Effect of virus-induced interferon on the antibody response of suckling and adult mice

Continued administration of potent virally induced mouse interferon (IFN type I) preparations to suckling mice resulted in an inhibition of the primary antibody response to sheep erythrocytes (14-day-old mice). When slightly older suckling mice were immunized (17 days old), a delay (about 2 days) in the kinetics of the response was observed, but the amplitude of the response was similar in both control and IFN-treated animals. In adult mice, potent IFN preparations did not inhibit the antibody response under a variety of experimental conditions (different doses of IFN, schedules of treatment, routes of injection, times of assay). Although IFN does inhibit the in vitro antibody response, we conclude that under most experimental conditions, injection of IFN type I is not immunosuppressive in adult mice. When administered after immunization, IFN clearly enhanced the primary antibody response. It is of interest that IFN, a product of viral infection, enhances in vivo those components of the immune system that have been implicated in the host response to viral infections (i.e. delayed-type hypersensitivity, natural killer cell and macrophage activity, expression of lymphocyte membrane molecules), and, as is shown here, the primary antibody response.     

Studies on the immunosuppressive properties of a pregnancy-associated alpha-macroglobulin.

A pregnancy-associated serum glycoprotein was shown to have an inhibitory effect on several in vitro methods of immunological assessment. This suppressing activity was evident at physiological concentrations and did not appear to be due to cytotoxicity. Transformation, induced by agents often regarded as preferential stimulators of T lymphocytes (concanavalin A, phytohaemagglutinin, allogeneic cells and tuberculin) was significantly depressed by the alpha-globulin. However, this phenomenon was much less evident when lipopolysaccharide or goat anti-human F(ab')2 serum was employed to selectively stimulate B-cells. The glycoprotein also blocked antigen-induced inhibition of leucocyte migration and caused a significant reduction in the number of lymphocytes binding sheep erythrocytes, in the spontaneous rosette test. It is proposed that the non-specific inhibitory activity of the alpha-macroglobulin depends upon some direct effect exerted on the lymphocyte itself and that it is levelled primarily at the cell-mediated immune response.     

Cellular source of interferons in the circulation of mice with delayed hypersensitivity.

The cellular origins of type I and type II interferons released into the circulation of mice with delayed hypersensitivity were investigated. We determined the effect of treatment with various immunosuppressive agents, including cyclophosphamide, cycloheximide, antithymocyte serum, and whole-body X-irradiation, on the release of interferons after intravenous injection of specific (old tuberculin) or nonspecific (lipopolysaccharide) stimuli. The results suggest that (i) a heterogeneous population of lymphocytes (T and B cells) produces type II interferon, (ii) type I interferon is produced by a different cell population, and (iii) type II interferon is produced de novo after challenge with old tuberculin of mice sensitized with Mycobacterium bovis BCG.     

Detection of human T-lymphocyte receptor for sheep erythrocytes in immunoregulatory α-globulin

Immunoregulatory α-globulin (IRA) has been shown to supress T cell-dependent immune responses including inhibition of E-rosette formation between human peripheral lymphocytes and sheep erythrocytes. The human lymphocyte receptors, responsible for the interaction with E, can be obtained in soluble form. Recent papers have indicated that these soluble E-receptors may play a regulatory role in the thymus-dependent immune response. In the present paper a possible relationship between IRA and soluble E-receptors was investigated. Absorption of IRA with sheep erythrocytes removed its inhibitory effect upon both E-rosette formation and lymphocyte response to phytohemagglutinin. In gel diffusion experiments a pattern of total identity was obtained between a component of IRA and soluble E receptors, revealed by an anti-E-receptor-specific antiserum (obtained by immunizing a sheep with autologous erythrocytes sensitized with human soluble E-receptors). These results indicate that IRA preparations contain soluble E-receptors. The possibility that E-receptors may be related, to some extent, to the inhibitory effects of IRA protein deserves further investigation.     

