Alternative activation of tumor-associated macrophages by IL-4

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression, and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ macrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.     

Alternative activation of tumor-associated macrophages by IL-4: Priming for protumoral functions

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ acrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.Key words: interleukin-4, tumor-associated macrophage, tumor microenvironment, cytokines, cathepsin proteases     

Virus-vs endotoxin-induced activation of liver macrophages

The response of liver macrophages (Kupffer cells) to distinct pathogenic material was investigated by comparing virus- and endotoxin-induced macrophage activation. Endotoxin-induced stimulation and induction with Newcastle disease virus (NDV) or Sendai virus led to the release of the same pattern of prostanoids characterized by a predominant production of prostaglandin E2 (PGE2). With respect to peptide mediators, hepatic macrophages secreted tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 after viral induction and endotoxin treatment, respectively. In response to viruses, however, much more interleukin-6 and TNF-alpha was detected than after endotoxin stimulation. Interferon type I (interferon-alpha/beta), on the other hand, was only detected in the supernatants of macrophages infected with viruses, but not of those exposed to endotoxin. This study also revealed that rat TNF-alpha exists in several soluble species, some of which are glycosylated.     

Interferon-gamma-induced apoptosis and activation of THP-1 macrophages

Apoptotic macrophages are frequently observed in human atherosclerotic lesions, and are considered to be involved in plaque instability in atherosclerosis. However, the molecular mechanism that promotes programmed cell death of macrophages in atherosclerosis remains to be elucidated. In this study, we investigated the effects of interferon-gamma (IFN-gamma), a cytokine secreted by activated T helper 1 (Th1) lymphocytes, on apoptotic cell death of THP-1 macrophages. Further we studied whether these apoptotic macrophages could be simultaneously activated in vitro and subsequently overgenerate monocyte chemoattractant protein-1 (MCP-1). When THP-1 macrophages were cultured with various concentrations of IFN-gamma, DNA synthesis was significantly decreased. IFN-gamma was found significantly to induce apoptotic cell death in THP-1 macrophages. RNase protection assay revealed that IFN-gamma up-regulated the mRNA levels of two pro-apoptotic molecules, tumor necrosis factor-alpha receptor 1 (TNFR1) and caspase-8, in THP-1 cells. Furthermore, TNF-alpha antibodies were found completely to neutralize the IFN-gamma-induced inhibition in DNA synthesis as well as apoptotic cell death in macrophages. IFN-gamma was found to activate these macrophages to stimulate MCP-1 production. The results suggest that IFN-gamma not only exerted apoptotic effects on macrophages, but also activated them and subsequently overgenerated MCP-1, and was thus involved in the development and progression of atherosclerosis.     

Alternative versus classical macrophage activation during experimental African trypanosomosis

African trypanosomes are extracellular parasites causing sleeping sickness to human or nagana to livestock in sub-Saharan Africa. To gain insight into factors governing resistance/susceptibility to these parasites, the immune responses in mice infected with a Trypanosoma brucei phospholipase C null mutant (PLC(-/-)) or its wild type counterpart (WT) were compared. We found that the T. b. brucei mutant inducing a chronic infection triggers the production of type I cytokines during the early stage of infection, followed by the secretion of type II cytokines in the late/chronic phase of the disease. In contrast, WT-infected mice are killed within 5 weeks and remain locked in a type I cytokine response. The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e. classically activated macrophages (type I) versus alternatively activated macrophages (type II) that are antagonistically regulated. Therefore, the phenotype and accessory cell function of macrophages elicited during WT and PLC(-/-) T. b. brucei infections were addressed. Results indicate that classically activated macrophages develop in a type I cytokine environment in the early phase of both WT and PLC(-/-) trypanosome infections. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated alternative macrophages. In parallel, we found that mice susceptible to Trypanosoma congolense infection, showing an exponential parasite growth until they die, have a higher level of type II cytokines in the early stage of infection than resistant animals controlling the first peak of parasitaemia. The levels of type I cytokines were comparable in both T. congolense-resistant and -susceptible mice. On the basis of these results, we propose that survival to African trypanosome infection requires a type I cytokine environment and classical macrophage activation in the early stage of infection, enabling mice to control the first peak of parasitaemia. Thereafter, a switch to type II cytokine environment triggering alternative macrophage activation is required to enable progression of the disease into the chronic phase. The possible role of the sequential activation of alternative macrophages in the late/chronic stage of infection in the increased resistance of mice to PLC(-/-) T. b. brucei will be discussed.     

