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.     

A hemopoietic cell line dependent upon a factor in pokeweed mitogen-stimulated spleen cell conditioning medium

A continously growing cell line (FMP1.1) has been isolated, which is dependent upon a factor in PWCM for both growth and survival. FMP1.1 appears to be a mast cell, since IgE receptors are present and their graunles react specifically with a mast cell granule stain. The factor in PWCM may be a glycoprotein and has biochemical properties in common with PWCM factors, which stimulate at least four other lineages of hemopoietic colonyforming cell. This line provides a pure population of cells as a model for studying molecular events of hemopoietic precursor cell growth and differentiation.     

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.     

Localization of human mononuclear cell interleukin 1

The detection and localization of interleukin (IL) 1 in human monocytes was carried out by flow cytometry using monoclonal antibodies to IL-1 alpha and IL-1 beta proteins. IL-1 alpha was detected on the surface of monocytes and the surface expression increased following lipopolysaccharide activation. No demonstrable IL-1 beta protein could be observed on the cell surface by antibody staining, while both IL-1 alpha and IL-1 beta could be visualized intracellularly by the appropriate monoclonal antibodies following acetone permeabilization of the monocytes. Further experiments with cell associated IL-1 revealed that most of the biological activity of human monocytes could be inhibited by affinity purified polyclonal antibodies to IL-1 alpha protein, whereas no inhibitory activity was observed with IL-1 beta specific antibodies. These data support the hypothesis that a differential localization of IL-1 alpha and IL-1 beta exists within human blood-derived monocytes.     

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.     

Regulatory role of microfilaments in the induction of T4 cell proliferation and interleukin 2 production

The role of microfilaments in human T4 cell proliferation and lymphokine production triggered via various pathways of activation was examined by investigating the effects of cytochalasins on these responses. The data demonstrate that the effects of cytochalasins vary depending on the nature of the stimulus and on the concentration of the cytochalasin. Concentrations of cytochalasin that would be expected to bind both the low and high affinity binding sites (5-20 microM), that represent cytosolic and surface actin filaments, respectively inhibited T4 cell proliferation regardless of the stimulus. T4 cell proliferation stimulated by antigen-bearing APC or anti-CD3 was inhibited much more markedly than responses stimulated by ionomycin and PMA. In contrast, concentrations of cytochalasin expected to bind only high affinity binding sites (0.125-1 microM), represented by surface actin filaments, enhanced T4 cell proliferation and interleukin 2 production stimulated by mAb to CD2, CD3, or class I major histocompatibility complex (MHC) molecules, but not those induced by mAb to the T cell receptor, paraformaldehyde fixed, or viable antigen-bearing APC, allogeneic APC, or ionomycin and PMA. The enhancing effect of cytochalasins on responses stimulated by cross-linking class I MHC molecules was studied in detail. Enhancement of T4 cell proliferation induced in this manner required that cytochalasin B was present between 4 and 18 hr of culture, but not before or after. The data demonstrate that T cell microfilaments play a number of roles in determining the magnitude of T cell responses induced by engaging specific cell surface receptors and imply that different components of the microfilament system exert opposing intrinsic regulatory effects on T cell function.     

Phorbol ester increases the level of interleukin 2 mRNA in mitogen-stimulated human lymphocytes

TPA alone did not induce the production of IL 2 in human tonsillar lymphocytes but enhanced the PHA-induced IL 2 production by seven-fold. That the effect of TPA was due to an increase in IL 2 mRNA was demonstrated by examining the amount of IL 2 mRNA translatable in Xenopus laevis oocytes, and by Northern blotting analysis using IL 2 cDNA as a probe. In these ways, it was shown that TPA alone did not induce any significant IL 2 mRNA synthesis, but when added together with PHA it increased the level of IL 2 mRNA by at least 10-fold, as compared with that induced by PHA alone.     

IL 2 production by mitogen-stimulated T cell subsets: helper-precursors are predominantly Lyt-2-

Although there is strong evidence that IL 2 can be secreted by at least some cells in both the Lyt-2- and Lyt-2+ T cell subsets, it is unclear whether lymphokine-secreting cells are present in equal frequencies in each population. We addressed this question by using a T cell mitogen, concanavalin A (Con A), that activates a large proportion of the cells of both subsets, as tested by their ability to grow in the presence of added conditioned medium. We found that, although Con A-responsive precursors of proliferating cells are at least as frequent in the Lyt-2+ set as in the Lyt-2- set, the frequency of Con A-responsive precursors of IL 2-secreting cells is much higher in the Lyt-2- population. Expressed as precursors per cell, lymphokine-secreting helpers are 17-fold more frequent in the Lyt-2- population. After adjusting for relative cell recoveries and for contamination, the data suggest that at least 97% (and more likely 99%) of the precursors for Con A-responsive IL 2-producing cells are in the Lyt-2- set. Thus, although the correlation between Lyt-2 surface antigen and helper function may not be absolute, our data support the idea that Lyt-2 does mark a functional distinction among T cells independent of antigen specificity.     

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.     

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by Semliki Forest virus

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.     

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 mixed 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.     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

The role of interleukin 6 in interferon-gamma production in thermally injured mice

Following traumatic injury, patients suffer from compromised immunity increasing their susceptibility to infection. Previous studies from this laboratory demonstrated that female BALB/c mice subjected to a 15% total body surface area (TBSA) scald injury exhibit a decrease in cell-mediated immunity 10 days post-burn. Studies described herein revealed that concanavalin A (Con A; 2 microg/ml)-stimulated splenocytes from sham treated animals produced 3557+/-853 pg/ml of IFN-gamma while splenocytes from burn injured animals released two-fold more cytokine (P<0.05). To determine whether leukocyte production of IFN-gamma was under the influence of macrophages, splenic macrophage supernatants generated from burned animals were incubated with splenic lymphocytes from sham and burn animals. The amount of IFN-gamma released by lymphocytes from sham animals increased when cultured with macrophages from burned mice (P<0.05). This suggests that the increase in IFN-gamma production by unfractionated splenocytes in burned mice relative to sham treated animals is macrophage-dependent. Macrophage supernatants from burned mice released twice as much IL-6 as supernatants from sham animals (P<0.05), and when IL-6 was blocked in vivo, the amount of IFN-gamma production in burned mice decreased to sham levels (P<0.05). Thus, IL-6 mediates IFN-gamma production following burn.     

The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R alpha-chain (IL-5Ralpha) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Ralpha-deficient (IL-5Ralpha(-/-)) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Ralpha. IL-5 stimulation of wild-type B-1 cells, but not IL-5Ralpha(-/-) B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Ralpha(-/-) mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.     

Human IgE synthesis in vitro by pokeweed mitogen-stimulated human lymphoid cells: verification with a reconfirmed epsilon-specific radioimmunoassay

This study was performed to address the controversy concerning human IgE biosynthesis in vitro induced by stimulation with pokeweed mitogen (PWM) or other agents. The controversy has focused on the specificity of reagents employed for quantitatively determining human IgE in culture supernatant fluids. Specifically, questions have been raised as to whether certain anti-human IgE antibody reagents possess anti-idiotypic reactivities, thereby resulting in reactions with Fab determinants of polyclonal immunoglobulins which would yield false-positive readings of IgE protein levels. We present a detailed analysis confirming that the goat anti-human IgE antibody designated GAHE(PS), which was initially isolated by affinity chromatography with the same IgE(PS) myeloma protein used for immunization, binds poorly, if at all, with IgG, IgA, or IgM immunoglobulins, even at excessive concentrations (100 micrograms/ml). Moreover, GAHE(PS) displayed no reactivity with Fab fragments of IgG or free L-chains prepared from pooled polyclonal IgG isolated from Cohn fraction II. A second GAHE reagent was prepared by purification by affinity chromatography on a second, completely unrelated IgE myeloma protein (DZA), which differed from IgE(PS) in light chain class, thereby resulting in a reagent, designated GAHE(DZA), which was completely devoid of any possible reactivity with L-chain or idiotypic determinants affiliated with IgE(PS). By utilizing both reagents, the studies presented here confirmed that PWM-stimulated human lymphoid cell cultures synthesize increased quantities of IgE, which can be detected in comparable amounts by both GAHE(DZA) and GAHE(PS) in supernatant fluids from such cultures. Because incorporation of the reversible protein synthesis inhibitor, cycloheximide, totally abolished the PWM-induced increases in IgE levels in such cultures, these results verify that such increases reflect de novo synthesis of human IgE as a result of PWM stimulation in vitro.