Polyclonal Antibody Secretion Research Articles

Macrophages prevent the differentiation of autoreactive B cells by secreting CD40 ligand and interleukin-6

AbstractActivation of the innate immune system promotes polyclonal antibody secretion to eliminate invading pathogens. Inherent in this process is the potential to activate autoreactive B cells and induce autoimmunity. We showed previously that TLR-stimulated dendritic cells and macrophages regulate B cell tolerance to Smith antigen, in part through the secretion of interleukin-6 (IL-6). In this manuscript, we show that neutralization of IL-6 fails to abrogate macrophage-mediated repression and identify soluble CD40 ligand (CD40L) as a second repressive factor secreted by macrophages. CD40L selectively repressed Ig secretion by chronically antigen-experienced (anergic) immunoglobulin transgenic and nontransgenic B cells but not by transiently stimulated B cells. The importance of macrophages in maintaining B cell tolerance was apparent in lupus-prone MRL/lpr mice. Compared with C57BL/6 mice, macrophages from MRL/lpr mice were significantly less efficient at repressing immunoglobulin secretion coincident with diminished IL-6 and CD40 ligand production. These data indicate that macrophages regulate autoreactive B cells by secreting repressive factors that prohibit terminal differentiation of B cells. The regulation of autoreactive B cells by macrophages is diminished in lupus-prone mice suggesting a role in autoimmunity.

Macrophages prevent the differentiation of autoreactive B cells by secreting CD40 ligand and interleukin-6

Activation of the innate immune system promotes polyclonal antibody secretion to eliminate invading pathogens. Inherent in this process is the potential to activate autoreactive B cells and induce autoimmunity. We showed previously that TLR-stimulated dendritic cells and macrophages regulate B cell tolerance to Smith antigen, in part through the secretion of interleukin-6 (IL-6). In this manuscript, we show that neutralization of IL-6 fails to abrogate macrophage-mediated repression and identify soluble CD40 ligand (CD40L) as a second repressive factor secreted by macrophages. CD40L selectively repressed Ig secretion by chronically antigen-experienced (anergic) immunoglobulin transgenic and nontransgenic B cells but not by transiently stimulated B cells. The importance of macrophages in maintaining B cell tolerance was apparent in lupus-prone MRL/lpr mice. Compared with C57BL/6 mice, macrophages from MRL/lpr mice were significantly less efficient at repressing immunoglobulin secretion coincident with diminished IL-6 and CD40 ligand production. These data indicate that macrophages regulate autoreactive B cells by secreting repressive factors that prohibit terminal differentiation of B cells. The regulation of autoreactive B cells by macrophages is diminished in lupus-prone mice suggesting a role in autoimmunity.

Polyclonal antibody secretion during acute graft-versus-host disease.

Spontaneous plaque-forming cells (S-PFC) were followed in 67 bone marrow transplantation (BMT) recipients and 41 controls. Patients with no acute graft-versus-host disease (GVHD) had decreased IgA and IgM S-PFC up to 7 weeks after BMT compared with controls. Patients with acute GVHD had increased IgG, IgA, and IgM PFC compared with controls and patients without GVHD during the first 4 weeks after BMT. The maximum number of S-PFC increased with increasing severity of acute GVHD. However, at diagnosis of GVHD there was no difference in S-PFC in patients who resolved their GVHD or in those who developed more severe GVHD. After 6 weeks, patients with acute GVHD had significantly decreased IgA and IgM S-PFC compared with normal. No major changes in S-PFC were induced during various infections. However, a patient who developed urticaria had a dramatic increase in S-PFC. Patients studied more than a year after BMT had reduced IgM S-PFC compared with controls. It is concluded that S-PFC are reduced after BMT, but markedly enhanced during acute GVHD.