Human adipose tissue macrophages display activation of cancer-related pathways

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.     

Viral activation of macrophages through TLR-dependent and -independent pathways

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.     

Mechanical fluid flow and surfactant-TA influence activation of macrophages

Summary We examined the effect of mechanical fluid flow and surfactant on macrophage aggregation as the indication of macrophage activation. Mechanical fluid flow enhanced aggregation in phorbol myristate acetate (PMA)-treated human myeloid leukemic cell lines (HL-60 cells), but had no effect on differentiation of PMA-treated HL-60 cells. Surfactant-TA (an artificial surfactant) inhibited fluid flow-induced aggregation, but had no effect on differentiation of PMA-treated HL-60 cells. Human alveolar macrophages spontaneously formed small aggregates without stimulation. This aggregation was enhanced by fluid flow and inhibited by surfactant-TA. Taken together, these data suggest that macrophage activation is affected by fluid flow and surfactants.     

Autocrine activation of cultured macrophages by brain-derived neurotrophic factor

To elucidate a significance of the expression of brain-derived neurotrophic factor (BDNF) in the activated microglia/macrophages of the injured central nervous system, we examined BDNF actions on or BDNF synthesis by macrophages cultured from the mouse peritoneal cavity. They synthesized BDNF and neurotrophin-3 (NT-3) in addition to expressing high-affinity neurotrophin receptors, full-length TrkB (FL), truncated TrkB (TK(-)), and TrkC, thus suggesting an autocrine influence of BDNF and NT-3. BDNF, but not NT-3, enhanced phagocytic activity and stimulated synthesis/secretion of interleukin-1beta in the same manner as lipopolysaccharide (LPS). Furthermore, there was a significant correlation of the phagocytic activity with the expression of BDNF or TrkB (FL). These results imply that the phagocytic activity of macrophages depends on BDNF synthesis and/or TrkB (FL) expression, suggesting that BDNF participates in the activation processes of macrophages by acting in an autocrine manner.     

Acetylcholinesterase activation of peritoneal macrophages is independent of catalytic activity

Summary 1. In diverse tissues, acetylcholinesterase appears to play a critical role in the functional state of cells completely dependent of cholinergic transmission. However, very little is known about the mechanisms and actual molecular structures mediating the fundamental interactions between this protein and the cellular membrane.2. In this study, peritoneal macrophages were used as a model system to study the possible interaction between acetylcholinesterase, acting in a non-cholinergic capacity, and the cellular membrane.3. When acetylcholinesterase was incubated with macrophages harvested from rat peritoneum, the rate of oxygen consumption was increased in a concentration-dependent manner that was independent of mitochondrial block with sodium cyanide. Furthermore, heat inactivation of enzymatic activity or application of BW 284C51 at a concentration which totally blocks catalytic activity did not eliminate the effect.4. In contrast, incubation with bovine serum albumin or butyrylcholinesterase actually retarded oxygen consumption.5. The effect of acetylcholinesterase depended on the presence of divalent cations and was inhibited by mannan andd-mannose, but notd-galactose. It is concluded that acetylcholinesterase can induce a respiratory burst in macrophages independent of its conventional catalytic site but involving either the mannose receptor of the monocyte-derived macrophage or a possible sugar binding site on acetylcholinesterase itself.     

PMA activation of macrophages alters macrophage metabolism of aggregated LDL

Aggregation of LDL may contribute to its retention in atherosclerotic lesions. Previously, we showed that aggregated LDL induces and enters surface-connected compartments (SCCs) in human monocyte-derived macrophages by a process we have named patocytosis. Aggregated LDL was disaggregated and released from SCCs of macrophages when exposed to human lipoprotein-deficient serum. The serum factor that mediated aggregated LDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. We now show that activation of macrophages with PMA inhibits plasmin-mediated release of aggregated LDL from macrophages. With macrophage activation, plasminogen released about 60% less cholesterol and 63% less TCA-insoluble (125)I-aggregated LDL than when macrophages were not activated. Electron microscopy showed that PMA did not cause SCCs to close, which could have trapped aggregated LDL within the SCCs and limited protease access to aggregated LDL. Rather, PMA decreased macrophage generation of plasmin by 61%, and stimulated lysosomal degradation of aggregated LDL by more than 2-fold. Degradation was mediated by protein kinase C, shown by the finding that degradation was inhibited by the protein kinase C inhibitor G?6976. PMA-stimulated degradation of aggregated LDL was associated with a 3-fold increase in cholesterol esterification, consistent with hydrolysis and re-esterification of aggregated LDL-derived cholesteryl ester. In conclusion, macrophage activation with PMA causes more of the aggregated LDL that enters macrophage SCCs to be metabolized by lysosomes. This results in more cholesterol to be stored in macrophages and less aggregated LDL to be available for plasmin-mediated release from macrophage SCCs.     

Interferon-gamma is required in activation of macrophages for tumor cytotoxicity

The participation of interferon-gamma in activation of murine macrophages for tumor cell lysis was investigated. Biochemically macrophage activation factor and interferon-gamma have not been separated. Antiviral titers correlated closely with macrophage activation in antigen- or mitogen-induced spleen cell supernatants. A monoclonal rat antibody that neutralized virus-induced interferon was also found to neutralize interferon-gamma in such supernatants. These monoclonal antibodies were coupled to CH-Sepharose 4B and used for absorption of antiviral activity from mitogen-induced spleen cell supernatants. Absorption of the interferon was paralleled by the reduction of the macrophage-activating capacity of the supernatants. Data from control absorptions supported the specificity of the absorption effect. These results indicate that interferon-gamma is required for activation of macrophages for tumor cell lysis. These results can be interpreted in two ways: (a) the monoclonal antibodies cross-react with interferon-gamma and with a mediator that is required for activation of macrophages for tumor cell lysis or (b) interferon-gamma itself is an essential cofactor for macrophage activation.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Phosphorylation and activation of hormone-sensitive lipase in isolated macrophages.

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesterol ester hydrolase activity in macrophages. Incubation of intact WEHI macrophages or mouse peritoneal macrophages leads to phosphorylation of HSL, which is increased by incubation with either dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine or okadaic acid. Correspondingly, these agents also activate neutral cholesterol ester hydrolase activity in intact WEHI cells. Regulation of mobilisation of esterified cholesterol in macrophages may be of antiatherogenic value, which this model system now allows us to investigate further.     

Luteolin, a food-derived flavonoid, suppresses adipocyte-dependent activation of macrophages by inhibiting JNK activation

Interaction between adipocytes and macrophages contributes to the development of insulin resistance in obese adipose tissues. In this study, we examined whether luteolin, food-derived flavonoid, could suppress the production of inflammatory mediators of the interaction between adipocytes and macrophages. Experiments using a coculture system of adipocytes and macrophages showed that luteolin suppressed the production of inflammatory mediators. In addition, activated macrophages were targets for the suppressive effect of luteolin. Luteolin inhibited the phosphorylation of JNK and suppressed the production of inflammatory mediators in the activated macrophages. The findings indicate that luteolin can inhibit the interaction between adipocytes and macrophages to suppress the production of inflammatory mediators, suggesting that luteolin is a valuable food-derived compound for the treatment of metabolic syndrome.     

Suppression of antibody-sensitized tumor cells by macrophages: insufficient supply or activation of macrophages within large tumors

Systemic administration of antibody against the C3H lymphosarcoma 6C3HED caused complete suppression of the growth of 10(5) tumor cells. When the size of the tumor inoculum was increased to 10(6) tumor cells, excess antibody was unable to cause complete suppression unless exogenous macrophages were added to the tumor inocula. The decreased effectiveness of antibody was shown to be caused by a local deficiency in the supply or activation of macrophages within the large tumor grafts and not by a systemic deficiency.