b. rhodesiense appear to differ in other mechanisms by which they affect human T-cell function. If occurring in infected hosts, the alterations that T. cruzi causes in the expression of TcR, CD4, CD8 and IL-2R--all molecules playing important roles in lymphocyte activation--could contribute to the development of the immunosuppression observed during the acute phase of Chagas' disease.     

Glucocorticosteroid enhancement of immunoglobulin synthesis by pokeweed mitogen-stimulated human lymphocytes.

We studied the role of the T lymphocyte in GCS enhancement of PWM-stimulated IgG synthesis by human peripheral blood mononuclear cells. Purified T or B lymphocyte subpopulations were pretreated with 10(-6) M prednisolone or recombined at various T:B ratios and 10(-6) M prednisolone was added. PWM-stimulated IgG synthesis was measured in the culture supernatants at 8 days by radioimmunoassay. Addition of prednisolone to cultures of autologous and allogeneic reconstituted mixtures of T and B lymphocytes resulted in enhancement of PWM-stimulated IgG synthesis. This effect was observed with constant and increasing numbers of lymphocytes in culture, independent of T:B ratio and occurred with purified B lymphocytes containing monocytes. Pretreatment of purified B lymphocytes containing monocytes but not purified T lymphocytes with prednisolone enhanced PWM-stimulated IgG synthesis in reconstituted mixtures of T and B lymphocytes. We propose that GCS enhancement of PWM-stimulated IgG synthesis by human mononuclear cells is independent of T lymphocyte regulation.     

Corticosteroid enhancement of immunoglobulin synthesis by pokeweed mitogen-stimulated human lymphocytes.

The effects of the addition in vitro of corticosteroid on pokeweed mitogen (PWM) induced Ig synthesis by human peripheral blood lymphocytes were studied. IgG in supernatants produced under standardized culture conditions was measured by double antibody radioimmunoassay. The addition of 10(-6)M prednisolone caused a remarkable enhancement of PWM-stimulated IgG synthesis beginning at day 4 of culture and increasing at a faster rate than that in cultures with PWM alone. 10(-6)M prednisolone resulted in a geometric mean enhancement of 5.6-fold of PWM-stimulated IgG synthesis in all twenty-five normal controls studied. This enhancement occurred up to 3 days after the addition of PWM. 10(-6)M and 10(-5)M prednisolone resulted in significantly greater enhancement of PWM-stimulated IgG synthesis than 10(-7)M prednisolone. Hydrocortisone, prednisolone, methylprednisolone, betamethasone and dexamethasone at 10(-6)M were all equally effective in the enhancement of PWM-induced IgG synthesis.     

Lysis of allogeneic human lymphocytes by nonspecifically activated T-like cells

In the generation of cytotoxic effector cells specific for influenza A virus-infected lymphocytes, three donors have given an unusual pattern of lytic activity, killing HLA-mismatched target cells. This has been analyzed in detail for one donor and one of the other two shows similar results. Activation only requires culture in medium between 1 and 4 days and parallels development of cell line K562-directed natural killer cells. Target lymphocytes do not need to be virus-infected and appear to be normal lymphocytes. The effector cells carry the surface markers T3 and T8 defined by OKT3/anti-Leu4 and OKT8/anti-Leu2a monoclonal antibodies, respectively. Unlike HLA class 1-restricted or -directed cytotoxic T cells, neither anti-Leu2a/nor anti-Leu4 blocked killing in the absence of complement. MHM23, a monoclonal antibody specific for the human lymphocyte function antigen, blocked lysis. The results indicate that these effector cells are related to cytotoxic T lymphocytes, but can lyse allogeneic target cells through a different recognition process. There is some specificity because autologous cells were not killed.     

Evidence that nonspecific inhibitors are induced by soluble products of activated human lymphocytes.

