Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose-dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant-active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.     

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr ⁵¹Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.     

Liposomal muramyl dipeptide therapy of experimental M5076 liver metastases in mice

Summary The effectiveness ofN-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) or of liposomes containing a lipophilic MDP derivative, MDP-glyceroyldipalmitate MDP-GDP in inhibiting the growth of M5076 reticulum cell sarcoma liver metastases in C57BL/6 mice has been determined. MDP (100 µg) or liposomal MDP-GDP (2.5 µmol containing 1 µg) were equally effective in inhibiting liver metastatic growth when given as a single treatment 3 days before tumor cell injection. Therapeutic treatment, initiated 3 days after tumor cell injection and continued for a period of 2 weeks, failed to inhibit metastatic growth. Activation of thioglycollate-elicited peritoneal macrophages or Kupffer cells in vitro with MDP or liposomal MDP-GDP resulted in the expression of tumoricidal activity against M5076 tumor cells. Adoptive cellular therapy with four injections of 2 × 10⁶ macrophages was ineffective: activation of the macrophages with either MDP or liposomal MDP-GDP prior to injection was effective in inhibiting liver metastatic growth. Incorporation of the macrophage toxin dichlorodimethylene diphosphonate within liposomes containing MDP-GDP abolished the ability of such liposomes to induce macrophage or Kupffer cell tumoricidal activity in vitro as well as the antitumor activity when administered 3 days before tumor cell challenge.     

Macrophage activation redirects yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis

Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ⁻ Yersinia pseudotuberculosis (Yptb). YopJ⁻ Yptb-induced macrophage death was dependent on caspase-1 activation, resulting in rapid permeability to small molecules, followed by membrane breakdown and DNA damage, and accompanied by cleavage and release of proinflammatory interleukin-18. Induction of caspase-1-dependent death, or pyroptosis, required the bacterial type III translocon but none of its known translocated proteins. Wild-type Yptb infection also triggered pyroptosis: YopJ-dependent activation of proapoptotic caspase-3 was significantly delayed in activated macrophages and resulted in caspase-1-dependent pyroptosis. The transition to susceptibility was not limited to LPS activation; it was also seen in macrophages activated with other Toll-like receptor (TLR) ligands and intact nonviable bacteria. Yptb infection triggered macrophage activation and activation of caspase-1 in vivo. Y. pestis infection of activated macrophages also stimulated caspase-1 activation. These results indicate that host signaling triggered by TLR and other activating ligands during the course of Yersinia infection redirects both the mechanism of host cell death and the downstream consequences of death by shifting from noninflammatory apoptosis to inflammatory pyroptosis.     

Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide.

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.     

The immunomodulating activity of new muramyl dipeptide derivatives in vitro.]

The action of some new MDP derivatives on functional activity of murine T-lymphocytes and macrophages was studied. The following tests have been used: proliferation of spleen cells in one-way allo-MLC; IL-1 and TNF production by peritoneal macrophages treated with the preparations. The most expressed enhancement of lymphocyte proliferative response in MLC has been exerted by beta C7H15 MDP and beta C16H33 MDP (stimulation indexes 31-69%). beta C7H15 MDP, beta C16H33 MDP and polyacrylamide-MDP (P-MDP) alone or in combination with LPS caused elevated secretion of IL-1 by macrophages. While beta C7H15 MDP was as active as MDP, beta C16H33 MDP and P-MDP manifested increased ability to stimulate IL-1 production in comparison with MDP. beta C7H15 MDP, beta C16H33 MDP, P-MDP and MDP induced similar level of TNF production by murine macrophages. However, simultaneous treatment of macrophages with beta C16H33 MDP and LPS resulted in more significant enhancement of TNF production than combination LPS + MDP.     

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.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Induction, in vivo and in vitro, of macrophage membrane interleukin-1 by adjuvant-active synthetic muramyl peptides

Recent evidence has shown that a membrane form of interleukin-1 (IL-1) serves as a necessary signal for antigen presentation, leading to T-cell activation. The synthetic immunostimulant muramyl dipeptide (MDP) is known to induce secretion of IL-1 and its adjuvant effect was found to be mediated through enhancement of T-helper cells. We have investigated the ability of MDP and 19 other adjuvant-active or -inactive MDP analogs and derivatives to induce membrane IL-1 in mouse peritoneal macrophages. Enhancement in vitro of membrane expression and secretion of IL-1 in fresh or aged cultures of macrophages was observed after stimulation with MDP or with adjuvant-active but not with adjuvant-inactive muramyl peptides. Administration in vivo of adjuvant-active doses of MDP or of any of 12 other active analogs induced high levels of macrophage membrane IL-1 detected by the lymphocyte-activating factor assay. This effect was not observed when 7 other adjuvant-inactive derivatives were used. Moreover, under conditions where MDP did not exert an adjuvant effect, this immunomodulator was found to be incapable of inducing the expression of macrophage membrane IL-1. These results demonstrate a very high correlation between the ability to induce membrane IL-1 and the adjuvant activity of muramyl peptides. The correlation was observed irrespective of other biological effects of the synthetic adjuvants such as pyrogenicity and/or anti-infectious activity.     

Modulation of the Mevalonate Pathway by Akt Regulates Macrophage Survival and Development of Pulmonary Fibrosis

Protein kinase B (Akt) is a key effector of multiple cellular processes, including cell survival. Akt, a serine/threonine kinase, is known to increase cell survival by regulation of the intrinsic pathway for apoptosis. In this study, we found that Akt modulated the mevalonate pathway, which is also linked to cell survival, by increasing Rho GTPase activation. Akt modulated the pathway by phosphorylating mevalonate diphosphate decarboxylase (MDD) at Ser⁹⁶. This phosphorylation in macrophages increased activation of Rac1, which enhanced macrophage survival because mutation of MDD (MDD_S96A) induced apoptosis. Akt-mediated activation in macrophages was specific for Rac1 because Akt did not increase activity of other Rho GTP-binding proteins. The relationship between Akt and Rac1 was biologically relevant because Akt^+/− mice had significantly less active Rac1 in alveolar macrophages, and macrophages from Akt^+/− mice had an increase in active caspase-9 and -3. More importantly, Akt^+/− mice were significantly protected from the development of pulmonary fibrosis, suggesting that macrophage survival is associated with the fibrotic phenotype. These observations for the first time suggest that Akt plays a critical role in the development and progression of pulmonary fibrosis by enhancing macrophage survival via modulation of the mevalonate pathway.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

Stimulation of Nonspecific Host Resistance to Infection Induced by Muramyldipeptides

The effect of muramyldipeptide (MDP), N-acetylmuramyl-l-alanyl-d-isoglutamine [MDP(Ala)], and its analogs on bacterial infection was studied using the experimental model of sepsis infection in mice. Injection of MDP(Ala) gave mice definitive protection against E. coli infection, but only partial protection against P. aeruginosa or K. pneumoniae infection. Several factors influencing the protective activity of MDP (Ala) on E. coli infection were studied, and it was demonstrated that the activity was induced by various routes of administration of MDP(Ala), including the oral route, and was markedly influenced by the bacterial inoculum size. It was also shown that the effective dose of MDP(Ala) was 100 μg per mouse for intraperitoneal, intravenous or subcutaneous injections and 1,000 μg per mouse when administered orally. Furthermore, the optimal interval between MDP-treatment and infection was 24 hr when the treatment was carried out before infection. Clearance of bacterial cells in blood was observed after E. coli infection in mice treated with MDP(Ala). The efficacy of MDP(Ala) and two analogs, N-acetylmuramyl-l-valyl-d-isoglutamine [MDP(Val)] and N-acetylmuramyl-l-seryl-d-isoglutamine [MDP (Ser)], was evaluated for the E. coli infection; MDP(Val) was proven to be slightly less active than MDP(Ala), and MDP(Ser) to be the least effective, although MDP(Val) or MDP(Ser) was reported to have higher adjuvanticity than MDP (Ala) for the development of delayed-type hypersensitivity.     

Effect of Muramyldipeptide,a Synthetic Bacterial Adjuvant,on Enzyme Release from Cultured Mouse Macrophages

Monolayer cultures of macrophages obtained by peritoneal lavage of normal or thioglycollate-stimulated mice spontaneously secreted lysosomal enzymes into the culture medium. When the elicited macrophages were cultured in the presence of muramyldipeptide (MDP), a 20–30% increase in the release of β-glucuro-nidase was consistently observed and the intracellular activity decreased to about 45% of that of control cells after 6–8 days' culture. A stimulatory effect of MDP on lysozyme secretion, though less profound, was also observed. In contrast, release of neither enzyme was stimulated in resident macrophages by the addition of MDP. A neutral α-glucosidase, which has recently been found to localize also in granules of macrophages, remained inside the cells and neither its activity nor its release was affected by the addition of MDP to either type of macrophages. A large amount of lactic dehydrogenase was released only when the resident, not the elicited, macrophages were cultured for 3–4 days and then phagocytosed zymosan.     

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg²⁺ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg²⁺ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.     

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.     

Dengue Virus Multiplication in Cultures of Mouse Peritoneal Macrophages: Effects of Macrophage Activators

Dengue-2 virus multiplied in cultures of methylcellulose-induced peritoneal macrophages of BALB/c mice. The in vitro-cultivated macrophages from dengue-1 virus-immune mice produced larger amounts of dengue-2 virus than did those from nonimmune controls. The effect of macrophage activators was examined by using nonimmune macrophages. Enhanced virus production was demonstrated in cultures of macrophages pretreated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The number of virus-infected cells in the pretreated cultures was estimated to be about 0.01% or less of the total macrophages. Continuous treatment of macrophages with PHA before and after virus inoculation brought about the most marked enhancement of dengue-2 virus multiplication. On the other hand, treatment with concanavalin A or pokeweed mitogen showed little effect on the multiplication of the same virus. Treatment with carrageenan, a specific macrophage blocking agent, markedly suppressed dengue-2 virus production in both dengue-1 virus-immune macrophages and LPS-treated macrophages. The indirect fluorescent-antibody (FA) technique revealed dengue-2 viral antigen in the cytoplasm of infected macrophages, and the FA-positive macrophages were more numerous in PHA-treated cultures than in untreated controls. The results obtained are discussed in relation to a possible role of activated monocytes/macrophages in the pathogenesis of dengue hemorrhagic fever.     

Macrophage and dendritic cell infiltration in head and neck squamous-cell carcinoma; an immunohistochemical study

A study was undertaken to help us reach a better understanding of the tumor-infiltrating pattern of lymphoid cells and in particular of monocyte-derived cells, namely the CD68⁺, acid-phosphatase-expressing scavenger macrophages and the MHC-class-II- and S100-antigen-presenting dendritic cells in head and neck squamous-cell carcinoma. In the stroma of the tumors distinctive small fields of lymphocytes were found, the T cell areas of these fields being intermingled with dendritic cells. Intra-epithelial dendritic cell infiltration was low. The infiltrative pattern of macrophages was similar to patterns described in earlier studies with substantial stromal invasion and inconsistent intra-epithelial invasion, but small granuloma-like structures of CD68⁺ macrophage-like cells, found in the stroma of tumors, have not been reported before. The histochemical localization of the tumor-infiltrated dendritic cells and macrophages supports the view that the former cells are involved in the sensitization to tumor antigens, whereas the latter cells are involved in tumor cytotoxicity/scavenging of tumor cell debris. Although it has been shown in the past that transmembranal (TM) factors (p15E-like factors) present in the serum and tumor of patients with cancer of the head and neck have suppressive effects on monocyte/macrophage/dendritic cell function, a relationship between the intensity of epithelial staining for TM factors and the infiltrative pattern of monocytes/macrophages/dendritic cells could not be demonstrated.     

Enhancement of B-cell stimulation by muramyl dipeptide through a mechanism not involving interleukin 1 or increased Ca2+ mobilization or protein kinase C activation

Muramyl dipeptide (MDP) enhanced mitogenic stimulation of mouse lymphocytes by polyclonal B cell activators (peptidoglycan, lipopolysaccharide, Staphylococcus aureus Cowan I cells, and pokeweed mitogen), but not by T-cell mitogens (phytohemagglutinin and concanavalin A). Only adjuvant-active MDP analogs were effective, whereas adjuvant-inactive MDP analogs, muramic acid, peptidoglycan pentapeptide, and low Mr digests of peptidoglycan were not. The half-maximal enhancement was seen at 5-10 microM MDP and occurred at both optimal and suboptimal concentrations of B cell mitogens. The enhancing effect of MDP was exerted on the B cells, since it was T cell- and macrophage-independent and was not mediated by IL-1. MDP was effective during the first 12 hrs of culture, and most strongly enhanced the mitogen-induced DNA synthesis, although significant enhancement of RNA synthesis and B cell differentiation into antibody-secreting cells was also observed. The enhancement of mitogenic response was not due to changed requirements for extracellular or intracellular Ca2+ or to increased activation of protein kinase C. These results demonstrate a novel immunoenhancing effect of MDP that should be useful in the studies on the mechanism of B cell activation.     

Regulation of Macrophage Motility by the Water Channel Aquaporin-1: Crucial Role of M0/M2 Phenotype Switch

The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl₂) increased by 2–3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1^-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1^-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues.