Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. I. Macrophage-mediated tumor cell lysis

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) were observed to have significantly increased tumoricidal activity. rIFN gamma had synergistic effects with cisplatin, LPS or MDP in activating macrophages. However, MDP showed much more pronounced synergism with cisplatin and LPS than with rIFN gamma. Supernatants collected from these activated macrophage monolayers also showed increased tumoricidal activity. Tumor cell lysis mediated by cisplatin-treated macrophages did not require priming with rIFN gamma though it may be necessary as a first signal for the increased macrophage activation with LPS and MDP.     

Synergistic activation by recombinant mouse interferon-gamma and muramyl dipeptide of tumoricidal properties in mouse macrophages

Mouse peritoneal macrophages were activated to become cytotoxic against B16-BL6 melanoma cells by the combination of subthreshold amounts of murine interferon-gamma (IFN-gamma; 0.1 to 10 U/ml) and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 0.001 to 10 micrograms/ml), but not by the combination of pH 2-treated IFN-gamma and MDP, heat-treated IFN-gamma and MDP, or IFN-gamma and the inactive stereoisomer of MDP, N-acetyl-muramyl-D-alanyl-D-isoglutamine (MDP-D). The encapsulation of intact IFN-gamma and MDP within the same liposome preparation was synergistic for macrophage activation. In contrast, the presentation of identical concentrations of IFN-gamma and MDP in separate liposome preparations did not activate macrophages. These data allow us to conclude that the encapsulation of genetically engineered IFN-gamma and synthetically produced MDP within the same liposome is highly efficient in producing synergistic activation of tumoricidal properties in mouse macrophages.     

The stimulating action of lipopolysaccharide and muramyl dipeptide on the activation of mononuclear cells in human peripheral blood by recombinant interleukin-2 in vitro

Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were effective in augmentation of killer cells generation from human peripheral blood mononuclear cells (PBMC) in response to recombinant human interleukin-2 (IL-2). Pretreatment of PBMC with combination of LPS and MDP resulted in most significant their proliferation stimulated by IL-2. Thus our results show the enhancement of PBMC sensitivity to IL-2 by action of LPS, MDP and most of all by their combination in vitro.     

Increasing the sensitivity of murine splenic cells to interleukin-2 after in vivo exposure to lipopolysaccharide, muramyl dipeptide and concanavalin A

Changes in sensitivity of the murine spleen cells to interleukin-2 (IL-2) after exposure to lipopolysaccharide (LPS), muramyldipeptide (MDP) and their combinations were studied. The possible effect of concanavalin A (Con A) administered in vivo on increasing sensitivity of the lymphoid cells to IL-2 was also studied. Exposure to LPS, MDP and their combinations led to an over 2-fold increase in responses of the spleen cells to the effect of IL-2 as compared to the controls. When Con A was administered to mice intravenously in a high dose, sensitivity of their spleen cells to IL-2 markedly increased in 18 hours.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Effect of cisplatin, rIFN-Y, LPS and MDP on release of H2O2, O2- and lysozyme from human monocytes in vitro.

Increased secretion of H2O2, O2- and lysozyme by human monocytes in vitro on treatment with cisplatin, rIFN-Y (interferon-Y), LPS (lipopolysaccharide) and MDP (muramyl dipeptide) is reported. It is suggested that increased production of these secretory products represent the activated state of monocytes. These in vitro activated monocytes could either kill the tumor cells via increased contact mediated cytolysis or cytolysis mediated via the release of the secretory products like H2O2, O2- and lysozyme.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Influence of interleukin-2 and interferon-gamma in murine schistosomiasis

Schistosoma mansoni-infected mice were administered at the time of parasite residency in the lung with recombinant murine interleukin (IL)-2 or interferon-gamma (IFN-gamma), to evaluate the impact of cytokines in host responses to primary schistosomiasis. S. mansoni lung-stage schistosomula did not affect plasma lipids levels in BALB/c, while elicited significant (p<0.05) increase in free fatty acids (FA) and decrease in cholesterol plasma levels in C57BL/6 and CD1 mice, and stimulated expression of mRNA for Th2 cytokines in BALB/c and Th1 cytokines in C57BL/6 and CD1 mice. Production of specific antibodies was negligible in the 3 strains. Interleukin-2 treatment elicited significant (p<0.001) decrease in triglycerides (TG) in CD1, and decrease in TG and cholesterol plasma levels and down-regulation of TNF-alpha mRNA expression in C57BL/6 mice. Induction of type 2 cytokines and/or IFN-gamma mRNA expression did not lead to increase in percentage of specific antibody responders in any mouse strain. Exogenous IL-2-related reduction in cholesterol plasma levels and TNF-alpha mRNA expression in C57BL/6 mice was associated with significant (p<0.05) decrease in adult worm recovery and egg count. Treatment with IFN-gamma elicited significant (p<0.05) free FA plasma levels increase in BALB/c and C57BL/6 and decrease in CD1 mice. Expression of type 2 cytokines mRNA was stimulated in BALB/c and CD1 mice, yet was not accompanied with increase in humoral responses. Exogenous IFN-gamma-related reduction in free FA plasma levels and IFN-gamma mRNA response, and up-regulation of TNF-alpha mRNA expression in CD1 mice were associated with significant increase in adult worm burden and egg load. The data were discussed in an attempt to define host factors predictive of resistance to schistosome infection.     

Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro

Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption.     

Stimulation by conditioned medium of L-929 fibroblasts, E. coli lipopolysaccharide,and muramyl dipeptide of candidacidal activity of mouse macrophages

A simple, quantitative assay method for microbicidal activity of phagocytic cells was devised using normal mouse peritoneal macrophages as effector cells and Candida parapsilosis as target cells. The macrophages were seeded in 96-multiwell tissue culture plates and infected with serially diluted Candida cells. Outgrowth of Candida cells in each well was estimated after a 48-hr incubation period. The maximum number of microbes killed on macrophage monolayers was then determined. The conditioned medium of L-929 cells (L-CM) influenced the fungicidal activity of the macrophages a great deal. An addition of L-CM, to 20% of the culture medium, stimulated the killing activity more than 128-fold, compared with no addition of L-CM. In the medium containing the L-CM macrophages spread very well on the plastic with several dendritic processes, whereas cells spread poorly and gradually cytolysed in the medium lacking L-CM. It was found that muramyl dipeptide at 100 μg/ml and E. coli lipopolysaccharide at 1–10 μg/ml stimulated the activity 4 to 16 times. An application of this method to destroying other kinds of microbes, measuring the activity of other phagocytes, and screening immunomodulators was discussed.     

In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Expression of murine interleukin-2 cDNA in E. coli and biological activities of recombinant murine interleukin-2

Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E. coli. Recombinant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.     

Lack of response of murine peritoneal macrophages to in vitro activation by muramyl dipeptide (MDP). I. Macrophage activation by MDP is species dependent

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Potentiation of direct antitumor cytotoxicity and production of tumor cytolytic factors in human blood monocytes by human recombinant interferon-gamma and muramyl dipeptide derivatives

Summary We investigated whether human peripheral blood monocytes isolated by centrifugal elutriation from healthy donors could be acitivated to become tumoricidal and release tumor cytolytic factor (TCF) subsequent to incubation with recombinant human interferon-gamma (r-IFN-) or a derivative of muramyl dipeptide (nor-MDP), or both. Blood monocytes incubated in endotoxin-free medium containing up to 1000 U/ml of r-IFN- or in medium containing less than 1 g/ml of nor MDP were not activated to lyse radiolabeled allogeneic human tumor cells. In contrast, the incubation of monocytes with various dose combinations of r-IFN- and nor-MDP generated significant direct cytotoxic activity as well as production of TCF. Preincubation of the r-IFN- and nor-MDP mixture with polymyxin B did not inhibit the synergism, thus ruling out the possibility that the process was due to endotoxin contamination. TCF harvested from monocyte culture supernatants was cytolytic against five allogeneic tumor targets, but not against a nontumorigenic cell line. Collectively, the data demonstrate that r-IFN- can prime human blood monocytes to allow their activation by synthetic nor-MDP.On leave from the Department of Internal Medicine, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770, Japan     