The role of prostaglandins in C3a-mediated suppression of human in vitro polyclonal antibody responses

Suppression of polyclonal antibody responses in human peripheral blood mononuclear cell cultures by human C3a appears to involve the release of endogenous prostaglandins from monocytes. C3a was found, under the experimental conditions employed, to activate the cyclooxygenase pathway of arachidonic acid metabolism with the release of large amounts of the prostaglandin E 2 species. Suppression of the protein A-induced polyclonal antibody response by C3a is abrogated by the prostaglandin synthesis inhibitor indomethacin. In addition, physiologic amounts of exogenous PGE 2 were able to inhibit polyclonal antibody secretion in a manner similar to the suppression observed when C3a was added to culture. These results suggest that C3a-induced release of prostaglandins could be a major element in immunosuppression induced by C3a.

Polyclonal antibody secretion in mouse spleen cells induced by staphylococcal phage lysate (SPL)

The effects of staphylococcal phage lysate (SPL) on polyclonal antibody secretion, and mitogenic activity of mice spleen cells were studied. 1) SPL, at a concentration of 30 μl/ml augmented mitogenic activity 1.9 times in mouse spleen cell cultures, however no enhancement of mitogenic activity was observed in B cell depleted, T cell cultures and in athymic nude mouse spleen cell cultures. 2) Augmentation of polyclonal antibody secretion by SPL was observed in athymic nude mouse spleen cell cultures, and did not enhance depleted of macrophages, by passage through Sephadex G-10. 3) By injection of 100 μl SPL, in vivo adjuvant activity against sheep red blood cells were augmented 2.4 times. As revealed above, SPL enhance both mitogenic activity and polyclonal antibody secretion of mouse spleen cells, but augmentation of mitogenic activity is not observed by SPL in T cell, or B cell cultures alone, existence of both T cell and B cell is required to mitogenic activity by SPL. Augmentation of polyclonal antibody secretion by SPL needs B cell, T cell and macrophages.

Phosphorylcholine on Streptococcus pneumoniae R36a is responsible for in vitro polyclonal antibody secretion by human peripheral blood lymphocytes.

We have shown that Pc on the C-polysaccharide of Streptococcus pneumoniae R36a is responsible for polyclonal PFC responses induced in vitro by this bacterium in humans. R36a grown in media containing EA instead of CL, and therefore having phosphorylethanolamine instead of Pc in their C-polysaccharide, were unable to induce substantial PFC responses. When EA-substituted bacteria were chemically conjugated with Pc, their ability to induce polyclonal PFC was restored. Specific removal of Pc from the surface of the bacteria by the use PLC also resulted in abrogation of the polyclonal antibody response. These data are consistent with our hypothesis that polyclonal activation resulting from R36a stimulation may be mediated by a recently described Pc-binding receptor that is distributed on the surface of a subpopulation of B lymphocytes in humans and mice.

Polyclonal activation of human B lymphocytes by Fc fragments. I. Characterization of the cellular requirements for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes.

Fc fragments derived from human immunoglobulin were found to be capable of inducing both a proliferative and polyclonal antibody response in human peripheral blood lymphocyte cultures. The cell population proliferating in response to Fc fragments belongs to the B cell lineage. Expression of polyclonal antibody formation requires the presence of both adherent monocytes and T cells. The role of the monocyte is to enzymatically cleave the Fc fragment into 19,000 mol wt Fc subfragments that are then able to induce polyclonal antibody secretion. Stimulation of polyclonal antibody production by Fc subfragments occurs in the absence of adherent monocytes but still requires the presence of T cells.

The use of hemolysis-in-gel assays to study polyclonal antibody secretion in bone marrow transplant recipients.

