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 activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-gamma (IFN-gamma)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and [3H]nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced by its encapsulation within multilamellar vesicles (MLV). Studies with [3H]nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-gamma-primed monocytes. The intracellular interaction of [3H]nor-MDP with IFN-gamma-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.     

Tumoricidal activation of murine alveolar macrophages by muramyldipeptide substituted mannosylated serum albumin

Rat and mouse alveolar macrophages have almost no spontaneous tumoricidal activity and are only slightly activated by muramyldipeptide (MDP). When MDP was carried by serum albumin, the activation was higher than with free MDP but only at high concentration. When MDP was bound to a neoglycoprotein (mannosylated serum albumin) - which binds to the sugar binding receptor at the macrophage cell surface and is actively endocytosed - the activation of rat or mouse alveolar macrophages is dramatically enhanced even at very low concentration of neoglycoprotein -bound MDP. Furthermore, neoglycoprotein -bound MDP injected i.v. or i.p. was found to be able to activate alveolar macrophages, the activity of which was maximal after 48 hours in mice and 72 hours in rats. Such conjugates have so potential values as new immunostimulant agents in cancer and parasite therapy.     

Induction of activated macrophages in C3H/HeJ mice by avirulent Salmonella

A single injection of viable Salmonella typhimurium SL3235, an avirulent organism blocked in the aromatic pathway, induced the generation of activated peritoneal macrophages in three different C3H mouse strains, including macrophage-defective C3H/HeJ mice. Macrophages obtained from immunized mice were cytotoxic for B16 melanoma cells, P815 mastocytoma cells, and TU-5 fibrosarcoma cells and microbicidal in vitro for the obligate, intracellular, protozoan parasite Leishmania major. The capacity of live SL3235 to activate C3H/HeJ macrophages contrasts with the failure of live Bacillus Calmette-Guérin to induce activated macrophages in this mouse strain. Although viable SL3235 were capable of fully activating cells of both normal and defective mice, a dose-dependent difference was observed in the number of organisms necessary for induction of tumoricidal macrophages in C3HeB/FeJ (normal) and C3H/HeJ (defective) animals. As few as 80 viable SL3235 were capable of activating C3HeB/FeJ macrophages whereas 5 X 10(4) organisms were required to activate C3H/HeJ macrophages. Maximal macrophage activation occurred 7 to 10 days after SL3235 inoculation in C3H/HeJ and C3HeB/FeJ mice. Acetone-killed cells of SL3235 had some but not all of the activity of the living Salmonella. A single in vivo injection of the nonviable preparation resulted in the induction of tumoricidal macrophages in C3HeB/FeJ but not in C3H/HeJ mice, even when tested over a wide dosage range. Injection of acetone-killed cells of SL3235 did, however, result in a population of primed macrophages in C3H/HeJ mice, as explanted cells could be induced to express activated macrophage effector activities after additional treatment in vitro with either LPS or IFN-gamma. Thus, in vivo administration of viable SL3235 is, by itself, capable of eliciting the full series of steps required for activation of C3H/HeJ macrophages, whereas killed SL3235 only provides signals sufficient to prime these defective macrophages for further activation in vitro. AI 15613     

Defective tumoricidal capacity of macrophages from A/J mice. I. Characterization of the macrophage cytotoxic defect after in vivo and in vitro activation stimuli.

Macrophages from A/J mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that activate cells from C3H/HeN mice. Peritoneal macrophages from A/J mice treated i.p. with viable Mycobacterium bovis, strain BCG, killed Corynebacterium parvum, or pyran copolymer fail to develop in vitro tumoricidal activity; varying the numbers of macrophages from treated mice added to target cells, or the dose and time of treatment, or the treatment schedule of these in vivo activation stimuli did not evoke cytotoxic activity. Moreover, cytotoxic activity by macrophages from A/J mice was not observed with any of four target cell lines derived from three different mouse strains. In vitro treatment of peritoneal exudate macrophages from A/J mice with lymphokine-rich supernatants, bacterial endotoxins, or T cell mitogens was also ineffective; varying the numbers of treated macrophages added to target cells, the dose of in vitro activation stimuli, or the time of treatment did not evoke cytotoxic activity. Thus, A/J mice exhibit a profound defect in macrophage tumoricidal capacity to both in vivo and in vitro activation stimuli over a wide range of experimental conditions.     

Prostaglandin E2 does not inhibit tumoricidal activity of mouse macrophages against adherent tumor cells

We determined whether endogenously produced PGE2 can down-regulate the tumoricidal properties of macrophages by a negative feedback mechanism. Peritoneal exudate macrophages or resident peritoneal macrophages of mice were incubated in medium (control) or in medium containing IFN-gamma and LPS. Activated macrophages were highly tumoricidal against syngeneic melanoma cells and secreted high levels of PGE2. Treatment with indomethacin or diclofenac sodium (voltaren) completely inhibited the production and secretion of PGE2 but not the tumoricidal activity of activated macrophages measured either immediately after activation or 1 to 3 days thereafter. Finally, the addition of exogenous PGE2 did not alter the ability of peritoneal exudate macrophages to respond to IFN-gamma or of LPS to produce high levels of tumor cell lysis. Collectively, these results show that PGE2 produced by activated macrophages is not a down-regulator of their tumoricidal activity against adherent tumor cells.     

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.     

Effect of systemic administration of mouse recombinant interferon-gamma on the lung tumor metastases in mice

The purpose of this study was to examine the effective anti-metastatic activity by multiple i.v. administrations of mouse recombinant interferon-gamma (IFN-gamma) against pulmonary metastases of 3LL or B16-BL6 melanoma cells after surgical excision of primary tumors. Multiple treatments with IFN-gamma reduced effectively the incidence of pulmonary tumor metastases. Repeated 4 consecutive treatment modalities with IFN-gamma showed remarkable reduction of lung tumor colonies, and also rendered alveolar macrophages (AM) cytotoxic against B16-BL6 cells. In contrast, 14 consecutive administrations of IFN-gamma at any doses (10(2) and 10(3) U/mouse) could not activate macrophages to become cytotoxic, but were effective in regressing metastases. Thus, antimetastatic activity of IFN-gamma may be due to the stimulation of host immune defense systems such as induction of tumoricidal macrophages, presumably the direct antiproliferative action to tumor cells, or both actions under the appropriate administration conditions. We found that the systemic administration of IFN-gamma under appropriate multiple treatment modalities results in the reduction of the lung metastases and can activate AM to become tumor cytotoxic at relatively low doses (10(2) U). High-dose IFN-gamma in the multiple administration schedule was also effective for the reduction of lung tumor colonies, but strongly suppressed the nonspecific immune function and could not activate tumoricidal properties of AM.     

Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Cytotoxicity of human peripheral blood monocytes

Native tumoricidal activity of human peripheral blood mononuclear cells was examined before and after their separation by counterflow centrifugation elutriation (CCE). Tumoricidal activity was found in the subpopulation of small mononuclear cells but not within the relatively pure subpopulation of large monocytes. Addition of lymphokine and/or lipopolysaccharide demonstrated that large monocytes were resistant to activation for tumor killing, in contrast to small mononuclear cells. However, cryopreservation or simply exposure to dimethyl sulfoxide (DMSO) rendered the large monocytes sensitive to activating agents without altering their unstimulated tumoricidal activity. Cryopreservation was not detrimental to small or large monocytes either in number or tumoricidal function but did decrease the number of large granular lymphocytes (LGL). The small mononuclear cell fraction was enriched for small monocytes to 80% by combining CCE with Percoll gradient separation. HNK-1 mouse monoclonal antibody against human LGL was used with complement to remove virtually all LGL from cryopreserved cells as judged by morphology and tumoricidal activity against K-562 human lymphoblastoid cells. Such treatment actually augmented rather than suppressed tumoricidal activity against P-815 mastocytoma cells. Therefore, we conclude that small monocytes but not large monocytes possess native tumoricidal activity distinct from that attributed to LGL or natural killer lymphocytes. Further, small monocytes are readily activated for tumor killing and can be cryopreserved without loss of tumoricidal activity.     

Selective activation of antitumor activity of macrophages by the delivery of muramyl dipeptide using a novel polynucleotide-based carrier recognized by scavenger receptors

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages.     

Macrophage activation: synergism between hybridoma MAF and poly(I). Poly(C) delivered by liposomes

High concentrations of a murine T cell hybridoma culture supernatant containing macrophage-activating factor (MAF) rendered resident mouse peritoneal macrophages cytotoxic for P815 mastocytoma cells. The capacity of the hybridoma-derived MAF (MAFH) to induce tumoricidal activity increased 10(3) to 10(4)-fold when the lymphokine was encapsulated into liposomes. Combinations of MAFH and poly(I) X poly(C) acted synergistically to render macrophages potently cytotoxic. Subthreshold (nonactivating) concentrations of free or liposome-encapsulated MAFH increased the potency of free poly(I) X poly(C) and liposome encapsulated poly(I) X poly(C). Either as free agent or encapsulated in liposomes, single-stranded poly(I) or poly(C) did not activate macrophages in the presence or absence of MAFH. Double-stranded poly(I) X poly(C) was thus required for macrophage activation and synergism with MAFH.     

Conditions controlling tumor cytotoxicity of rat liver macrophages mediated by liposomal muramyl dipeptide

Activation of rat liver macrophages with free and liposome-encapsulated muramyl dipeptide (MDP) to a tumorcytotoxic state was characterized by employing various experimental conditions. Macrophage-mediated tumor cytotoxicity was determined using two standard assay systems: a [methyl-3H]thymidine release assay to measure the extent of tumor cell lysis and a [methyl-3H]thymidine incorporation assay to measure the combined effects of tumor cell lysis and stasis. The extent of cell lysis was not affected by the ratio of macrophages to tumor cells within the ratio range of 30:1 to 5:1, provided that the macrophages form a confluent monolayer. Tumor cell lysis, however, was significantly influenced by macrophage density; a low macrophage density for example resulted in a low percentage of tumor cell lysis. Tumor target cells used in this study, i.e., C26 adenocarcinoma, B16 melanoma and P815 mastocytoma, differed in their susceptibility towards macrophage-mediated cell lysis, whereas no differences were observed with respect to tumor cell stasis. Non-tumorigenic cell lines such as human fibroblastic cells and LLC monkey kidney cells were not lysed by activated macrophages, although proliferation of these cells was markedly inhibited. Additionally, the effects of liposomal lipid composition on macrophage activation were studied. With a basic composition of phospholipid/cholesterol/dicetylphosphate, we used either egg-yolk, dipalmitoyl-, distearoyl- or dihexadecylphosphatidylcholine as the bulk phospholipid constituent. Although these liposomes display a widely different susceptibility to lysosomal phospholipase activities, we could not detect any significant difference in either the extent or the duration of the tumoricidal activity induced by MDP encapsulated in these different types of liposomes.     

Imbalanced accumulation of ribosomal RNA in macrophages activated in vivo or in vitro to a cytolytic stage

Previous studies have shown that peritoneal murine macrophages activated in vivo and in vitro to a tumoricidal stage have a depressed rate of RNA synthesis. In attempting to clarify the differences in RNA metabolism between noncytotoxic and tumoricidal macrophages, we have studied the relative accumulation of various species of RNA in macrophages activated in vivo and in vitro with the use of agarose gel electrophoresis. Macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines (LK) and traces of lipopolysaccharide (LPS) have an imbalanced accumulation of mature ribosomal RNA (rRNA), with a decreased accumulation of 28S rRNA compared to 18S rRNA. In contrast, macrophages primed in vitro with LK free of detectable endotoxins that exhibit suppressive rather than tumoricidal activity do not manifest a decreased 28S:18S rRNA ratio. The conclusion that the decreased 28S:18S rRNA ratio was associated with the activation of macrophages to a cytolytic stage was supported by the finding that cytotoxic macrophages activated in vivo by i.p. injection of Propionibacterium acnes (formerly designated C. parvum) also demonstrated a decreased accumulation of 28S comparable with that observed in in vitro-activated macrophages. Moreover, activated macrophages that lost their cytolytic activity upon prolonged in vitro culture had an augmented accumulation of 28S rRNA. These results provide the first direct evidence that the expression of cytolytic activity is associated with modulation of a specific class of RNA. The unbalanced accumulation of rRNA appears to be a late molecular event in the activation process occurring during the transition from primed to cytotoxic macrophages, because inflammatory and primed macrophages had normal rRNA accumulation. A model of macrophage activation accounting for these results is proposed.     

Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells

Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr 51Cr release or 111In release assay. Both purified monocytes (greater than 99% monocytes) and natural killer (NK)-enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NK-depleted lymphocytes did not lyse the melanoma targets. Culture supernatants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 51Cr release assay. Treatment of the monocytes with anti-Leu 1 1b and anti-Leu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leu 1 1b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-gamma (rIFN-gamma), but not lipopolysaccharide, into the 51Cr release assay or pretreatment of monocytes with rIFN-gamma significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFN-gamma from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Differential effects of liposome-incorporation on liver macrophage activating potencies of rough lipopolysaccharide, lipid A, and muramyl dipeptide. Differences in susceptibility to lysosomal enzymes

We investigated the in vitro activation of rat liver macrophages to a tumor-cytotoxic state with muramyl dipeptide (MDP), rough LPS (Re-LPS) and lipid A in both a free and liposome-encapsulated form. The tumor cytotoxic state of the liver macrophages was determined with a [methyl-3H]thymidine release assay using C26 colon adenocarcinoma cells as target cells. As was shown previously, the encapsulation of MDP within multi-lamellar phospholipid vesicles greatly enhanced the activating potency of the drug; by contrast, encapsulation of Re-LPS or lipid A significantly reduced the activation of macrophages as compared to the free form of these agents. At a dose of 1 ng of free Re-LPS per ml a significant induction of tumor cell lysis was observed whereas a maximal level was obtained at a concentration of approximately 10 ng/ml. By encapsulation of Re-LPS in liposomes the activating potency diminished 20- to 100-fold. The minimal concentration required to induce detectable macrophage activation with free lipid A was 10 ng/ml, while liposome-encapsulated lipid A did not induce any detectable tumor cell lysis up to a concentration of 200 ng/ml. After a 1-h pre-incubation with a lysosomal fraction from rat liver at pH 4.8, the macrophage-activating potency of Re-LPS and lipid A was diminished by up to 95% whereas MDP remained fully active under these conditions. We conclude that, due to endocytic uptake of liposome-incorporated Re-LPS and lipid A and subsequent intralysosomal degradation, these immunomodulators are inactivated with respect to their potency to activate liver macrophages to tumor cytotoxicity.     

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     

Comparative analysis of the priming effect of human interferon-gamma, -alpha, and -beta on synergism with muramyl dipeptide analog for anti-tumor expression of human blood monocytes

Freshly isolated human peripheral blood monocytes from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the cytotoxic state by incubation in vitro with either des-methyl muramyl dipeptide (norMDP; minimal effective dose, 0.5 micrograms/ml) or recombinant human interferon-gamma (rIFN-gamma; minimal effective dose, 1 U/ml). A combination of subthreshold concentrations of these agents (norMDP, 0.5 micrograms/ml; rIFN-gamma, 10 U/ml) also induced significant cytotoxicity, indicating that the effects of norMDP and rIFN-gamma in monocyte activation are synergistic. Natural human IFN-gamma (nIFN-gamma) and norMDP also had similar synergistic effects. Pretreatment of rIFN-gamma with anti-IFN-gamma antibody completely inhibited its synergistic effect with norMDP in monocyte activation. Because pretreatment of rIFN-gamma and norMDP with polymyxin B did not interfere with their effects in monocyte activation, the preparations were not contaminated with lipopolysaccharide. Moreover, because pretreatment of monocyte monolayers with anti-Leu-11b antibody (anti-natural killer (NK) cell antibody) and complement did not interfere with the synergistic effects of norMDP and rIFN-gamma, whereas pretreatment with anti-Leu-M1 antibody (anti-monocyte antibody) caused complete inhibition of their effects, the observed tumor cytotoxicity of monocyte-rich monolayers was probably not due to a small number of adherent NK cells, but to the stimulation of the monocytes. Natural and recombinant IFN-alpha and IFN-beta at concentrations of greater than or equal to 100 U/ml also induced tumoricidal activity of monocytes, but unlike IFN-gamma, their effects were additive with norMDP, and they had less priming effect than IFN-gamma when they were added before norMDP to monocytes. These findings suggest that recombinant human IFN-gamma has much more synergistic potential with norMDP than IFN-alpha or IFN-beta, and this synergism of rIFN-gamma and norMDP for monocyte activation could be of clinical value in treatment of disseminated malignant diseases, because these compounds are readily available at standardized concentrations.     

Activation of the production of cytotoxic factors by mouse spleen cells exposed to the combined action of lipopolysaccharide and muramyl dipeptide in vitro

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.