Stimulation of an enhanced in vitro immune response by a synthetic adjuvant, muramyl dipeptide.

Various subcellular bacterial fractions are known to enhance immune responses and serve as potent adjuvants. Muramyl dipeptide (MDP), a synthetic adjuvant mimicking a component of mycobacterial cell walls, enhances humoral immunity to soluble antigens and can increase macrophage cytotoxicity toward mastocytoma cells in vitro. In the present study MDP was found to enhance the hemolytic antibody plaque response of normal mouse spleen cells in vitro to SRBC at a level equal to or greater than that induced by Escherichia coli lipopolysaccharide. Furthermore, MDP was found to enhance the antibody response to SRBC nonspecifically in unimmunized spleen cell cultures, suggesting that similar to LPS the synthetic dipeptide may induce a generalized clonal expansion of committed lymphocytes and thus serve as a "polyclonal activator." MDP also enhanced the immune responsiveness of normal splenocytes to suboptimum concentrations of SRBC, indicating that this material may be useful in enhancing immunity in situations where there would normally be a poor immune response.     

Activation of the production of the tumor-necrosis factor by the combined action of lipopolysaccharide and muramyl dipeptide in vitro and in vivo

The effect of bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP) and their combination on the production of tumour necrosis factor by spleen cells in vitro and on tumour regression in vivo has been studied. TNF activity was detected in spleen cell supernatants and serum of mice treated with drugs, using L929 cells as targets. The combination of LPS and MDP was more effective in TNF production than each of the drugs used alone in vitro and in vivo. The injection of LPS and MDP to A/Sn mice with subcutaneous nodes of sarcoma SA-I resulted in total tumour necrosis. The treatment of mice with these drugs in water solutions was more effective, however, more toxic than the administration of LPS-treated splenocytes in MDP solution.     

Production of H2O2 by alveolar macrophages in experimental allergic alveolitis

Experimental extrinsic allergic alveolitis (EAA) was induced in guinea pigs with Saccharopolyspora rectivirgula. Bronchoalveolar lavages were performed before inducing EAA (day 1, BAL 1), on day 23 (BAL 2), and on day 48 (BAL 3). The number of cells/ml in lavage fluid was increased at BAL 2 (4.79 x 10(6) and BAL 3 (4.29 x 10(6)) compared with BAL 1 (0.56 x 10(6)). The number of major cell types increased simultaneously, neutrophil becoming the predominant cell type over alveolar macrophages (AM). The production of H2O2 by AM was measured at the different phases of EAA. Adherent AM were either non-stimulated or triggered with phorbol myristate acetate (PMA), zymosan. S. rectivirgula opsonized with normal guinea pig serum (SRNS), or S. rectivirgula opsonized with guinea pig anti-S, rectivirgula serum (SRAS). Stimulated AM produced larger quantities of H2O2 than unstimulated cells, PMA being the most potent stimulus. At day 1, AM stimulated with S. rectivirgula and zymosan produced similar quantities of H2O2. After the induction of the disease, AM stimulated with S. rectivirgula produced larger quantities of H2O2 than with zymosan. Production of H2O2 by AM stimulated with S. rectivirgula or PMA, respectively, stayed the same at day 1 and 23, but increased sharply for both stimuli at day 48. There was no difference between H2O2 production by AM triggered with SRNS or with SRAS.(ABSTRACT TRUNCATED AT 250 WORDS)