Cytotoxic Activity Of Macrophages Research Articles

Analyzing the role of cancer-associated fibroblast activation on macrophage polarization.

Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFβ. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.

Macrophage cytotoxic activity and its role in the tumor pathogenesis

Macrophages, natural killers and T cells play the central role in tumor cells destruction. The purpose of this review is to summarize the state-of-the-art perspectives of the interplay between tumor cells and tumor stroma leading both to the formation of a macrophage population incapable of effective antitumor activity and to the selection of tumor cells resistant to macrophage cytotoxicity. Macrophages are highly versatile cells that can both stimulate the inflammatory response (type 1 macrophages, M1) and suppress it (type 2 macrophages, M2). Tumor-associated macrophages (TAMs) are considered the main regulator of the antitumor immune response and usually have anti-inflammatory properties, that is, they belong to M2 type. Tumor cells are able to affect macrophages, "reprogramming" them to perform an immunosuppressive function. In addition, TAMs stimulate angiogenesis and remodelling of the extracellular matrix necessary for metastasis. Recently, more and more studies have been published describing a mixed TAMs phenotype with characteristics of both M2 and M1. M1 is characterized by production of pro-inflammatory cytokines, reactive oxygen species, bactericidal and cytotoxic activity. M1 can destroy tumor cells both directly and indirectly by attracting other cells. Despite the mechanisms of direct cytotoxic activity are quite variable, their effectiveness is largely dependent on the properties of a particular tumor. The cytotoxic activity of macrophages is a powerful factor that inhibits tumor initiation and progression. However, in some cases, it is not sufficient to control the tumor process. Activation of the cytotoxic activity of TAMs is one of the strategies to use macrophages for cancer treatment. Understanding the mechanisms of macrophage cytotoxic activity and specific patterns of its manifestation in a tumor environment is of critical importance for better effectiveness of existing cancer treatments and development of promising methods for tumor immunotherapy.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity

BackgroundTumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in...

Targeting CD47 and Autophagy Elicited Enhanced Antitumor Effects in Non-Small Cell Lung Cancer.

CD47-specific antibodies and fusion proteins that block CD47-SIRPα signaling are employed as antitumor agents for several cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in non-small cell lung cancer (NSCLC). SIRPαD1-Fc, a novel CD47-targeting fusion protein, was generated and was found to increase the phagocytic and cytotoxic activities of macrophages against NSCLC cells. During this process, autophagy was markedly triggered, which was characterized by the three main stages of autophagic flux, including formation and accumulation of autophagosomes, fusion of autophagosomes with lysosomes, and degradation of autophagosomes in lysosomes. Meanwhile, reactive oxygen species and inactivation of mTOR were shown to be involved in autophagy initiation in SIRPαD1-Fc-treated cells, indicating a probable mechanism for autophagy activation after targeting CD47 by SIRPαD1-Fc. Inhibition of autophagy enhanced macrophage-mediated phagocytosis and cytotoxicity against SIRPαD1-Fc-treated NSCLC cells. In addition, simultaneously targeting both CD47 and autophagy in NSCLC xenograft models elicited enhanced antitumor effects, with recruitment of macrophages, activated caspase-3, and overproduction of ROS at the tumor site. Our data elucidated the cytoprotective role of autophagy in CD47-targeted therapy and highlighted the potential approach for NSCLC treatment by simultaneously targeting CD47 and autophagy. Cancer Immunol Res; 5(5); 363-75. ©2017 AACRSee related Spotlight by Kaufman, p. 355.

High dilutions of antimony modulate cytokines production and macrophage – Leishmania (L.) amazonensis interaction in vitro

BackgroundIn previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. AimsTo know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. MethodologySpreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. ResultsCells treated with AC 30cH (10−58M) and AC 200cH (10−398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time. ConclusionsBoth dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.

Abstract 3631: Modulation of monocyte- and macrophage-mediated antibody-dependent cell phagocytosis and cytotoxicity (ADCP/ADCC) by Fc engineering of therapeutic antibodies