Polyclonal antibody secretion was measured as direct plaque-forming cells (PFC) against fluorescein isothiocyanate coupled sheep red blood cells (FITC-SRBC) or in an indirect assay using protein A coupled SRBC and anti-sera against human IgG, IgA, and IgM. Eighty individuals who were recipients of bone marrow transplants and 66 healthy controls were studied. Lymphocytes from patients studied during the first three months (short-term patients) had deficient B-cell function in both assays compared to normals. In healthy controls the direct assay only detected about 4% of the IgM producing B cells found in the indirect assay. PFC in long-term patients were not different from that of controls except for patients with chronic graft-versus-host disease (GVHD) who had a deficient IgM production. Patients with acute GVHD had unusual high numbers of IgG PFC after Staph. aureus activation (p less than 0.001). Short-term patients with infections had increased (p less than 0.01) IgG and IgA after Staph. aureus activation. PFC assays performed in three patients with grafts from HLA-nonidentical donors showed an increase in the background cultures for IgG PFC (p less than 0.025) and an increased IgG PFC after Staph. aureus stimulation (p less than 0.01) compared to patients with HLA-identical donors.

Alloreactive cloned T cell lines. II. Polyclonal stimulation of B cells by a cloned helper T cell line.

A cloned helper T cell line, L2, has been derived that promotes the proliferation of cytolytic T cells with the concomitant expression of cytolytic activity. L2 helper cells also cause a polyclonal stimulation of B cells in the absence of antigen, as measured by the number of total plaque-forming cells (PFC) as well as those generated toward TNP or SRBC. The L2 helper cell function is dependent on cell number, is radioresistant, and is mediated via soluble factor(s). Maximum levels of PFC were generally observed after 4 days of culture, with IgM-secreting B cells being predominantly induced in these cultures. Both the proliferation of and stimulation by L2 helper cells are activated by Misa determinants. F1 progeny from a mating of CBA/N and DBA/2 (Mlsa) that did not bear Mlsa (NDF1) were nonstimulatory, whereas those F1 animals bearing Mlsa (DNF1) induced L2 helper-cell proliferation and function. L2 helper-cell recognition of Mlsa determinants present on the responding B cell surface was not required for polyclonal helper-dependent stimulation in vitro. L2 cells stimulated polyclonal antibody secretion in syngeneic as well as allogeneic B cells. Since Mls antigens are not expressed on T cells, these results indicate that regulatory T cell circuits are initiated by non-T cells, possibly B cells.

Polyclonal antibody secretion induced in human mixed lymphocyte cultures. II. No direct activation of B cells.

Antibody secreting B cells were measured as plaque forming cells (PFC) in a hemolysis-in-gel assay using fluorescein isothiocyanate (FITC) coupled SRBC as targets or protein A coupled SRBC as targets and developing antisera. Peak antibody secretion occurred on day 5 and the highest number of PFC was seen when mitomycin treated stimulator cells: responder cells were used in a ratio of 1:4. The number of PFC in MLC was not correlated to the DNA synthetic response. Antibody secretion in MLC was found to be of IgM, IgG and IgA classes. Significant numbers of PFC in MLC from blood lymphocytes were detected with the protein A technique, but not using FITC-SRBC targets. Compared to spleen cells, fewer PFC were stimulated in blood lymphocytes. B cells alone, enriched by rosetting of T cells, did not respond by antibody secretion or DNA synthesis in MLC. When Lipopolysaccharide (LPS) was added to MLC an additional effect was seen on the number of PFC which may indicate that distinct B cell subpopulations are activated in MLC by LPS.

In vitro characterization of immunological responsiveness of uremic patients.

Uremic patients are thought to have deficient immune reactivity. The mechanisms for immunosuppression are not known. We have studied various in vitro immune response parameters in lymphocytes from uremic patients and from healthy controls. Using polyclonal activating substances, it was found that PHA and LPS responses were reduced in cells from the patient group compared to the control group (p < 0.05). Furthermore, MLC responsiveness using pooled stimulator cells and polyclonal antibody secretion induced by Staphylococcus aureus bacteria in vitro were reduced in the patients (p < 0.05). No differences with regard to proportions of T/B cells in blood were noted between the two groups. No correlation was found between responses of individual cells to different activating substances, with the exception of PHA and ConA. However, low responses to PHA were usually accompanied by a general low responsiveness. Patients were further subdivided into groups according to the type of dialysis treatment, peritoneal dialysis (PD) or hemodialysis (HD), and to the duration of the hemodialysis period (< and > 1 year). Patients treated with PD showed impaired T cell reactivity with loer PHA responses compared to the HD patients (p < 0.05). Between the HD groups there were no significant differences in mitogen or MLC responses. We believe that the differences between PD and HD were due to the facts that the PD patients were older and not in the same nutritional state as the HD patients.