Cultures of lymphocytes stimulated by polyclonal mitogens or specific antigens exhibit an initial wave of cell proliferation followed by a decline which has been explained by the activation of suppressor T cells. This article presents the first results aiming at testing the possibility that the suppressor cells are activated by soluble products released by stimulated lymphocytes. It was found that the supernatants of PHA-activated human lymphocytes stimulate lymphocytes to DNA synthesis with peak responses occurring on day 5 independent of the cell concentration in the cultures. In contrast, the kinetics of lymphocyte responses to phytomitogens or in the mixed lymphocyte culture were found to be dependent on cell concentration; peak responses were delayed at decreasing cell concentrations. The results support the view that certain lymphokines released in stimulated lymphocyte cultures induce suppressor cells and that biologically active concentrations are reached earlier at high cell densities.     

Regulation of immunoglobulin secretion by T lymphocytes in human malaria

In vitro studies were carried out on the nature of immunoglobulin synthesis and secretion by peripheral blood mononuclear leukocytes (PBMLs) and on the function of T and B cells from malaria patients. The mean values of secreted IgG and IgM concentrations of 22 malaria patient PMBLs were significantly higher than those of 20 normal PBMLs. When the suppressor T cell activity and the function of B cells in response to suppressor T cells were assayed by the cell co-culture technique, it was found that there was a decrease in suppressor T cell activity and the B cell function in response to normal suppressor T cells in malaria patients. The defects of these T and B cell functions may play some role in the immunological abnormalities seen in some malaria patients.     

Free immunoglobulin light chain synthesis by human foetal liver and cord blood lymphocytes.

Immunoglobulin (Ig) synthesis by immature B lymphocytes from human foetal liver and cord blood has been investigated. Seven out of fifteen preparations of foetal liver cells and eight out of eleven cord bloods synthesized Ig detectable by biosynthetic labelling. All cultures of foetal lymphocytes with detectable Ig synthesis secreted free light chain. Two of these cases also synthesized free mu heavy chain. Cord blood lymphocyte synthesis patterns were variable ranging from free light chain as the only secreted Ig product to balanced synthesis of heavy and light chains. No cord blood cultures synthesized detectable free mu chains. Free Ig-light-chain synthesis appears to be associated with normal immature B lymphocytes and the results are discussed in relation to the B-cell differentiation pathway.     

Selective suppressive effects of Trypanosoma cruzi on activated human lymphocytes.

The acute phase of Chagas' disease is accompanied by immunosuppression. To explore the underlying mechanism(s), we used an in vitro culture system in which the capacities of activated human peripheral blood mononuclear cells to express interleukin-2 receptors (IL-2R) and proliferate are markedly inhibited in the presence of Trypanosoma cruzi, the etiologic agent. The present work was designed to define the earliest time at which T. cruzi-induced suppression is manifested in terms of IL-2R expression on the cell surface and establish whether expression of other lymphocyte activation markers is also suppressed by the parasite. We found that expression of IL-2R by human peripheral blood mononuclear cells cocultured with T. cruzi and stimulated with either phytohemagglutinin or anti-CD3 (a monoclonal antibody specific for an epitope of the T cell receptor complex T3-Ti) was significantly suppressed as early as 12 h after culture initiation. Both the percentage of IL-2R+ cells and the surface density of IL-2R, measured by flow cytometry, were affected. However, expression of EA1, a human lymphocyte activation antigen known to be expressed 4 to 6 h after stimulation, was not altered by T. cruzi whether phytohemagglutinin or anti-CD3 was used. On the other hand, expression of transferrin receptors (TfR), which first occurs between 20 and 24 h after lymphocyte activation, was markedly suppressed by T. cruzi. This effect was denoted by significant reductions in both the percentage of TfR+ cells and the cell surface density of TfR whether phytohemagglutinin or anti-CD3 was used as the mitogen and was observed at all test times, i.e., at 24, 48, 72, and 96 h. Because expression of IL-2R and TfR is required for lymphoproliferation but that of the EA1 lymphocyte activation marker is apparently not, these results are consistent with the possibility that T. cruzi, at a relatively early stage during lymphocyte activation, selectively affects certain key events on which clonal expansion is dependent. Inhibition of IL-2R and TfR expression by the parasite might play a role in causing the suppressive effects associated with acute Chagas' disease.     