Aberrant secondary antibody responses to sheep erythrocytes in rabbits with experimental syphilis.

Rabbits infected with Treponema pallidum have strikingly depressed in vivo immunoglobulin G responses to sheep erythrocytes. To gain further insight into the nature of this suppression, the immune responses of splenic and peripheral blood lymphocytes from infected rabbits to sheep erythrocytes were studied in vitro. Spleen cells from rabbits that had been sensitized with sheep erythrocytes during active syphilis had greatly decreased immunoglobulin M and G responses after in vitro incubation with sheep erythrocytes, when compared to the results obtained with cells from sensitized uninfected animals. Suppressor cells could be demonstrated in peripheral blood lymphocytes of control rabbits 6 months after sensitization with sheep erythrocytes; these cells could be removed by nylon wool filtration. When primary sensitization with sheep erythrocytes was carried out during active syphilis, these suppressor cells were not detectable in peripheral blood lymphocytes 6 to 9 months later. These findings provide further evidence that induction of immune responses may be abnormal early in treponemal infection and may help to explain the failure of the host to produce antibodies which eradicate the organism during the first 2 to 3 months of infection.     

In Vivo Immunosuppressive Effects of Recombinant Ovine Interferon-tau (Trophoblastin): r.oTP (r.oIFN-τ) Inhibits Local GVH Reaction in Mice (PLN Assay), Prevents Fetal Resorptions,and Favors Embryo Survival and Implantation in the CBA/J × DBA/2 Mice Combination

PROBLEM : Ovine trophoblastin protein, be it natural or recombinant (oTP, r.oTP), a member of the tau interferon family (r.oIFN-τ), has been shown to possess immunosuppressive properties in vitro. It acts as a cytostatic agent across species. Indeed, it was immunosuppressive when tested on human and murine lymphocytes in a variety of in vitro immune assays, as it is also on syngenic (ovine) lymphocytes. METHODS : In the present paper, we first verified that this property to act across species also occurred in vivo assays; r.oTP was able to down regulate a local GVH reaction assay (PLN assay) in mice. We then took advantage of these properties of r.oTP to investigate its in vivo effects during murine pregnancy as there is no ovine equivalent of the murine CBA/ J × DBA/2 resorption prone mating combination. RESULTS : When given in the postimplantation period, r.oTP drastically boosted resorptions in the CBA/J × DBA/2 matings, as did murine recombinant gamma interferon. However, the same r.oTP treatment in the peri-implantation period resulted in a reduction in resorptions in this spontaneous abortion system. CONCLUSION : The data suggested that r.oTP might have acted more by favouring implantation and embryo survival than by preventing the resorption process itself. The mechanisms possibly underlying these effects, as well as the putative uses of r.oTP evolving from these data, are discussed.     

Immunosuppressive activity of Phaseolus coccineus and Phaseolus vulgaris extracts in mice.

Lectin mixtures from 10 samples of Phaseolus coccineus and Phaseolus vulgaris beans were used as immunosuppressive agents in the sheep red blood cell (SRBC) antibody response of the mouse. Some bean samples contain lectins capable of inducing several times better suppression of humoral immunity than concanavalin A and phytohemagglutinin tested before in the same immune system. High dilutions of the 10 bean extracts were shown to agglutinate mouse, rabbit and sheep erythrocytes in vitro but the titers and "specificities" did not correlate with the immunosuppressive potency and selectivity displayed by the same extracts in mice. Two immunosuppressive bean extracts, assayed in lymphocyte cultures, were found to stimulate DNA synthesis of murine spleen T cells. This interaction appears to be a necessary step for the reduction of antibody synthesis to SRBC in the whole animal.     

In vitro production and cellular origin of murine type II interferon.

Antigen-specific type II interferon was produced in vitro by harvesting supernatants of spleen cell cultures from Swiss-Webster mice sensitized with Mycobacterium bovis strain BCG and challenged with old tuberculin. Treatment of C3H mouse spleen cell cultures with appropriate anti-Ia, anti-IgG, anti-Thy-1 or anti-Ly-2,3 sera resulted in a significant decrease in production of type II interferon. Removal of nylon wool adherent cells or cells with histamine receptors by column chromatography similarly caused reduced production of type II interferon. Recombination of spleen cell cultures treated with anti-Ia and anti-Thy-1 sera or of cells treated with anti-IgG and anti-Thy-1 resulted in restored production of type II interferon. Interferon production was also restored by combination of cells passed through histamine columns with anti-Ia treated cells, or those passed through nylon wool columns with anti-Thy-1 treated cells. Anti-Ly-1 serum treatment had no effect on interferon production. Removal of plastic-adherent cells or cells that had phagocytosed carbonyl iron also decreased interferon production, suggesting that macrophages were also involved in type II interferon production. Recombination of non-adherent spleen cells with anti-Ia and anti-Thy-1 sera treated spleen cells, however, did not restore interferon production, suggesting that other cells in addition to macrophages are depleted by the adherence procedure. These findings indicate that type II interferon is produced by suppressor or cytotoxic (Ly-2,3+) T lymphocytes in co-operation with one or two additional cell types: (i) B lymphocytes, and (ii) macrophages.     

Plasmacytoid dendritic cells prime IFN-gamma-secreting melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions

Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8(+) lymphocytes specific for the melan-A(26-35) epitope to become IFN-gamma producing cells in vitro. In addition, we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of cutaneous lymphocyte antigen and L-selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of most primary cutaneous melanomas in vivo and that type I IFN-containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor-specific T cell responses and affect tumor growth via soluble factors.     

Evidence for the presence of a low molecular-weight activator of suppressor monocytes (LASM) in dialysates of T lymphocytes.

Lysates of peripheral blood T lymphocytes from healthy individuals were found to contain a low molecular-weight peptide that inhibited phytohaemagglutinin-induced DNA synthesis in vitro by autologous or allogeneic peripheral blood mononuclear cells. The peptide was dialysable, partially heat stable, resistant to trypsin, RNase, and DNase but not to pronase, and was not part of the membrane receptor involved in rosette formation by T lymphocytes with sheep erythrocytes. It was found to act through monocytes, inducing the synthesis of second mediator responsible for the inhibition of lymphocyte DNA synthesis. This inducer of inhibition, designated as "low molecular-weight activator of suppressor monocytes' (LASM), may have a role in the depression of cellular immune response seen in various pathological conditions involving the destruction of T lymphocytes.     

Effect of free radical scavengers on superoxide dismutase (SOD) enzyme in patients with alcoholic cirrhosis

The in vitro and in vivo effects of three hepatoprotective antioxidants (silymarin, (+)cyanidanol-3 and 4-amino-5-imidazole-carboxamide-phosphate) on the expression and activity of superoxide dismutase (SOD) enzyme were studied in erythrocytes and lymphocytes from patients with alcoholic cirrhosis. In vitro incubation with the drugs in a concentration corresponding to the usual therapeutic dosage markedly increased (i) the SOD expression of lymphocytes as measured by flow-cytofluorimetry following staining with monoclonal anti-Cu, Zn-SOD-antibody and FITC-conjugated anti-mouse Ig, as well as (ii) erythrocyte and lymphocyte SOD activities. In vivo treatment also restored the originally low SOD activity and expression of the patients' lymphocytes and erythrocytes. The data indirectly suggest that antioxidant activity might be one of the important factors in the hepatoprotective action of these agents.     

