Edward L. Morgan

Regulation of Immune Response by Components of the Complement Cascade and Their Activated Fragments

An activation of the complement system results in generation of a variety of cleavage products that mediate such phenomena as chemotaxis, phagocytosis, anaphylaxis, acute shock, numerous inflammatory reactions, acute allergic reactions, and increased vascular permeability [reviewed in 35, 36]. In addition, complement and its activated components have been reported to interact with the immune system and modulate the immune response [reviewed in 85]. That these activated components play an important role in the regulation of immune responses is not surprising since (1) complement along with antibody is responsible for tissue localization of antigen [16], (2) it is activated by antibody-antigen interactions [reviewed in 56], (3) it is involved in lymphokine production [40, 45, 83] and suppression [62], (4) the activated components of complement are most likely present in vivo in the microenvironment of the interacting cells of the immune system [85], (5) some of the components of complement are synthesized by lymphocytes [82], (6) genetic deficiencies in complement components have been associated with impairment of the immune system [57], and (7) receptors for complement are present on the surface of lymphocytes and macrophages [reviewed in 9, 76]. In addition, numerous reports suggest that cells involved in the immune response are activated by complement components and their fragments, and that such activation can modulate the immune response. Since the majority of the fragments that are involved in modulation of lymphocytes and their responses are generated upon activation of either C3 or C5, it appears that the activation of complement via both the classical and alternative pathway is equally important in immunoregulation, since the activation of complement by either pathway involves the cleavage of C3 and C5 to their active fragments [reviewed in 14].

Mechanisms of leukocyte regulation by complement-derived factors.

Progress over the past five years has drawn attention to the fact that the anaplylatoxins are important factors in both leukocyte activation and regulation events. The C5 anaphylatoxin has been proposed to play major role in leukocyte aggregation and adherence phenomenon. Influences of C5a on the leukocyte may lead to clinical manifestations such as respiratory distress syndrome after trauma or postpump syndrome after cardiopulmonary bypass, both effects derived from leukocyte sequestration. Many other clinical conditions involving repeated transient sequestration of leukocytes, particularly in the pulmonary vasculature, may eventually be recognized as a complication of systemic complement activation. Dramatic pathologic changes observed in the lungs of animals exposed to either C3a or C5a emphasizes the potential damage that these factors may exert via cellular activation mechanisms (Huey et al., 1983). More recent evidence that the anaphylatoxins are potent immunoregulatory factors under in vitro conditions suggests a physiologic role for these humoral factors in nonspecific modulation of the immune response. It is an attractive hypothesis to suggest that once activated, complement is capable of relaying information to the cellular immune system via the anaphylatoxins. Other components of the complement system have long been known to exert regulatory influences on the immune system, and perhaps molecular description of such entities as the C3d-K fragment will serve to unravel this seemingly more complex effector system. In any case, as our understanding of both the chemical and biologic nature of factors derived from blood complement components advances, it has become apparent that a major function of complement may be to modulate the immune response. We have already demonstrated that these factors are selective both for cell type and for eliciting a variety of cellular responses. From this, one can infer that manipulation of the cellular events will eventually be possible. Hence a therapeutic value may be realized once involvement of these complement factors under in vivo conditions is better characterized.

Suppression of human B lymphocyte activation by ß-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.

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.

Human C3a-mediated suppression of the immune response: II. Suppression of human in vitro polyclonal antibody responses occurs through the generation of nonspecific OKT8+ suppressor T cells

C3a-mediated suppression of Ig secretion in human PBL cultures occurs through the activation of suppressor T cells. Incubation of T cell-enriched populations derived from peripheral blood or tonsil results in the activation of nonspecific OKT3+8+ suppressor T cells capable of inhibiting Fc fragment-induced Ig secretion in fresh autologous PBL cultures. Generation of OKT8+ suppressor T cells by C3a requires the interaction of T cells, adherent cells, and C3a. Activation of the suppressor-T-cell pathway is initiated early in culture. Incubation of the T-cell-enriched populations with C3a for 0.5 hr results in functional, transferable suppressor cells. Maximum suppression was achieved when C3a was allowed to interact with the cell population for 1-2 hr.

The immune response in aged C57BL/6 mice: I. Assessment of lesions in the B-cell and T-cell compartments of aged mice utilizing the Fc fragment-mediated polyclonal antibody response

Abstract The Fc fragment-mediated polyclonal antibody response was utilized to assess B-cell, T-cell, and macrophage reactivity in aged C57BL/6 mice. Spleen cells from aged (28–30 months) mice were found to be deficient in their capacity to proliferate and produce polyclonal antibody in response to Fc fragments when compared to adult (2–3 months) controls. Since T cells are required for the Fc-induced polyclonal antibody response, T cells from aged mice were assessed for their ability to restore the polyclonal antibody response in T-cell-depleted adult spleen cell populations. Aged T cells were not as effective as adult T cells in restoring the antibody response. The T-cell requirement in the Fc-induced polyclonal response has been shown to be replaceable by the Fc-stimulated T-cell replacing factor (Fc)TRF. T cells derived from aged mice were unable to produce (Fc)TRF to the level of adult cells. In addition to a defect in the T-cell compartment a lesion exists in the B-cell compartment of aged mice as well. Adult T cells were not capable of restoring the polyclonal antibody response of aged B cells any higher than aged T cells indicating a B-cell defect. Moreover, when a direct B-cell activator, Fc subfragment, was employed, the aged B cells were not stimulated to the level of adult controls. To test the ability of aged macrophages to function as accessory cells in the polyclonal response, macrophage-depleted adult spleen cells were mixed with aged or adult macrophages and the response measured. The results indicate that aged macrophages restore the polyclonal antibody response as efficiently as their adult counterpart.

Fibronectin augments anti-CD3-mediated IL-2 receptor (CD25) expression on human peripheral blood lymphocytes

The extracellular matrix (ECM) is composed of a number of macromolecules that promote cell adhesion, cell migration, and differentiation. Receptors for these molecules have been identified and belong to a superfamily of cell surface proteins, collectively known as the integrins. In this study, we show that the matrix protein fibronectin (FN) acts synergistically with immobilized anti-CD3 antibody to promote proliferation of total human peripheral blood lymphocytes (HPBL) in the absence of exogenous IL-2. Proliferation was inhibited by both the alpha 5 beta 1 and alpha 4 beta 1 recognition peptides. ARG-GLY-ASP (RGD), and GLU-ILE-LEU-ASP-VAL-PRO-SER-THR (EILDVPST), respectively. Expression of CD25 (IL-2 receptor) was significantly higher on cells cultured on anti-CD3 and FN, indicative of T-cell activation. Additionally, cells cultured on immobilized anti-CD3 and FN for 3 days showed increased adhesion to FN and increased forward light scatter/side scatter profile. Synthesis of both IL-1 and to a lesser extent IL-2 was elevated in supernatants from cultures containing both anti-CD3 and FN. These data are consistent with published reports which demonstrate that ECM proteins can act as costimulants of lymphocyte proliferation. Finally, our results show that cells cultured on anti-CD3 antibody and FN have an activated phenotype and that cytokines may be involved in this process.

Lymphocyte activation by the Fc region of immunoglobulins.

The Fc region of Ig is required for numerous biological effector functions which include: opsonization, anaphylaxis, C fixation, catabolism of the Ig molecule, FcR binding, and immune regulation. To this latter point, the cellular and subcellular events involved in immune regulation by IC and Fc fragments of Ig have been the focus of numerous investigations. Characterization of cyanogen bromide cleavage fragments from a human IgG1 myeloma protein indicates that one biologically-active site is found in residues 335-357 of the CH3 domain of the molecule. Synthesis of the biologically-active region resulted in a peptide, termed p23, which stimulates mouse and human B cells to secrete polyclonal Ig and activates AA metabolic pathways. In contrast to these findings, p23 is unable to induce B cell proliferation or IL-1 secretion from macrophages. Analysis of data obtained with overlapping peptides, based on p23, suggests that the minimal active sequence needed for B cell differentiation is leu-pro-pro-ser-arg (residues 351-355). In contrast, only p23 or p23 minus the carboxyterminal glu356 and glu357 were able to induce PGE release. Release of biologically-active peptides derived from the Fc region of Ig into the cellular microenvironment may form the nucleus of a nonspecific in vivo immunoregulatory network. The specificity of peptide regulatory activities could reside in their effectiveness at high concentrations in the cellular microenvironment. The interaction of Fc region peptides with receptors on B cells, T cells, and macrophages/monocytes could result in a dynamic control of immune reactivity.

Bifunctional lymphocyte regulation by human Fcγ 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.

Antagonistic regulatory properties of the Fc region of immunoglobulin

Coculture of human peripheral blood mononuclear cells with Fc fragments of human IgG, or the synthetic Fc region-derived peptide, p23, results in the release of oxidative products of arachidonic acid. Prostaglandin E was the major arachidonic acid metabolite found in the culture supernatants. Induction of polyclonal antibody production by Fc fragments and p23 is influenced by the concomitant production of prostaglandin E in culture. Addition of prostaglandin synthetase inhibitors, indomethacin and aspirin, to human peripheral blood mononuclear cell cultures resulted in a significant increase in the amount of polyclonal antibody produced. Moreover, addition of exogenous prostaglandin E to these cultures abrogated the ability of indomethacin to enhance Fc fragment-induced polyclonal antibody production. These results suggest that Fc fragments possess bifunctional immunoregulatory properties.