Abstract Therapeutic monoclonal antibodies contribute to elimination of tumor cells by different mechanisms including blocking of receptor signaling, induction of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and induction of adaptive immunity. FcγR-expressing monocytes and macrophages have recently been identified as important immune effectors contributing to the activity of monoclonal antibodies in vivo. Therefore, we analyzed the FcγR-dependent effector functions mediated by both cell subsets upon treatment with glycoengineered (GE) antibodies (having enhanced FcγRIIIa (CD16a) binding affinity), wild-type (WT), and Fc-mutated antibodies that lack binding to FcγRs (P329G LALA mutation). Trastuzumab, cetuximab, rituximab and ofatumumab were used as WT antibodies, whereas GE trastuzumab, GA201 (targeting EGFR) and GA101 (targeting CD20) were used as GE antibodies. To better reflect the plasticity of myeloid lineage cells we evaluated the activities on classical and non-classical/intermediate monocytes, M1 and M2c macrophages. We found that GE and WT antibodies displayed comparable binding and induced similar monocyte/macrophage-mediated ADCC and ADCP in absence of unspecific endogenous IgGs. Interestingly, in their presence, i.e. in a situation that more closely mimics physiological conditions, GE antibodies displayed significantly superior binding and induced higher levels of ADCC/ADCP compared to WT antibodies. Data collected by imaging further indicated that the degree of phagocytized cells is higher and faster in presence of GE antibodies and that M2c macrophages perform serial phagocytosis by engulfing 2-3 tumor cells. Fc-mutated P329G LALA variant showed no binding and no functional activity. A direct comparison of the three anti-CD20 antibodies (GA101, rituximab and ofatumumab), pointed out that GA101 mediated significantly stronger elimination of tumor cells further underscoring the relevance of combination of both glycoengineering and cell death induction for monocyte/macrophage-mediated killing of tumor cells. These data show, for the first time that, in addition to enhancing NK cell-mediated cytotoxicity (ADCC), glycoengineering also enhances monocyte and macrophage cytotoxic and phagocytic activities through enhanced binding to FcγRIIIa under conditions that more closely resemble the physiological setting. Citation Format: Herter Sylvia, Christian Klein, Martina Birk, Thomas Weber, Pablo Umana, Marina Bacac. Modulation of monocyte- and macrophage-mediated antibody-dependent cell phagocytosis and cytotoxicity (ADCP/ADCC) by Fc engineering of therapeutic antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3631. doi:10.1158/1538-7445.AM2014-3631

Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Polyacetylenes from Bidens bipinnata L. and their biological activities

Two new polyacetylenes, (2S) (5E,11E)-tridecadiene-7,9-diyne-1,2,13-triol (1) and (6E,12E)-3-oxo-tetradecadiene-8,10-diyne-14-hydroxyl-1-O-β-d-glucopyranoside (2) along with four known polyacetylenes (3–6), were isolated from the whole plant of Bidens bipinnata L. Their structures were elucidated by spectroscopic data. All compounds were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced IL-1 and tumor necrosis factor (TNF-α) production in macrophage (RAW264.7) cells and cytotoxic activities on human cervical carcinoma (Hela), human hapetocelluar carcinoma (HepG-2), and human breast carcinoma (MCF-7) cell lines.

Susceptibility of rhabdomyosarcoma cells to macrophage-mediated cytotoxicity

The prognosis of advanced stage rhabdomyosarcoma (RMS) is still sobering. In recent years, outcome has not been further improved by conventional therapy. Therefore, novel treatment options such as macrophage-directed immunotherapy have to be investigated. The aim of this study was to analyze the phagocytosis of RMS cells by macrophages and to modulate the susceptibility using monoclonal antibodies and cytotoxic drugs. Expression of the macrophage activating ligand calreticulin and CD47, the counterpart of the inhibitory receptor SIRPα, was analyzed with Affymetrix mRNA expression arrays and immunohistochemistry on 11 primary RMS samples. Results were verified in two RMS cell lines using flow cytometry and immunocytochemistry. Macrophage cytotoxic activity was quantified by a MTT colorimetric assay in co-culture experiments of RMS cells with monocyte-derived, GM-CSF stimulated macrophages. Gene expression analysis and immunohistochemistry revealed a high expression of CD47 and calreticulin in alveolar and embryonal RMS tissue specimens. Extracellular expression of CD47 on RMS cell lines was confirmed by flow cytometry, whereas calreticulin was exclusively detected in the endoplasmatic reticulum. After co-culturing of RMS cells with macrophages, viability dropped to 50–60%. Macrophage-mediated cytotoxicity was not influenced by a blocking antibody against CD47. However, susceptibility was significantly enhanced after pre-treatment of RMS cells with the anthracycline drug doxorubicin. Furthermore, translocation of calreticulin onto the cell surface was detected by flow cytometry. The immunologic effect of doxorubicin may improve the efficacy of adoptive cellular immunotherapy and chemotherapy of childhood RMS.