Cytogenetic effects of cis-platinum(II)diamminedichloride in vivo

The chemotherapeutic agent cis-platinum(II)diamminedichloride (cis-PDD) has been shown to be mutagenic, teratogenic, and carcinogenic. We determined the cytogenetic effects of cis-PDD on human and rabbit lymphocytes in vitro and on rabbit marrow cells, lymph node cells, and lymphocytes in vivo. Lymphocyte cultures from two humans and one rabbit were treated in vitro with cis-PDD. For in vivo studies, five New Zealand white rabbits were given iv injections of cis-PDD. Posttreatment blood samples were withdrawn for analysis and rabbits were sacrificed at either 6 or 24 hr for cytogenetic analysis of marrow and node cells. Sister chromatid exchange (SCE) analysis of human and rabbit metaphases from lymphocytes treated in vitro showed that rabbit lymphocytes are more sensitive to SCE induction by cis-PDD. Significant increases in SCE were observed in lymphocyte cultures obtained as early as 1 hr post treatment from injected rabbits. Analysis of node, marrow, and lymphocyte metaphases from injected rabbits showed a high number of chromosome aberrations in these cells with bone marrow showing a delayed response to treatment. These results indicate that cis-PDD is clastogenic in hematopoietic tissues in vivo and that SCE methodology may be useful in monitoring patients receiving cis-PDD therapy.     

Immunologically specific production of interferon in cultures of rabbit blood lymphocytes: association with in vitro tests for cell-mediated immunity.

Lymphocytes of animals with delayed hypersensitivity produce mediators of cellular immunity when challenged in vitro with specific antigen. Among these are macrophage migration inhibitory factor (MIF) and interferon (IF). Nonspecific mitogens also induce the production of these lymphokines. In the following study leukocytes and column-purified lymphocytes of the same peripheral blood sample from tuberculin (purified protein derivatives [PPD])-sensitive rabbits were concurrently cultured in medium alone or with PPD. Supernatants of 1- and 4-day lymphocyte cultures were assayed for MIF. Supernatants of 1-, 2- to 4- and 5- to 7-day leukocyte cultures were assayed for IF by inhibition of cytopathic effect of vesicular stomatitis virus on rabbit kidney cultures. In the presence of PPD, normal lymphocytes did not produce MIF, but lymphocytes from sensitized animals did (8/8 animals), after 1 and 4 days of culture. Leukocytes from normal animals produced little or no IF when cultured with or without PPD. Leukocytes from sensitized animals cultured in medium alone produced little IF. However, when cultured with PPD they produced significant amounts of IF on day-1 (6/8 animals) and day-2 to day-4 (4/8) animals. There was no correlation between relative amounts of MIF and IF produced by cultures of respective cells from individual animals. Rabbit IF produced or released in vitro appeared in significant and maximum amounts by 24 h coincident with the time release of significant amounts of another mediator of cellular immunity, MIF.     

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells

Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.     

Immunosuppressive Activity of α 2H-'isoferritin' as Compared to Normal Crystallized Ferritin

The suppressive activity of α 2H-isoferritin was studied in vivo and in vitro.
α 2H-isoferritin inhibits the hemolysin synthesis in mice and the T independent and T dependent antibodies induced in vitro.
The immune response of normal human lymphocytes is suppressed as shown by lymphoblastic transformation induced by mitogen (PHA), antigens (tuberculin and Candidin), mixed lymphocyte cultures.
The inhibitory effect is proportional to the quantity of added protein. The normal ferritin has no effect. The role of the carbohydrate component is discussed.     

Determinants of the ability of malignant fibroma virus to induce immune dysfunction and tumor dissemination in vivo

The relationship of virus-induced immunological dysfunction and tumor dissemination was studied using two related tumor-causing leporipoxviruses: malignant fibroma virus (MV) and Shope fibroma virus (SFV). Recombinant viruses, produced by transferring MV's 10.7 kb BamHI C fragment to SFV, replicate in lymphocytes and suppress lymphocyte function in vitro. Those recombinants that replicate in lymphocytes and suppress lymphocyte function in vitro share about 3.5 kb from MV's C fragment. Some recombinants mimic MV in producing immune suppression and disseminated virus infection in vivo. Other recombinants, even some that are highly immunosuppressive in vitro (e.g. R71), only variably induce immune suppression in vivo, and do not cause disseminated disease. A segment of DNA from MV that transfers to Shope fibroma virus almost all of MV's virulence in vivo was identified.     