Polyclonal Antibody Secretion Induced in Human Mixed Lymphocyte Cultures

In mixed leucocyte culture (MLC), using human spleen cells or thoracic duct lymphocytes, antibody secretion was induced, measured as plaque-forming cells (PFC) in a haemolysis-in-gel assay with fluorescein isothiocyanate (FITC)-coupled sheep erythrocytes (SRBC) as targets. Peak antibody secretion was seen on day 5. Using protein-A-coupled SRBC as targets and developing antisera, antibody secretion in MLC was found to be of IgM, IgG and IgA type. There was no correlation between the number of PFC against FITC-SRBC in MLC and DNA synthesis. Supernatants from MLC failed to induce antibody secretion.

Optimal Conditions for Polyclonal Antibody Secretion and DNA Synthesis in Human Blood and Spleen Lymphocytes by Lipopolysaccharide

Lipopolysaccharides from E. coli (LPS) stimulated increased DNA synthesis and high-rate antibody secretion in human lymphocytes from blood and spleen. Peak DNA synthesis by 10 or 100 microgram/ml LPS was seen on day 6 for blood lymphocytes. Using 500 microgram/ml of LPS the peak response was reached on day 5 and with 0.1--1 microgram/ml the maximal stimulation was on day 7. For spleen cells LPS 100 microgram/ml gave a peak stimulation on day 4 or 5. Antibody secretion, measured as plaque-forming cells (PFC) against fluorescein isothiocyanate-coupled sheep erythrocytes in a haemolysis-in-gel assay, was maximal on day 5 in blood lymphocytes and on day 3 or 4 in spleen cells. The highest cell viability and antibody secretion after stimulation with LPS was obtained in 16 mm diameter multiwell plates usng 2.5 x 10(6) cells in 1 ml of medium containing 10% AB serum absorbed with SRBC. Feeding of cultures was essential for stimulation of blood lymphocytes but not for spleen cells.

Shared antigenic determinants by mitogen receptors and antibody molecules to the same thymus-independent antigen.

The antibody response to dextran B1355 is thymus independent, and in high responder mice, over 90% of the antibodies carry the idiotype of an alpha-1,3 binding myeloma protein (J558). The present experiments demonstrate: (a) dextran B1355 is a B-cell mitogen both in a strain which carries the J558 idiotype on antibodies and in a low-responder strain which does not express that idiotype on antibodies to dextran; (b) anti-idiotypic antibodies to J558 recognize a dextran-specific surface receptor on 10--15% of all splenic B cells in those two strains as well as in all strains so far tested; (c) as shown by inhibition experiments such surface receptors cross-react with J558, and (d) anti-idiotypic antibodies are mitogenic for spleen cells of both strains resulting in B-cell proliferation and maturation to polyclonal antibody secretion.

