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.

Regulation of Fc fragment-induced murine spleen cell proliferation.

Murine splenic lymphocytes proliferate in response to supernatant material derived from Fc fragment-pulsed splenic adherent cells. The stimulatory supernatant results from the interaction of Fc fragments with adherent cells or adherent cell supernate. Isolation of the stimulatory material in the supernate by Sephadex chromatography revealed that the mitogenic component was a cleavage product of Fc with a mol wt of approximately 14,000. The spleen cell type responsible for the generation of mitogenic Fc subfragments appears to be a macrophage. Unstimulated macrophages release an active supernate without being exposed to Fc fragments. The supernate of unstimulated macrophages apparently contain an enzyme which is capable of cleaving Fc fragments into the 14,000-mol wt mitogenic molecules. The spleen cell population induced to proliferate in response to the adherent cell supernate is present in T-cell depleted and Sephadex G-10 filtered cell preparations. Depletion of cells bearing immunoglobulin on their surfaces results in a reduced proliferative response to the mitogenic supernatant material indicating that it is probably a B cell.

The requirement for adherent cells in the Fc fragment-induced proliferative response of murine spleen cells.

The proliferative response of mouse B lymphocytes induced by Fc fragments was found to be dependent upon an adherent cell population. The adherent cell is esterase positive, irradiation resistant, and not susceptible to lysis by anti-thymus serum and complement. The mechanism(s) by which Fc fragments induce B-cell proliferation could be the result of the interaction of Fc with both B cells and adherent cells or with adherent cells which then release factors that trigger the B cells to proliferate. Spleen cells from the C3H/HeJ mouse were shown to be unable to respond to Fc fragments. The addition of adherent cells from either C3H/St or C3H/HeN mice to adherent cell depleted C3H/HeJ cells enabled them to respond to Fc, indicating the defect was in the adherent cell population.

Enhancement of T lymphocyte functions by Fc fragments of immunoglobulins. I. Augmentation of allogeneic mixed lymphocyte culture reactions requires I-A- or I-B-subregion differences between effector and stimulator cell populations.

Fc fragments derived from human Ig were found to be capable of enhancing T cell-mediated, antigen-induced proliferative and mixed lymphocyte culture responses. Maximum enhancement occurred when suboptimal amounts of antigen or suboptimal numbers of stimulator cells were employed. Augmentation of the allogeneic mixed lymphocyte culture reaction requires an I-A and/or I-B subregion difference between effector and stimulator cell populations. Although a significant proliferative response was observed with K- or D- region differences, Fc fragments were unable to enhance the response. The T cell population acted upon by Fc fragments in the potentiation of these responses bears the Lyt-1(+)23(-) phenotype.

Anaphylatoxin-mediated regulation of the immune response. I. C3a-mediated suppression of human and murine humoral immune responses.

The C3a fragment of the third component of complement was found to have immunosuppressive properties. C3a is capable of suppressing both specific and polyclonal antibody responses. In contrast, C3a had no effect on antigen- or mitogen-induced B or T cell proliferative responses. The carboxy-terminal arginine is essential for C3a to exhibit its immunosuppressive properties. The serum carboxypeptidase inhibitor 2-mercaptomethyl-5-guanodinopentanoic acid, which prevents cleavage of the terminal arginine that would produce C3ades Arg-77, allowed us to assay the effects of C3a on in vitro immune response systems where serum is required. When the terminal arginine is removed from C3a, the resulting C3ades Arg-77 molecule is nonsuppressive. Helper T lymphocytes are the target of C3a-mediated suppression of the immune response. Substitution of T cells by soluble T cell factors was found to abrogate the C3a suppressive activity.

Regulation of the immune response. I. The potentiation of in vivo and in vitro immune responses by Fc fragments.

Fc fragments derived from human and murine Ig were found to be potent adjuvants when administered with antigen. Both the in vivo and in vitro anti- sheep erythrocytes (SRBC) responses were significantly enhanced by Fc fragments. The adjuvant effect was shown to be extremely dependent upon the dose of antigen used, with the greatest enhancement occurring when suboptimal doses of antigen are employed. The anti-genicity of the Fc molecule was not related to its adjuvanticity because homologous Fc was as potent an adjuvant as heterologous Fc. Moreover, human Fc fragments enhanced anti-SRBC responses in mice which were tolerant to human gamma globulin.

Synthetic Fc peptide-mediated regulation of the immune response. I. Characterization of the immunomodulating properties of a synthetic 23-amino acid peptide derived from the sequence of the CH3 domain of human IgG1.

A synthetic 23-amino acid peptide derived from the CH3 domain of human IgG1 was found to be a potent adjuvant as well as a polyclonal activator. The Fc peptide was found to enhance human and murine humoral, and T cell-mediated immune responses. Moreover, in vivo administration of Fc peptide enhanced murine natural killer cell activity. The synthetic Fc peptide was found to be more potent, on a molar basis, than native Fc fragments in inducing polyclonal antibody production and potentiating immune responses.

Lymphocyte phenotype and function in pseudolymphoma associated with Sjögren's syndrome.