The immunological significance in the guinea-pig of in vitro transformation of lymph node, spleen and peripheral blood lymphocytes

Cells from lymph nodes or spleen, or peripheral blood leucocytes of immunized guinea-pigs were cultured in the presence of antigens or phytohaemagglutinin. Significant incorporation of tritiated thymidine occurred in a variable proportion of the experiments with lymphocytes from each of the three sources. Cells taken from animals that had been immunized with sheep erythrocytes with adjuvant, and which showed strong delayed hypersensitivity, and from animals immunized intravenously with sheep erythrocytes, which failed to show delayed hypersensitivity reactions, both responded to sheep erythrocytes in vitro.

Cells from guinea-pigs immunized with complete Freund's adjuvant alone, which showed strong delayed hypersensitivity to tuberculin PPD, gave more positive responses in vitro than did cells taken from animals which received an intravenous injection of tuberculin PPD before the adjuvant. These animals showed no or weak delayed hypersensitivity reactions (immune deviation).

The immunological significance of the in vitro proliferative reaction is discussed.

     

Effects of the newer antifungal agents (bifonazole, ICI 195, 739 and amorolfin) on in vitro phagocytic, lymphocytic and natural-killer cell responses

Amphotericin B and some of the imidazole drugs have been shown to suppress certain neutrophil and lymphocyte functions both in vitro and in vivo. We present here the in vitro effects of: amorolfin, a morpholine derivative; the imidazoles clotrimazole and ketoconazole; the N-substituted imidazole bifonazole and a triazole (ICE 195, 739), on neutrophil and lymphocyte function. All of these drugs inhibited neutrophil random migration, chemotaxis and hexose monophosphate shunt activity. The effects of the drugs on neutrophil adherence, deoxyglucose transport and beta-glucuronidase release were variable while lysozyme release was unaffected. Natural Killer cell cytoxicity was depressed by all drugs tested except for amorolfin. Mitogen-induced lymphocyte blastogenesis was suppressed by all the antifungal drugs tested. Similar results were obtained using the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. The mechanism of action of these drugs on these cell functions remains unknown, there may be a correlation between their effects on fungi and their effects on leukocytes. Clearance of systemic fungal infection is heavily dependent on integrity of the cellular immune system and it is clearly undesirable that antifungal drugs have immunosuppressive properties. Further studies are required to determine the in vivo and clinical relevance of our observations.     

Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.     

In vivo and in vitro effects of lead on humoral and cell-mediated immunity.

The humoral and cell-mediated immune responses of murine lymphocytes exposed to lead in vivo and in vitro were investigated. In vivo Pb was administered via the drinking water (0 to 10 mM) for 1 to 10 weeks. In vivo exposure of the mice to Pb did not alter significantly their plaque-forming cell response to sheep erythrocytes; however, their susceptibility to Listeria infection was reduced significantly with Pb dosages of greater than 0.4 mM. Although the in vivo plaque-forming cell responses did not appear to be altered, in vitro assessment of the reactivity of these in vivo Pb-exposed lymphocytes indicated that intermediate doses enhanced, but a high dose (10 mM) was suppressive. The 10 mM in vivo Pb dose suppressed the in vitro plaque-forming cell response, the mixed-lymphocyte culture response, and lipopolysaccharide-induced proliferation, but it did not affect concanavalin A- or phytohemagglutinin-induced proliferation. Interestingly, in vitro Pb exposure (10(-6) to 10(-4) M) of murine spleen cells caused an enhancement of most activities even though these in vitro concentrations of Pb were slightly above the in vivo concentrations. Direct in vitro Pb effects on the lymphocytes could be measured, and Pb consistently enhanced humoral and cell-mediated immunity.