Impairment of mitochondrial respiratory control by activated lymphocytes.

Mitochondria isolated from rat livers were used as a source of antigens capable of producing in vitro activation of lymphocytes that had previously been sensitized in vivo with the same antigen. Such lymphocytes were shown to have an injurious effect on target mitochondria, as was demonstrated by a new biochemical approach based on the study of the inhibition by these cells of mitochondrial respiratory control. A somewhat milder injurious capacity was also observed in normal and sensitized lymphocytes activated in vitro with the mitogen PHA. The lateration of the respiratory control of mitochondria by lymphocytes correlated with an increase in DNA synthesis in these cells.     

Bacterial lipopolysaccharide-induced immunoglobulin synthesis by human blood lymphocytes partially depleted of monocytes.

Bacterial lipopolysaccharide (LPS), a potent polyclonal B cell activator in rodents has not been found to be a consistent activator of human peripheral blood mononuclear cells (PBM). Since LPS activates monocytes to become suppressor cells, we asked whether depletion of monocytes would enhance the ability of LPS to induce in vitro activation and immunoglobulin synthesis and secretion by human B lymphocytes. Addition of 50 micrograms/ml LPS for 7 days to PBM cultures failed to induce a significant increase in IgM and IgG synthesis as measured by radioimmunoassay of culture supernatants. However, after partial depletion of adherent cells, the non-adherent cell population (NAC) produced large amounts of IgM and IgG (IgM: 696 ng/10(6) PBM vs 4236 ng/10(6) NAC, P less than 0.005; IgG: 68 ng/10(6) PBM vs 922 ng/10(6) NAC, P less than 0.02). The LPS-induced response was found to be T cell dependent and could be readily suppressed by the addition of autologous adherent cells. Addition of indomethacin to LPS-stimulated PBM did not result in increased Ig secretion. The poor response of human blood B cells to LPS may be due to the suppressive effect of activated monocytes.     

AMLR and MLR stimulating activity of human T lymphocytes activated in vitro by soluble HLA-DR antigens.

We have examined the allogeneic mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) stimulating activities of T cells precultured in vitro with soluble allogeneic or autologous HLA-DR antigens. These cells (Ts) are known to suppress the human MLR: this suppression is specific in that it occurs only when stimulator cells have the same HLA-DR antigen as that used to induce differentiation of suppressor cells. Ts cells express new membrane specificities; they can be separated by immunoabsorption into two populations: Ts enriched (Tx+; with suppressive activity) and Ts depleted (Ts-; with helper function). In the present study, we have demonstrated that both Ts cell subsets activated by soluble HLA-DR alloantigens are able to stimulate both MLR and AMLR. Ts cells activated by soluble autologous HLA-DR antigens are able to stimulate MLR, but not AMLR.     

Surface markers on human activated T lymphocytes. III. High-affinity E-rosette receptors

High-affinity E-rosette receptor (EhR) is suggested to be an activation marker of human T lymphocytes. The present study has been undertaken to estimate the dependence of EhR expression upon the T cell activation. To this aim the expression of EhR on T cells stimulated with PHA was examined. Nonsynchronized and arrested in G1 phase of cell cycle cultures of T lymphocytes were employed. The kinetics of expression of either de novo induced or reexpressed EhR was estimated by using two T cell subsets (originally EhR- and EhR+). In accordance with opinion of others, our results have shown that EhR is an activation marker of human T cells. Moreover, we have found that EhR is the marker of early and late activation stage because: its expression is induced in G1 phase of cell cycle, it continues to increase with the time of mitogen stimulation and the maximal level of EhR expression coincides with the time of the maximal RNA and DNA synthesis. Both induced de novo and reexpressed Eh receptors display the same expression kinetics.     

gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans.

Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non-major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens.