Michael G. Goodman

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].

Modulation of lymphocyte activation: II. Alteration of intracellular cyclic nucleotide concentrations by an oxidation product of arachidonic acid☆

Abstract Murine spleen cells incubated with arachidonic acid oxidized with lipoxidase (AA/L) do not respond to mitogenic stimulation. Analysis of the components of AA/L separated by high-pressure liquid chromatography has previously demonstrated that purified 15-hydroperoxyarachidonic acid, the predominant product generated, is the seat of the inhibitory activity observed. Correlative studies were undertaken to determine the relationship between the pervasive inhibitory effect of AA/L and the modulation of intracellular cyclic nucleotide concentration. AA/L elevated intracellular levels of cGMP in murine spleen cells cultured under serum-free conditions and caused a similar (two-fold) increment in intracellular levels of cyclic AMP. These are the first cellular activities observed in this laboratory that are not subject to inhibition by treatment of cells with AA/L. Therefore, studies were conducted to determine if AA/L-mediated inhibition of lymphocyte mitogenesis could be mimicked by elevation of intracellular nucleotide levels by pharmacologic agents. Attempts to recreate this inhibitory phenomenon by use of such agents indicated that inhibition could not be induced by modulation of cyclic AMP alone, cyclic GMP alone, or of both together. Similarly, when the same stimuli of cyclic nucleotide accumulation were employed to determine if modulation of intracellular cyclic nucleotide content could counteract the inhibitory effect of AA/L, these agents did not interfere with AA/L-mediated inhibition. The capability of the cyclic nucleotide agonists to elevate levels of cAMP and cGMP was confirmed by direct assay. Thus, although cyclic nucleotides may play an essential, adjunctive role in inhibition of lymphocyte mitogenesis, their modulation is not of itself sufficient either to evoke or to interfere with the establishment of a state of profound inhibition.

Distinct effects of dual substitution on inductive and differentiative activities of C8-substituted guanine ribonucleosides

The current studies compare the inductive and differentiative properties of 8-mercaptoguanosine with those of 7-methyl-8-oxoguanosine. 7-Methyl-8-oxoguanosine (7m8oGuo) is a new member of the family of C8-substituted guanine ribonucleosides, the first such biologically active compound described that differs from guanosine other than by the specific substituent at the 8 position. Like 8MGuo, 7m8oGuo stimulates proliferation selectively in B lymphocytes. However, 7m8oGuo possesses greater activity than 8MGuo as a mitogen and greater potency as an adjuvant for humoral immune responses. Thus, as a B-lymphocyte mitogen, 7m8oGuo induces quantitatively greater [3H]TdR uptake than does 8MGuo, but with the same concentration optimum. As an adjuvant for in vitro antibody responses, however, 7m8oGuo achieves the same degree of immunoenhancement as 8MGuo but at approximately 10-fold lower concentrations, that is, the dose-response profile has been shifted to the left. Moreover, whereas the mitogenic responses to 8MGuo and 7m8oGuo exhibit parallel kinetic profiles, the adjuvant activity of 7m8oGuo arises earlier and persists later than does that of 8MGuo. These results are interpreted in terms of two distinct intracellular pathways: one mediating mitogenesis and the other adjuvanticity.

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1. 1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.

Enhancement of the in vivo antibody response by an 8-derivatized guanine nucleoside☆

The capacity of the C8-substituted guanine ribonucleosides to enhance the in vivo humoral immune response to the protein antigen, human gamma globulin (HGG), in A/J mice was evaluated. It has been shown recently that the C8-substituted guanine ribonucleosides are a new class of potent adjuvant for humoral immune responses to the sheep erythrocyte antigen. The current studies extend these findings to HGG with 8-bromoguanosine (8BrGuo), a representative of this group of nucleosides. The adjuvant activity of 8BrGuo in this system is highly dose and time dependent. Although 8BrGuo enhanced responses when injected either early (Day 0) or late (Day 4 or 5) after immunization, its administration on Day 1 or 2 most often led to no enhancement, suggesting that 8BrGuo may act on two events separated by a resistant stage in an ongoing immune response. The plaque-forming cell (PFC) response to HGG was enhanced optimally at doses as low as 1 mg 8BrGuo/mouse administered either on the day of immunization or 4 days thereafter. In contrast, however, serum anti-HGG antibody concentration assayed by enzyme-linked immunosorbent assay (ELISA) was enhanced only at doses of 10 mg or more, injected on the day of immunization, but doses as low as 1 mg were effective on Day 4. 8BrGuo was also an effective adjuvant when injected after antigen administration in incomplete Freunds adjuvant or when administered by several different routes (intraperitoneal, subcutaneous, oral).

Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on membrane-directed versus intracellular activators

Immunoregulatory effects of the oxidative metabolites of arachidonic acid (AA) on proliferation of B-lymphocytes were assessed in a serum-free culture system. Activation of B cells by membrane-directed ligands and intracellular activators was regulated by AA metabolites in very distinct fashions. Thus exogenous cyclooxygenase products (particularly prostaglandins E1 and E2) amplified the response to anti-immunoglobulin antibodies, whereas lipoxygenase products damped this response. In contrast, B cell activation with 8-mercaptoguanosine (an intracellular activator) was inhibited by cyclooxygenase products and remained relatively unaffected by several lipoxygenase products tested. This pattern of results was confirmed in studies with pathway inhibitors. Moreover, when liberation of endogenous AA was induced by stimulation of phospholipase A2 activity with melittin, inhibition of the response to each class of activator was counteracted with the appropriate pathway inhibitor. Results suggest that the two major groups of AA oxidation products function as a system of counterbalancing regulatory influences, serving to modulate B cell activation at the plasma membrane and to downregulate B cell activation at the intracellular level.

Enhancement of immunostimulatory activity by dual substitution of C8-substituted guanine ribonucleosides : correlation with increased cytokine secretion

Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine). All of the compounds enhance natural killer (NK) activity, lymphocyte proliferation, and antibody production in dose-dependent fashion. However, the potency and maximal activity of the disubstituted analogues are considerably greater than those of the monosubstituted analogues. Spleen cells stimulated for 48 h with the disubstituted compounds produce immunoreactive interleukin (IL) 1α, IL-6, tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ). Monosubstituted analogues induce lower quantities of IL-6, TNFα, and IFNγ and fail to induce detectable levels of IL-1α. Total IFN activity, assessed by viral inhibition assay, is also lower for the monosubstituted analogues. Augmentation of antibody secretion by B cells is diminished for neither mono- nor disubstituted compounds upon incubation with anti-cytokine antibodies. In contrast, anti-IFNαβ markedly reduces the effects of monosubstituted analogues on NK activity but has less marked effects on NK induction by the disubstituted compounds. A similar pattern of differences is seen for lymphocyte proliferation. Thus, although the analogues induce synthesis of several cytokines, to date only IFNαβ appears directly involved in enhancement of NK activity and lymphocyte proliferation. The present data do not, however, exclude the existence of an autocrine stimulatory mechanism not susceptible to inhibition by anti-cytokine antibodies.

Induction of interleukin 1 activity from macrophages by direct interaction with C8-substituted guanine ribonucleosides☆

Production of an IL-1-like activity in cultures of irradiated splenic adherent cells can be elicited by the C8-substituted guanine ribonucleosides 8-bromoguanosine (8BrGuo) and 8-mercaptoguanosine (8MGuo). This report constitutes the first evidence for activation of a non-lymphocytic cell type by these agents to secrete an immunologically-active mediator. The secreted activity is mitogenic for murine thymocytes, co-stimulates these cells synergistically in the presence of concanavalin A, and co-stimulates low cell density cultures of purified B-cells in the presence of anti-mu antibodies. Production of this activity increases in a dose-dependent manner as the concentration of nucleoside is increased, both in cultures of splenic adherent cells and of the macrophage cell line P388D1. The P388D1 results indicate that this effect of the nucleoside is not mediated by another cell type, but can proceed by direct nucleoside-cell interaction. Optimal amounts of IL-1-like activity are produced after about 24 h of culture. Anti-IL-1 antibodies that neutralize the biologic activity of an IL-1 standard also eliminate the IL-1-like activity induced by 8BrGuo. These antibodies, however, fail to alter the magnitude of the primary humoral immune response to sheep erythrocytes amplified by 8BrGuo. These data indicate that C8-substituted guanosines, known intracellular stimuli for B-lymphocytes, can also induce non-lymphocytic cells (including a macrophage-like cell line) to elaborate an active principle which exhibits IL-1-like activity. These nucleosides thus are apparently able to elicit secretion of monokines, lymphokines and immunoglobulin from macrophages, T-cells and B-cells, respectively.

Enhancement of T Cell Proliferation and Differentiation by 8-Mercaptoguanosine

Recent studies of the mechanisms by which cellular interactions govern lymphocyte proliferation and differentiation have resulted in discovery of the lymphokines and elucidation of their modes of action. These molecules are peptides or proteins, whose amino acid sequence and physico-chemical properties have been difficult to characterize due to the difficulty in achieving high degrees of purity in the preparations under study. Recently, however, we have described the potent immunobiologic activity of a novel class of molecule, the C8-subsituted guanine ribonucleosides. The observation that these synthetic guanosine derivatives are mitogenic for murine splenic lymphocytes without participation of cyclic GMP1 was followed by description of their capacity to induce polyclonal synthesis and secretion of immunoglobulin in both murine and more recently human B lymphocytes. The mitogenic activity of these nucleoside derivatives was shown to be exerted intracellular2, predominantly on a subpopulation of mature B lymphocytes and to a lesser degree on a subset of immature B cells3. In addition, these agents exhibit powerful adjuvant effects both invitro and invivo and provide a T cell-like inductive signal for B cells that enables them to generate antigen-specific responses to T cell-dependent antigens in the absence of T cells4.

Demonstration of T-Cell-Dependent and T-Cell-Independent Components of 8-Mercaptoguanosine-Mediated Adjuvanticity

Abstract The contribution of T cells to potentiation of humoral immunity by the C8-substituted guanine ribonucleosides and the origin of the increased numbers of antigen-responsive B cells generated consequent to their action have been investigated. Augmentation of the antigen-specific antibody response by these nucleosides, exemplified by 8-mercaptoguanosine (8MGuo), can be separated into T-cell-dependent and T-cell-independent components, both by use of the T-cell tropic immunosuppressive agent, cyclosporin A, and by experiments using separated populations of T and B cells. Augmentation of adjuvanticity by T cells is hypothesized to involve a B-cell subpopulation not otherwise subject to the action of 8MGuo. This subpopulation could potentially arise by either of two mechanisms, one whereby preexisting antigen-specific B cells undergo clonal expansion, and one in which cells not normally participating in the response are recruited in the absence of clonal expansion. Although the former mechanism makes a minor contribution to adjuvanticity, the latter mechanism appears to be the dominant one, insofar as models in which 8MGuo-induced proliferation fails to occur (such as after irradiation, or in the SJL mouse) nonetheless exhibit strong adjuvant effects. Analysis of precursor frequency of antigen-specific B cells indicates that for each mature, antigen-responsive B cell present in adult murine spleen, an average of four additional cells can be recruited by the conjoint actions of antigen and 8MGuo. One group subject to such recruitment is the immature antigen-specific B cell, whose degree of functional maturity is accelerated in the presence of antigen and 8MGuo.