Lymph node (LNL) and salivary gland lymphocytes (SGL) from three patients with pseudolymphoma and primary Sjögren's syndrome (1(0)SS) were characterized with monoclonal antibodies to demonstrate (a) a predominance of T cells (greater than 80%) reactive with anti-T cell antibodies OKT4 (greater than 70%) and OKT8 (less than 20%); (b) a high prevalence of activation antigens (greater than 50% of cells reactive with antibody OKT10 and anti-Ia antibody); (c) polyclonal B cells (8-15% of all cells expressing kappa or lambda); and (d) a specific B cell subset defined by reactivity with antibody B532 that was not present in their peripheral blood. In vitro functional studies showed that both SGL and LNL provided T helper activity for immunoglobulin synthesis and that this activity could be abolished by treatment with antibody OKT4 plus complement. The SGL and LNL exhibited little natural killer, antibody-dependent cellular cytotoxicity, or cytotoxic T cell activity. Normal karyotype was observed in SGL, LNL, and peripheral blood lymphocytes (PBL) from these patients. These findings indicate that pseudolymphoma in 1(0)SS results from the infiltration of salivary glands and extraglandular tissues by nonneoplastic T helper cells. Monoclonal antibodies provide an important tool to distinguish pseudolymphoma from non-Hodgkins (B cell) lymphomas that have a markedly elevated incidence in 1(0)SS patients. Our finding of T helper cells in pseudolymphoma tissues supports the hypothesis that chronic stimulation of B cells by helper T cells leads to eventual escape of a malignant B cell clone.

Role of Fc receptors in lymphocyte activation: deficiency in T- and B-lymphocytes from aged animals.

Fc fragments derived from human immunoglobulin possess the ability to induce B-cell proliferation, polyclonal antibody responses, and augment cell-mediated and T-dependent humoral responses. However, aged animals display much lower responses to Fc fragment stimulation. Proliferation and polyclonal antibody synthesis are reduced two-five-fold in aged animals compared to the young-adult responses. Furthermore, Fc fragments are unable to potentiate plaque-forming cell (PFC) development in aged animals. Aged B-lymphocytes are deficient in responding to Fc fragments, as their admixture with young-adult T-cells does not restore polyclonal antibody formation. T-cells from aged animals are also ineffective in promoting the polyclonal response when mixed with young-adult B-cells. The T-cell lesion has been further defined as a deficiency in production of an Fc fragment induced T-cell-replacing factor [(Fc)TRF]. The inability of Fc fragments to potentiate anti-SRBC responses in aged animals is also due to a T-cell defect which can be corrected by supplementation with interleukin-2 (IL-2).

Regulation of human B lymphocyte activation by opioid peptide hormones. Inhibition of IgG production by opioid receptor class (mu-, kappa-, and delta-) selective agonists.

Opioid peptides have been reported by many laboratories to modulate in vitro and in vivo cell-mediated and humoral immune responses. However, less attention has been afforded to the class or classes of opioid receptors involved in these immunomodulatory effects. Previous studies by this laboratory indicated that beta-endorphin and methionine-enkephalin were potent inhibitors of Staphylococcus aureus, Cowen strain I (SAC)-induced IgG production by human B lymphocytes. Results obtained from the present studies indicate that, at pharmacological concentrations, mu-, delta-, and kappa-receptor-selective agonists are potent inhibitors of SAC-induced IgG-secreting cells (IgG-ISC) by human B lymphocytes. Moreover, the suppression of IgG-ISC formation was reversed by mu-, delta-, and kappa-receptor class-selective antagonists, [D'Tic]cTAP, ICI 174,864, and nor-BNI, respectively. These findings are in agreement with other studies showing that more than one class of receptors are involved in opioid peptide-mediated immunoregulation. Additional studies indicated that all three class-selective receptor agonists were found to suppress SAC-induced IL-6 production in intact PBMC cultures. As observed for suppression of IgG-ISC formation, inhibition of IL-6 production was found to be reversed by the appropriate receptor class-selective antagonist. These results support the hypothesis that one mechanism of opioid peptide-mediated inhibition of antibody production is via the down regulation of cytokine synthesis.

Suppression of human B lymphocyte activation by beta-endorphin.

The effects of beta-endorphin (beta-E) and contained peptides were investigated for their ability to regulate Staphylococcus aureus (SAC)-induced immunoglobulin secretion by human B lymphocytes. Co-culture of beta-E with SAC-stimulated peripheral blood-derived mononuclear cells, under serum-containing or serum-free conditions, resulted in a dose-dependent inhibition of immunoglobulin-secreting cell (ISC) formation. When the same cultures were assessed for class-specific Ig formation it was found that IgG-ISC were suppressed to a greater extent that IgA-ISC or IgM-ISC. In contrast to these results, beta-E was found to be unable to suppress SAC-induced lymphocyte proliferation. To map the suppressive activity associated with beta-E, truncated peptides based on the beta-E sequence were assessed for biological activity. The results indicated that peptides containing the N-terminal region of beta-E suppressed ISC formation. Moreover, methionine-enkephalin (beta-E 61-65) was found to be effective in suppressing ISC formation. beta-E-mediated suppression of IgG-specific ISC formation appears to involve classical receptor-ligand interaction as evidenced by the ability of naloxone to block suppression of ISC formation.

Bifunctional lymphocyte regulation by human Fc gamma fragments and a synthetic peptide, p23, derived from the Fc region.

Fc fragments of human IgG1 and the synthetic peptide, p23, representing residues 335-357 in the CH3 domain of IgG1 were able to increase levels of secreted Ig in murine spleen cell cultures. B cell activation by Fc gamma fragments was macrophage- and T cell-dependent whereas activation by p23 was only T cell-dependent. Induction of Ig secretion by both stimulators was influenced by endogenous oxidative products of arachidonate, as evidenced by the augmentation of Ig levels in cell cultures treated with indomethacin (IM), a prostaglandin (PG) synthetase inhibitor. Both Fc gamma fragments and p23 were able to induce the release of PGE from splenic adherent macrophages and, in the former case, the release was inhibited by either IM or aspirin. Moreover, addition of either exogenous PGE1 or PGE2 reduced the levels of secreted Ig in Fc gamma fragment- or p23-stimulated cell cultures. These data suggest that B cell activation by Fc gamma fragments is influenced by the concomitant induction of suppressive PG.

Bioactive complement fragments in immunoregulation.

Several fragments derived from complement components have been identified as potent effector substances in in vitro assays that measure cell proliferation and antibody synthesis. The anaphylatoxin C3a suppresses the immune response but fails to influence T- or B-cell proliferation. The factor C5a augments both antibody production and antigen-induced, but not mitogen-induced, T-cell proliferation. C3a-mediated suppression occurs through the activation of a suppressor T-cell cascade with macrophage collaboration. C5a-mediated enhancement, depending upon the in vitro system studied, acts at the level of the helper T cell and/or macrophage. A fragment generated from treating iC3b with kallikrein (c3d-K) has aided in defining a structural region of the C3b molecule that can influence the level of circulating leukocytes. The factor C3d-K is also capable of suppressing both specific and non-specific T-cell proliferative responses and mitogen-induced B cell growth. The mechanism of C3d-K action is defined as a direct effect on "activated" T cells, even though IL-2 synthesis of treated cells is diminished. The effect of C3d-K is long lasting, non-reversible and requires only a short exposure to the target cell.

Trabecular bone density and menstrual function in women runners.

Osteoporosis results in decreased bone mineral mass and reduced trabecular bone density. Although its etiology remains unknown, studies have revealed differential changes in the bone mineral densities of postmenopausal women, anorexic women, and amenorrheic female athletes. Correlations have also been made between estrogen deficiency and osteoporosis in both premenopausal and postmenopausal women. In order to examine the possibility of osteopenia, a group of 36 female runners between the ages of 15 and 44 years were evaluated for bone mineral density, menstrual function, and dietary habits. Serum calcium, phosphorus, and parathyroid hormone (PTH) levels were also determined for each participant, as were complete blood counts. Using dual photon absorptiometry, all participants underwent a 20 minute scan of the lumbar spine with specificity to the L1-14 vertebrae. The 36 subjects included 19 oligomenorrheic and 17 eumenorrheic women. Results of bone density analyses revealed that the oligomenorrheic runners had significantly lower calibrated bone mineral density (CBMD) than their eumenorrheic counterparts (P less than or equal to 0.01). Likewise, the PTH levels of the oligomenorrheic runners were also significantly lower (P less than or equal to 0.01). Analysis of dietary logs revealed no significant differences between the dietary habits, the calcium intake, or the caloric intake of the two groups. The data from this study indicate that there is a relationship between reduced serum PTH levels and the oligomenorrheic state. The loss of the protective effect of estrogen in the oligomenorrheic runners possibly contributed to their reduced bone mineral densities and could be a contributing factor in osteopenia.

Modulation of the immune response by anaphylatoxins.

Bioactive C3a and C5a fragments derived from the human complement compounds C3 and C5, respectively, possess immunoregulatory activities. C3a and C5a differentially influence in vitro immune function. C3a was found to be a potent suppressor of antigen-specific and polyclonal antibody responses. In contrast, C3a was unable to suppress antigen-or mitogen-induced B and T cell proliferation. Analyses of synthetic peptides based on the sequences of C3a revealed that the carboxy-terminal region of the molecule is responsible for immunosuppression. C3a-mediated suppression occurs through the activation of a nonspecific suppressor T cell pathway. In contrast to the results obtained with C3a, C5a was found to augment both in vitro humoral and cell-mediated immune responses. Regulation of immune function by complement components may form part of an in vitro nonspecific immunoregulatory network.

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 E2 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 PGE2 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.

The requirement for the Fc portion of antibody in antigen-antibody complex-mediated suppression.

The role of the Fc portion of antibody in immune complex-induced suppression was studied in vivo and in vitro. BSA pheasant anti-BSA complexes, formed in antigen excess (Ag1Ab1), were found to suppress both responses to BSA and SRBC. When complexes were formed with F(ab')2 fragments of pheasant anti-BSA, no suppression was observed, indicating that the Fc piece was indeed essential for the induction of Ag-Ab complex-mediated suppression.