Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

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.     

Regulation of Nitric Oxide Production by Murine Peritoneal Macrophages Treated in Vitro with Chemokine Monocyte Chemoattractant Protein 1

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl- -arginine (L-NMMA), a specific inhibitor of the -arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated -arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

Necrotic tumor cells oppositely affect nitric oxide production in tumor cell lines and macrophages

Nitric oxide (NO) is a highly reactive free radical with profound tumoricidal activity, produced by both macrophages and tumor cells. While it has been postulated that necrotic tumor cells can augment macrophage anti-tumor action, we investigated the effect of tumor cell necrosis on NO synthesis and viability of L929 fibrosarcoma and C6 astrocytoma cell lines. The presence of necrotic tumor cells dose-dependently reduced NO production in IFN-gamma stimulated L929 cells, and rescued them from NO-dependent autotoxicity. This effect was mediated through soluble products, since it was completely preserved after blocking the contact between the necrotic and live cells. On the other hand, apoptotic tumor cells were unable to suppress IFN-gamma-triggered NO release and subsequent decrease of cell respiration in L929 cultures. Similar results were obtained with C6 astrocytoma cell line. This down-regulation of NO synthesis in response to necrotic cell products was not specific for tumor cell lines, since necrotic tumor cells markedly suppressed NO production in cytokine-stimulated primary fibroblasts and astrocytes. In contrast, both murine and rat peritoneal macrophages readily increased their basal or IFN-gamma-induced NO production when incubated with necrotic tumor cells. Taken together, these results suggest that tumor cell necrosis might promote or restrict tumor growth through suppression or enhancement of NO synthesis in tumor cells and macrophages, respectively, with net effect presumably depending on the extent of macrophage infiltration.     

Restoration of macrophage tumoricidal activity by bleomycin correlates with the decreased production of transforming growth factor β in rats bearing KDH-8 hepatoma cells

 To explore the mechanisms of immuno-modulatory activities of bleomycin, we investigated interferon γ (IFNγ) mRNA expression, tumor necrosis factor α (TNFα) production, nitric oxide (NO) production and macrophage tumoricidal activities in rats bearing KDH-8 hepatoma cells, which secreted a large amount of transforming growth factor β (TGFβ), and these processes in KDH-8 tumor-bearing rats treated with bleomycin. We found that IFNγ mRNA expression, TNFα production, NO production and macrophage cytotoxic activities were lower in the KDH-8-bearing rats than in normal rats. On the other hand, low-dose bleomycin restored the macrophage cytotoxic activities, NO production, IFNγ mRNA expression and TNFα production in the KDH-8-bearing rats. In vitro experiments showed that KDH-8-derived TGFβ decreased the IFNγ mRNA expression and TNFα production in splenocytes, and NO production in peritoneal macrophages. These results suggest that low-dose bleomycin restored the cytokine production and macrophage tumoricidal activities in the KDH-8-bearing rats by decreasing KDH-8-derived TGFβ. Received: 14 October 1996 / Accepted: 22 July 1997     

Tumoricidal responses in vitro of peritoneal macrophages from conventionally housed and germ-free nude mice

Peritoneal macrophages from untreated nude mice were nonspecifically cytotoxic to tumor cells in vitro and were more responsive to chemotactic stimuli than macrophages from normal mice or from phenotypically normal littermates of nude mice. Tumoricidal and chemotactic responses of activated macrophages from nude mice were quantitatively comparable to responses of macrophages from BCG-infected normal mice. Peritoneal macrophages from germ-free nude mice, however, were not tumoricidal in vitro. These observations suggest that environmental stimuli, rather than thymic deficiency per se, induced activated macrophages in nude mice.     

AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms

Chronic obstructive pulmonary disease (COPD) is characterized by intense lung infiltrations of immune cells (macrophages and monocytes). Lipopolysaccharide (LPS) activates macrophages/monocytes, leading to production of tumor necrosis factor α (TNFα) and other cytokines, which cause subsequent lung damages. In the current study, our results demonstrated that AS-703026, a novel MEK/ERK inhibitor, suppressed LPS-induced TNFα mRNA expression and protein secretion in RAW 264.7 murine macrophages, and in murine bone marrow-derived macrophages (BMDMs). Meanwhile, TNFα production in LPS-stimulated COPD patents’ peripheral blood mononuclear cells (PBMCs) was also repressed by AS-703026. At the molecular level, we showed that AS-703026 blocked LPS-induced MEK/ERK activation in above macrophages/monocytes. However, restoring ERK activation in AS-703026-treated RAW 264.7 cells by introducing a constitutive-actively (CA)-ERK1 only partially reinstated LPS-mediated TNFα production. Meanwhile, AS-703026 could still inhibit TNFα response in ERK1/2-depleted (by shRNA) RAW 264.7 cells. Significantly, we found that AS-703026 inhibited LPS-induced nuclear factor κB (NFκB) activation in above macrophages and COPD patients’ PBMCs. In vivo, oral administration of AS-703026 inhibited LPS-induced TNFα production and endotoxin shock in BALB/c mice. Together, we show that AS-703026 in vitro inhibits LPS-induced TNFα production in macrophages/monocytes, and in vivo protects mice from LPS-induced endotoxin shock. Thus, it could be further studied as a useful anti-inflammatory therapy for COPD patients.     

Tumor Suppressor WWOX Contributes to the Elimination of Tumorigenic Cells in Drosophila melanogaster

WWOX is a >1Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, we find that WWOX is a modulator of TNFα/Egr-mediated cell death. We found that altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFα/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of which we have previously shown can also be modified by altered WWOX activity. We now hypothesise that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. We have further shown that WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.     