Abstract 1520: Beta-endorphin neuron transplants suppress mammary tumor growth and progression via neuroimmune activation

Abstract A review of studies that evaluated psychological factors and outcome in cancer patients suggested an association between certain psychological factors and the growth and progression of cancer. It has been suggested that the effect of stress on the immune system may in turn affect the growth of some tumors. We have previously shown that a set of hormone secreting nerve cells in the hypothalamus, called beta-endorphin (BEP) neurons, plays a role in regulating stress response and immune function. Hence, we tested whether activation of BEP neurons reduces stress hormone levels and prevents the N-nitroso-N-methylurea (NMU)-induced mammary tumor burden and malignancy rate. To test this we differentiated rat neural stem cells from the hypothalamus into BEP neurons in culture, which were later transplanted into the paraventricular nuclei (PVN) of the hypothalamus of 90 day old Sprague-Dawley female rats, which were injected with MNU 4 weeks prior to cells transplantation. Control rats were transplanted with cortical cells or not operated. Animals were palpated every week to check for tumor growth for additional 12 weeks. Tumor length and width were measured with a calibrator. Sixteen weeks after MNU injection, animals were sacrificed, tumors were collected, and slices of tumors were put in formalin and processed for histological staining for pathology. Additionally, the impact of cell transplants on plasma levels of stress hormone corticosterone and cytokines interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α) in plasma and the levels of natural killer (NK) cell and macrophage cytotoxic activities in peripheral blood and spleen were evaluated. When the BEP neurons were transplanted into the PVN of an adult rat, they integrated well with the surrounding cells and produced BEP and it precursor gene proopiomelanocortin. We found that BEP implanted animals have significantly lower tumor incidence, tumor volume, tumor number and malignancy rate. Rats with BEP neuron transplants also showed reduced plasma corticosterone levels, increased NK cell and macrophage activities in the spleens and peripheral blood mononuclear cells, elevated plasma levels of anti-inflammatory cytokine INF-γ and decreased levels of inflammatory cytokine TNF-α. These results identify a protective role of BEP neurons against MNU-induced mammary tumor (animal model for breast cancer) growth and progression via increasing the innate immune function and reducing the inflammatory milieu. (Supported by NIH Grant AA015718). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1520. doi:10.1158/1538-7445.AM2011-1520

Immunotoxicity of dibromoacetic acid administered via drinking water to female B6C3F1 mice

Dibromoacetic acid (DBA) is a disinfection by-product commonly found in drinking water as a result of chlorination/ ozonation processes. The Environmental Protection Agency estimates that more than 200 million people consume disinfected water in the United States. This study was conducted to evaluate the potential immunotoxicological effects of DBA exposure when administered for 28 days via drinking water to B6C3F1 mice, at concentrations of 125, 500, and 1000 mg/L. Multiple endpoints were evaluated to assess innate, humoral, and cell-mediated immune components, as well as host resistance. Standard toxicological parameters were unaffected, with the exception of a dose–responsive increase in liver weight and a decrease in thymus weight at the two highest exposure levels. Splenocyte differentials were affected, although the effects were not dose–responsive. Exposure to DBA did not significantly affect humoral immunity (immunoglobulin M [IgM] plaque assay and serum IgM anti-sheep erythrocyte titers) or cell-mediated immunity (mixed-leukocyte response). No effects were observed on innate immune function in either interferon-γ-induced in vitro macrophage cytotoxic activity or basal natural killer (NK)-cell activity. Augmented NK-cell activity (following exposure to polyinosinic-polycytidylic acid) was decreased at the low dose, however the effect was not dose–responsive. Finally, DBA exposure had no effect on resistance to infection with either Streptococcus pneumoniae or Plasmodium yoelii, or challenge with B16F10 melanoma cells. With the exception of changes in thymus weight, these results indicate that DBA exposure resulted in no immunotoxic effects at concentrations much larger than those considered acceptable in human drinking water.

