The immunologic properties of Legionella cytolysin

Immunogenic properties of cytolysin were studied in experiments on guinea pigs. Preliminary immunization with cytolysin led to the suppression of response to ConA in lymphocytes not adhering to nylon wool and to the stimulation of response to Legionella antigens in lymphocytes adhering to nylon wool. For a month after infection with L. pneumophila the suppression of the proliferative activity of lymphocytes in the spleen of the immunized animals in response to ConA and Legionella antigens was observed, while in the lungs transitory suppression of response to ConA and Legionella antigens was followed by the restoration and then stimulation of proliferation in response to T-cell mitogen and specific antigens. The data obtained in these experiments indicate the capacity of cytolysin for modulating the development of immune response.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Effects of the methanol extraction residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. III. Consequence of prior sensitization to MER

Mice repeatedly immunized with the methanol extraction residue fraction of tubercle bacilli (MER) in incomplete Freund's adjuvant produced high titers of circulating antibodies against MER, as assessed by the enzyme-linked immunosorbent assay (ELISA) method. Spleen cells derived from these animals failed to respond to the usual nonspecific immunopotentiating influence of MER on the primary production of antibodies (generation of specific plaque-forming cells) in vitro to sheep red blood cells. The defect was expressed by B lymphocytes and splenic macrophages, but not by splenic T lymphocytes or peritoneal exudate macrophagic cells. Impaired responsiveness by spleen cells from MER-immunized animals to nonspecific immunostimulation was also expressed with regard to another, unrelated biological response modifier, lipopolysaccharide. There was no impairment of responsiveness to polyclonal mitogenic stimulation. Possible mechanisms of the effects described are discussed.     

The effect of ceramic fibers on the immune system

Male Sprague-Dawley rats were treated by intratracheal instillation with 1 mg/animal of refractory ceramic fibers. Intratracheal exposure to ceramic fibers led to significant changes of immune response. Results of proliferative activity of spleen lymphocytes showed significantly decreased proliferative activity of T-cells in response to mitogens phytohemagglutinin and concanavalin A in animals given ceramic fibers in comparison with control rats. Similarly, T-dependent B-cell response to pokeweed mitogen was significantly suppressed. Spontaneous proliferative activity of lymphocytes in non-stimulated spleen cell cultures did not differ in exposed and control rats. No significant changes were found among groups in percentage of phagocytic blood polymorphonuclear leukocytes and percentage of cells with respiratory burst.     

The cryopreservation of immunocompetent cells

Lymphoid cells sensitized against tumor cells in vivo were removed from sheep and mice, frozen and thawed, and then assayed for retention of cytotoxic function. Sheep lymphocytes efferent from immunized nodes have been shown previously to be cytotoxic owing to the production of antibody and these cells retained approximately 50% of their activity after freezing. The specific cytotoxicity of sensitized mouse spleen cells has been shown to be due to thymus-derived lymphocytes and such cells totally retained specific cytotoxic action after freezing. However, spleen cells also generally exhibited a degree of nonspecific cytotoxic action which disappeared on freezing and was attributed to the selective elimination of glass-adherent cells by the freezing process.     

Regulation of parasite antigen-induced T cell growth factor activity and proliferative responsiveness in Brugia pahangi-infected jirds

Previous studies have demonstrated that the induction of immunoregulatory mechanisms in the spleens of Brugia pahangi-infected jirds is correlated with the onset of microfilaremia. This study investigated the relationship between production of a factor with IL-2-like activity and the regulation of T cell-mediated responses in jirds experimentally infected with B. pahangi. A factor present in culture supernatants of mitogen-stimulated jird lymphocytes supported the proliferation of murine CTLL cells and provided the basis for an IL-2 assay. Mitogen induced proliferative responses and IL-2 production of spleen cells but not lymph node cells from pre-patent and microfilaremic jirds were suppressed. Both B. pahangi Ag-induced proliferative responsiveness and IL-2 production of spleen cells from microfilaremic jirds were also suppressed relative to lymph node cells from the same animals or spleen cells from B. pahangi immunized or prepatent jirds. Depletion of histamine receptor-bearing cells restored the ability of spleen cells from microfilaremic jirds to produce significant levels of IL-2. In addition, in add-mixture experiments, spleen cells from microfilaremic jirds suppressed Ag-induced IL-2 production by cells from either B. pahangi- or KHL-immunized jirds. Exogenous IL-2 failed to reconstitute the suppressed Ag-induced proliferative response of spleen cells from microfilaremic jirds. This study demonstrates that the down-regulation of immune responses in B. pahangi infection is a cell-mediated event and is associated with an inability to produce IL-2.     

Mechanisms of suppressed cell-mediated immunity and impaired antigen-induced interleukin 2 production in granuloma-bearing mice

Pulmonary granulomas were induced in BALB/c mice immunized with methylated bovine serum albumin in complete Freund's adjuvant by the intratracheal injection of plain agarose beads or beads conjugated to specific antigen. Large hypersensitivity granulomas developed around antigen-coupled beads in immunized animals. Smaller but still prominent granulomatous reactions developed around plain beads in immunized mice. In nonimmunized animals, both plain and antigen conjugated beads produced very small granulomas. Granuloma formation in sensitized animals was associated with suppressed delayed-type hypersensitivity reactions induced by the footpad injection of specific and nonspecific antigens. Lymph node cells from sensitized granuloma-bearing mice with cutaneous anergy showed suppressed specific and nonspecific antigen-induced proliferative responses in vitro. These cells also showed suppressed interleukin 2 production in response to specific antigen. Although no soluble suppressive factor was detected in granuloma extracts, suppressor cells were found in lymph nodes of granuloma-bearing mice, which could inhibit antigen-induced production of interleukin 2 by lymph node cells from immunized mice. Antigen-specific immunoglobulin G antibody production was not suppressed in immunized granuloma-bearing mice. Previous studies from our laboratory have demonstrated migration inhibition factor and interleukin 1 activities in aqueous extracts prepared from granuloma-bearing lungs of immunized mice. These results and the findings reported here indicate that granuloma formation and the associated anergy observed in this system are primarily expressions of cell-mediated immunity; selective suppression of in vivo and in vitro expressions of cell-mediated immunity in granuloma-bearing mice may be due to impaired antigen-induced interleukin 2 production; and such impairment is caused by suppressor cells.     

The role of cellular proliferation in the induction of experimental autoimmune thyroiditis (EAT) in mice

Experimental autoimmune thyroiditis (EAT) can be induced in susceptible strains of mice by injection of mouse thyroglobulin (MTg) and adjuvant. Lymphocytes from immunized mice develop a proliferative response to MTg which generally correlates with the development of EAT. We utilize a cell transfer system wherein spleen cells from CBA/J mice primed with MTg and lipopolysaccharide (LPS) in vivo are activated by culture with MTg in vitro to transfer EAT to naive recipients. In vivo priming of CBA/J mice is required to develop an antigen specific proliferative response to MTg. This response is optimal between 48 and 90 hr of culture at an MTg concentration of 125-250 micrograms/ml. The correlation between proliferation and transfer of EAT is not absolute as primed Balb/c X CBA/J F1 and AKR lymphocytes do not proliferate detectably in response to MTg but can be activated to transfer EAT; primed Balb/c lymphocytes neither proliferate nor transfer EAT. Proliferation per se is not sufficient to activate cells to transfer EAT as culture with nonspecific mitogens is not effective in activating primed CBA/J spleen cells to transfer EAT. However, lymphoblasts generated during in vitro culture of primed CBA/J spleen cells with MTg are responsible for transfer of EAT; small lymphocytes are ineffective. We conclude that antigen specific proliferation in response to MTg is essential in activating lymphocytes in vitro to transfer EAT.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Immunovac-VP-4, immunomodulator decreases immunosuppression and enhances the cytotoxic activity induced by the cytostatic Cisplatin

The influence of the vaccine Immunovac-VP-4, prepared from the antigens of opportunistic microorganisms, on the proliferative and cytotoxic activity on peripheral blood mononuclears (PBMN) from healthy donors in vitro and on spleen cells of CBA mice in vivo during their incubation with Cisplatin was studied. VP-4 produced a dose-dependent, stimulating effect on the proliferative potential of PBMN and, when used in the highest of all tested doses (20 microg/ml), increased the Cisplatin-suppressed proliferative activity of PBMN in 9.4-fold. VP-4 increased the cytotoxic activity of PBMN on tumor line cells K-562 (38,4 to 60.1%) and increased the cytotoxic effect of Cisplatin (68.18 to 87.56%). A single injection of VP-4 to mice stimulated the proliferative activity of spleen cells, studied ex vivo, units and partially restored their cytostatic-suppressed activity. The cytotoxic action of the spleen cells of immunized mice on tumor line cells YAC-1 was twice as great as that of spleen cells taken from intact animals and potentiated the cytotoxic action of Cisplatin. The mechanism of increasing the proliferative activity and cytotoxic effect of monomuclears under the influence of vaccine VP-4 is seemingly linked with the synthesis of cytokines, influencing the lymphokine-activated cytotoxicity of lymphocytes.     

The role of myelin lipids in experimental allergic encephalomyelitis. III. Transfer of suppression from guinea pigs recovering from EAE, induced by myelin basic protein--galactocerebroside complexes

Juvenile strain 13 guinea pigs were immunized with myelin basic protein (MBP) combined with galactocerebrosides (MBP + GC) or with total myelin lipids without GC [MBP + (TL-GC)] in CFA. Control animals received dinitrophenylated-ovalbumin (DNP-OA) in CFA, CFA or IFA alone. The animals injected with MBP + GC showed a higher rate of recovery from the first EAE episode (83%) than those treated with MBP + (TL-GC) (50%). With the exception of the group treated with IFA alone, all animals were refractory to EAE following rechallenge with MBP in CFA 90 days after the first exposure. The in vitro proliferative response to MBP, of peripheral blood lymphocytes (PBLs) derived from guinea pigs freshly sensitized to MBP in CFA, was drastically suppressed in the presence of PBLs from animals injected with MBP + GC. Upon transfer to normal syngeneic recipients, spleen cells from MBP + GC-treated animals completely suppressed the clinical and histological manifestations of EAE following recipient challenge with MBP in CFA. Cell-free supernatants from PBLs and spleen cells of strain 13 guinea pigs treated with MBP + GC inhibited lymphocyte proliferation to MBP, of allogeneic responder cells, and spleen cell supernatants completely suppressed the induction of EAE upon transfer to allogeneic recipients. Suppression could not be transferred with cells from other treated groups. These results suggest that animals immunized with MBP + galactocerebrosides in CFA develop suppressor cells that may be in part responsible for the recovery from the first EAE episode and for protection against rechallenge with MBP in CFA. Their cell-free supernatants act in an MHC-nonrestricted fashion. These results do not rule out an additional protective mechanism since all animals exposed to CFA were refractory to rechallenge despite lack of demonstrable suppressor cell activity.     

Effect of preparations of protein Yop encoded by Yersinia pestis calcium-dependence plasmid on mice

The impact of the preparations of Y. pestis secreted proteins Yop (YopH-M, YopB, YopD-N, YopE) on mice immunized with 3 s.c. injections was studied. Though these proteins failed to protect the animals from plague, they stimulated the immunobiological transformation in the immunized animals. YopB and YopD-N were found to have the highest immunobiological activity with respect to mice. The preparation of YopB induced the production of the highest titers of specific antibodies and stimulated cell-mediated immune response. The injection of YopD-N to mice led to a considerable decrease in the proliferative capacity of splenocytes in vitro in response to stimulation with nonspecific mitogen ConA, as well as to pathological changes in the kidneys.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

Bioaccumulation of metals from a nickel smelter waste in P and F1 generations of exposed animals. II. Modulation of immune processes

A group of female Chinchilla rabbits was exposed through inhalation to the metal aerosol derived from dumped waste of a nickel smelter. The experiments were carried out in a field exposure station. Increased levels of tissue immune complexes were found in the myocardium and lungs of P females, whereas F1 rabbits (exposed both prenatally and 6 weeks postnatally) from the same group of P females had significantly elevated serum circulating immune complexes as compared to controls. In P rabbits, nonspecific serum tumoricidal activity was increased by 8.2%, while in F1 animals the increase was by 14%. Transplantation immunity was examined in a group of inbred Lewis rats following the transplantation of a skin allograft from the ear of inbred Berlin-Druckrey rats. The mean time of allograft survival in animals following i.v. administration metal dust suspension 2 days prior to transplantation, was prolonged as compared to controls. On day 22 after allograft transplantation, lactate dehydrogenase activity was found to be reduced in peripheral lymphocytes, and the liver and spleen weight proved to be diminished. These findings suggest a modulating effect of the metal dust from a nickel smelter regarding nonspecific serum tumoricidal activity and transplantation immunity as well as immune complex formation.     