Discovery of a Natural Product-Like iNOS Inhibitor by Molecular Docking with Potential Neuroprotective Effects In Vivo

In this study, we applied structure-based virtual screening techniques to identify natural product or natural product-like inhibitors of iNOS. The iNOS inhibitory activity of the hit compounds was characterized using cellular assays and an in vivo zebrafish larvae model. The natural product-like compound 1 inhibited NO production in LPS-stimulated Raw264.7 macrophages, without exerting cytotoxic effects on the cells. Significantly, compound 1 was able to reverse MPTP-induced locomotion deficiency and neurotoxicity in an in vivo zebrafish larval model. Hence, compound 1 could be considered as a scaffold for the further development of iNOS inhibitors for potential anti-inflammatory or anti-neurodegenerative applications.     

Stimulation and inhibition of neoplastic cell growth by tumor promoter-treated macrophages

Macrophages treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent inflammatory and tumor-promoting agent, can have the diametrically opposed functions of contact-mediated tumor cytotoxicity and release of soluble clonal proliferation factor(s) for tumor cells. In vitro TPA treatment of macrophages at 1.0 ng/ml induced prostaglandin E₂ release and morphological changes analogous to cell activation. In addition, conditioned medium from macrophages pulsed with TPA enhanced M109 carcinoma colony formation in vitro. Although macrophages were not rendered tumoricidal by TPA in vitro, cytotoxic macrophages were recovered from mice following ip treatment with TPA at 1–100 μg/kg. This indicated an indirect pathway for the activation of macrophages by TPA. The very weak tumor promoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate lacked effects on macrophages at all doses tested. The possibility that macrophage secretions (e.g., prostaglandin E₂, angiogenesis-stimulating factor(s), and clonal proliferation factor(s) for carcinogen-triggered cells) may be involved in the tumor promotion process is discussed.     

Abnormalities in Osteoclastogenesis and Decreased Tumorigenesis in Mice Deficient for Ovarian Cancer G Protein-Coupled Receptor 1

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1''s role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.     

Leishmania infantum Modulates Host Macrophage Mitochondrial Metabolism by Hijacking the SIRT1-AMPK Axis

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.     

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

Macrophage activation for tumor cytotoxicity: genetic variation in macrophage tumoricidal capacity among mouse strains.

Development of activated tumoricidal macrophages following Mycobacterium bovis, strain BCG infection in vivo or lymphokine treatment in vitro was examined with more than 20 mouse strains. Peritoneal macrophages from 8 of 22 strains failed to develop tumoricidal capacity by 7 days after intraperitoneal BCG infection. Macrophages from 6 of 6 in vivo nonresponder strains also failed to develop tumoricidal capacity after in vitro treatment with lymphokines. Identification of nonresponder mouse strains should provide a useful resource for analysis of intermediary reactions in macrophage activation.     

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B‐cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL–tumor contacts in vivo. In the therapeutic settings of anti‐CD19 CAR T cells, the majority of CAR T cell–tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti‐tumor responses in vivo.     

Candida albicans Suppresses Nitric Oxide Generation from Macrophages via a Secreted Molecule

Macrophages and neutrophils generate a potent burst of reactive oxygen and nitrogen species as a key aspect of the antimicrobial response. While most successful pathogens, including the fungus Candida albicans, encode enzymes for the detoxification of these compounds and repair of the resulting cellular damage, some species actively modulate immune function to suppress the generation of these toxic compounds. We report here that C. albicans actively inhibits macrophage production of nitric oxide (NO). NO production was blocked in a dose-dependent manner when live C. albicans were incubated with either cultured or bone marrow-derived mouse macrophages. While filamentous growth is a key virulence trait, yeast-locked fungal cells were still capable of dose-dependent NO suppression. C. albicans suppresses NO production from macrophages stimulated by exposure to IFN-γ and LPS or cells of the non-pathogenic Saccharomyces cerevisiae. The NO inhibitory activity was produced only when the fungal cells were in direct contact with macrophages, but the compound itself was secreted into the culture media. LPS/IFNγ stimulated macrophages cultured in cell-free conditioned media from co-cultures showed reduced levels of iNOS enzymatic activity and lower amounts of iNOS protein. Initial biochemical characterization of this activity indicates that the inhibitor is a small, aqueous, heat-stable compound. In summary, C. albicans actively blocks NO production by macrophages via a secreted mediator; these findings expand our understanding of phagocyte modulation by this important fungal pathogen and represent a potential target for intervention to enhance antifungal immune responses.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

Subpopulations of human T lymphocytes. II. Effect of thymopoietin, corticosteroids, and irradiation.

Lymphoid cells from normal SJL/J mice gave high proliferative responses but failed to develop cytotoxic activity to γ-irradiated cells from syngeneic transplantable reticulum cell sarcomas (X-RCS). In spite of a vigorous in vivo proliferative response to X-RCS, cytotoxic activity was never generated to detectable levels in vivo. After repeated injections of X-RCS, spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitro upon co-culture with X-RCS. This immunity was T-cell mediated and specific for RCS although it did not distinguish between different transplantable RCS lines. SJL/J mice also developed resistance to RCS growth after injection of X-RCS, which showed a transient RCS-line-specific component. (SJL/J × C₅₇B1/6)F₁ mice showed 60% less RCS growth than did SJL/J mice, and their lymphoid cells gave slightly lower proliferative responses than did cells from SJL/J mice, whereas (SJL/J × BALB/c)F₁ mice showed little tumor growth, and their spleen cells proliferated only minimally to X-RCS. B10.S mice allowed moderate RCS growth. Cytotoxic activity was generated in co-cultures with X-RCS of immunized F₁ spleen cells even after a single immunization in vivo but not in cultures of normal F₁ cells with X-RCS.