Activation of human B and T lymphocytes by protein A of Staphylococcus aureus

Protein A from Staphylococcus aureus, in soluble form or coupled to Sepharose beads, acts as a polyclonal B cell activator (PBA) for human lymphocytes in blood and spleen. PBA activity was demonstrated in spleen cells by the ability of protein A to induce the formation of intracellular immunoglobulin synthesis and to activate polyclonal antibody secretion demonstrated against fluorescein isothiocyanate-coupled sheep erythrocytes in a modified hemolysis in gel assay. More plaqueforming cells (PFC) were seen in unseparated cells than in purified B cells. In blood lymphocytes, only few PFC were activated by soluble protein A. Protein A increased DNA synthesis in blood and spleen cells. At a concentration of 100 microgram/ml the peak response was on day 4 or 5, but at 1 microgram/ml the peak response occurred later. On day 4 of culture, high mitogenic activity was seen in unseparated lymphocytes or mixtures of separated B and T cells, whereas in enriched B and T cell suspensions activity was low. On day 7, however, DNA synthesis in both the enriched B and T cells was higher than in mixtures of B and T cells. Protein A stimulated DNA synthesis in thymus cells with a peak response on day 6. It is concluded that protein A alone or as an IgG complex can activate both B and T cells, though the mechanism of activation is not known and may be different for B and T cells.

Polyclonal Antibody Secretion in Human Lymphocytes Induced by Killed Staphylococcal Bacteria and by Lipopolysaccharide

Preparations of Staphylococcus aureus strains Cowan 1 and Wood 46 and of lipopolysaccharide (LPS) were found to act as polyclonal B-cell-activating substances for human splenic and blood lymphocytes. All three substances induced polyclonal antibody secretion in blood and spleen cell cultures, as tested against fluorescein isothiocyanate-coupled sheep erythrocytes by a modification of the local hemolysis-in-gel assay. Antibodies were of IgM class, as shown by inhibition of plaque formation by anti-IgM but not by anti-IgG or anti-IgA antisera. All these substances also consistently induced the formation of intracellular immunoglobulin and increased DNA synthesis in stimulated spleen cells. In blood lymphocytes Staph. aureus Cowan 1 induced a consistent increase in DNA synthesis, whereas Staph, aureus Wood and LPS often gave low or no increase in DNA synthesis. Peak antibody formation was observed on day 3 in spleen cells and on day 6 in blood lymphocyte cultures. Stimulation into high-rate immunoglobulin secretion occurred with all PBAs also in B-cell-enriched cell suspensions but not in T-cell-enriched cells. Optimal responses were, however, always noted in unseparated cell suspensions. It is concluded that preparations of killed bacteria can be useful tools for the clinical evaluation of both specific and nonspecific antibody-forming ability in cells from different groups of patients.

Genetic Control of B‐Cell Responses

The responsiveness of spleen cells from C3H/HeJ and C3H/Tif mice to lipopolysaccharide (LPS) was compared. Around 1,000-fold higher concentration of LPS were required to minimally activate HeJ cells, as compared with Tif high-responder cells, to both proliferation and polyclonal antibody secretion. However, HeJ cells did respond to higher LPS concentrations (100 and 1000 mug/ml). This selective pattern of responsiveness to LPS was also observed in the polyclonal responses of strains to LPS in vivo. Furthermore, the unresponsiveness of HeJ spleen cells was found to depend on a pure B-cell defect in the capacity to interact and/or generate triggering signals on interaction with LPS. Thus, adherent cells, thymus-derived lymphocytes, serum factors, and other non-specific conditions inherent in spleen cell suspensions of low-responder mice were not responsible for suppressing a putative B-cell response to LPS. The present findings are compatible with the possibility that the defect in C3H/HeJ B cells reflects the absence of a structure on the cell surface membrane that is functionally responsible for mediating LPS triggering.

Genetic control of B-cell responses. I. Selective unresponsiveness to lipopolysaccharide.

Spleen cells from C3H/HeJ mice fail to develop both proliferative responses and increased polyclonal antibody secretion in the presence of concentrations of the B-cell mitogen lipopolysaccharide that are optimal for the induction of B-cell responses in conventional strains. This unresponsiveness is selective for lipopolysaccharide, since C3H/HeJ spleen cells respond normally to two other polyclonal B-cell activators-dextran-sulphate and purified protein derivative of tuberculin. These findings are interpreted as indicating a selective defect in the B-cell subpopulation that responds to lipopolysaccharides in conventional strains.