Immunogenicity and adjuvanticity of lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila was found to be a potent antigen and inducer of antibody with strong adjuvant activity for related and unrelated antigens such as sheep erythrocytes by in vivo and in vitro systems. The LPS was also a potent stimulator of blastogenic responses by spleen cells from normal mice as well as from mice immunized with inactivated whole cells of Legionella. It strongly stimulated production of interferon and interleukin 1. These results indicate that the LPS of Legionella may be an important immune regulator in the host response.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

Legionella pneumophila-induced immunostimulation and blastogenesis of normal mouse spleen cells in vitro

Cell-free sonic extracts prepared from Legionella pneumophila serogroup 1 were found to enhance the uptake of [3H]thymidine by normal mouse spleen cell cultures in vitro and also stimulate an enhanced antibody response to sheep erythrocytes, both in immunized and nonimmunized cultures. Increased background antibody responses to other erythrocyte species also occurred, indicating that the Legionella antigen was a polyclonal B cell activator. A purified cell wall component with physicochemical properties relatively similar to endotoxin, but without toxicity for mice, was found to have mitogenic activity for normal mouse spleen cells and immunostimulatory properties for anti-erythrocyte antibody response. Heating the sonicate or the purified somatic antigen for 10 min diminished immunoenhancing activity but had little effect on mitogenic properties. These results point to the complex effects of Legionella-derived antigens on normal lymphoid cell function and indicate that antigens derived from Legionella have marked immunomodulatory properties.     

Detection of nonspecific cytotoxicity in graft-versus-host reaction as a function of target cell type

The cytotoxic activity of spleen cells from mice undergoing graft-versus-host (GVH) reaction on ⁵¹Cr-labeled target cells was studied under in vitro conditions. Among normal tissues used as target cells, skin fibroblasts proved to be most sensitive to the nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction, whereas kidney cells or macrophages were insensitive to these nonspecific cytotoxic effects. Of the two murine neoplastic target cells used, Sarcoma 1 cells were susceptible to these nonspecific cytotoxic effects whereas mastocyoma cells were resistant. However, the target cells which were insensitive to the nonspecific cytolytic effects, were lysed specifically by the spleen cells from animals specifically sensitized. Therefore, both specific and nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction could be detected with appropriate targets. These results provide a basis for reconciliation of several apparently contradictory results, reported in the literature, concerning the specificity of the cytotoxic effects of specifically sensitized lymphocytes.     

Distinctive cytokinetics of blastogenic responses by spleen cells fromLegionella pneumophila-sensitized mice

Legionella pneumophila, the etiologic agent of respiratory pneumonia and systemic infections of man and some experimental animals, was studied in regard to the ability of these bacteria to induce blastogenic responses by spleen cells from normal vs sensitized mice. Antigens from this organism, including whole cell vaccine, an outer membrane extract, and a purified lipopolysaccharide-rich antigen, induced blastogenesis of normal spleen cells with peak responses on day +3 in vitro, similar to the blastogenic responses of spleen cells from the same animals exposed to the plant mitogens phytohemagglutinin and Concanavalin A, or the nonspecific bacterial antigenEscherichia lipopolysaccharides coli (LPS). Spleen cells from mice vaccinated with killedLegionella or infected with a sublethal dose of these bacteria 3–4 weeks or more previously evinced increased blastogenic responses to theLegionella antigens but not to the nonspecific mitogens or theE. coli LPS. The spleen cells from legionellae-sensitized mice evinced not only heightened blastogenic responses on day +3 of culture but also heightened responses during day +5 of culture. Spleen cells from sensitized mice showed less responses to the nonspecific plant mitogens orE. coli LPS on day +5 of culture. These results support the view that, after sensitization of mice with a bacterial antigen such asL. pneumophila, spleen cells respond in a specific heightened blastogenic manner toLegionella antigen, and this response has a higher magnitude and is more prolonged than the non-specific responses of cells from normal mice.     

Nitric-oxide-dependent systemic immunosuppression in animals with progressively growing malignant gliomas

The role of nitric oxide (NO) and adherent spleen cells in systemic immunosuppression developing in animals carrying malignant glioma isografts was analyzed. Rats harboring a subcutaneous glioma isograft for 3 weeks were immunized with glioma cells genetically engineered to express IFN-gamma. One week later spleen cells were tested for immune responsiveness in vitro. A decreased cytotoxic activity of NK-cells and T-cells compared to tumor-free animals immunized in parallel was shown. Spleen cell proliferative responses to tumor cells, SEA, and anti-CD3 were all significantly suppressed, as was the production of IFN-gamma and IL-10. Plastic adherent spleen cells from tumor-bearing rats suppressed the SEA-induced proliferative response and the production of IFN-gamma and IL-10 by nonadherent spleen cells from tumor-free rats. A major part of this suppression appears to be dependent on the production of NO because suppression was efficiently counteracted in vitro by the NO-synthase inhibitor N-nitro-l-arginine methyl ester. Moreover, a significantly increased level of nitrite in culture supernatants correlated with the observed suppression. We conclude that the systemic immunosuppression associated with growing gliomas is in part mediated by mechanisms dependent on NO overproduction in adherent spleen cells.