Abstract 3820: Inhibition of M1 macrophages may aid the conversion process to M2, which may aid tumor survival and growth

Abstract Macrophages perform a variety of functions, from phagocytosis to the repair and remodeling of damaged tissues. Two phenotypes have been proposed: M1 and M2. The M1 macrophage performs phagocytosis of tumor cells and releases pro-inflammatory cytokines. The M2 macrophage acts as a support cell by promoting the healing of damaged cells by angiogenesis and tissue remodeling. The transition from M1 to M2 may be facilitated by tumor environments. To further study the relationship between cancer and macrophages we developed a live-cell migration assay, which measures cancer's ability to recruit macrophages. We hypothesize that cancer cell lines have varied abilities to recruit macrophages and convert them into M2, and that this ability corresponds to the aggressiveness of the cell line. Macrophages were derived from the human mononuclear phagocyte cell line U937, after addition of phorbol 12-myristate 13-acetate (PMA). These U937 cells were incubated with three adenocarcinomas of varying aggressivity. The adenocarcinomas were MDA-MB-435, MDA-MB-231, and MCF7. The U937 cells were separated from the three cell lines by 3.0 µm trans-permeable polycarbonate membranes (Corning Inc.) which allowed the U937 cells to migrate to the cell lines. After 1 hour non-migratory U937 cells were isolated and counted by means of a hemocytometer. The control without cancer caused 73.86% of U937 cells to remain, MDA-MB-435 69.28% (N=24, P=0.29), MCF7 61.09% (N=24, P<0.01), and MDA-MB-231 55.83% (N=24, P<0.01). This data was then compared with expression levels of macrophage Migration Inhibition Factor (MIF) analyzed from Real Time PCR data. These samples consisted of monocytes, from human blood, which were co-incubated with the cell lines for 48 hours. The cells were separated by 0.4 µm trans-permeable polycarbonate membranes (Corning Inc.) to prevent cells from coming into contact and allowing signal exchange. The RT-PCR data correlated with the migration data. The amount of MIF increased 7.56 fold, (N=9) in MDA-MB-435 when compared to controls, 3.13 fold for MCF7 (N=9), and 1.33 fold for MDA- MB-231 (N=9). This data suggests that the more aggressive the cancer the more MIF emitted and the greater the potential to inhibit macrophages. Previous studies have shown Interleukin-10, (IL-10), IL-6, IL-4, and prostaglandin F2 (PGF2) to be released from tumors and inhibit the cytotoxic activity of macrophages. This exposure to IL-10 has been suggested to convert M1 macrophages to a M2 phenotype. The migration inhibition observed from this study might be the initial step in the conversion process from M1 to M2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3820.

The effects of a cyclooxygenase-2 (COX-2) expression and inhibition on human uveal melanoma cell proliferation and macrophage nitric oxide production

BackgroundCyclooxygenase-2 (COX-2) expression has previously been identified in uveal melanoma although the biological role of COX-2 in this intraocular malignancy has not been elucidated. This study aimed to investigate the effect of a COX-2 inhibitor on the proliferation rate of human uveal melanoma cells, as well as its effect on the cytotoxic response of macrophages.MethodsHuman uveal melanoma cell lines were transfected to constitutively express COX-2 and the proliferative rate of these cells using two different methods, with and without the addition of Amfenac, was measured. Nitric oxide production by macrophages was measured after exposure to melanoma-conditioned medium from both groups of cells as well as with and without Amfenac, the active metabolite of Nepafenac.ResultsCells transfected to express COX-2 had a higher proliferation rate than those that did not. The addition of Amfenac significantly decreased the proliferation rate of all cell lines. Nitric oxide production by macrophages was inhibited by the addition of melanoma conditioned medium, the addition of Amfenac partially overcame this inhibition.ConclusionAmfenac affected both COX-2 transfected and non-transfected uveal melanoma cells in terms of their proliferation rates as well as their suppressive effects on macrophage cytotoxic activity.

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 microM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a (51)Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.

Brucella abortus: inmunidad, vacunas y estrategias de prevención basadas en ácidos nucleicos

SUMMARY Brucellosis, a disease caused by the intracellular facultative bacteria Brucella abortus , is a widely distributed zoonosis throughoutthe world, affecting mainly cattle and causing sterility in males and abortion in pregnant females. Protection against B. abortus relieson the activation of cellular immunity involving lymphocytes T CD4 + , subset Th1, secreting gamma-interferon (γ-INF), a cytokinestimulatings macrophage bactericidal activity and cytotoxic activity of lymphocytes TCD8 + , which are able to lyse Brucella infectedcells. The main antigenic components of Brucella are lipopolysaccharide (LPS) and proteins, especially superoxide dismutase (SOD)with demonstrated immune potential. Brucellosis spreading is prevented with vaccines using attenuated or inactivated strains of B.abortus , such as strains 19, 45/20 and RB51. On the other hand, several investigators are making efforts to obtain immunity usingantigenic structures of Brucella as subcellular vaccines and recently, genetic vaccines based on DNA and RNA molecules. The aimof this review is to give a current overview about brucellosis, its pathogenicity and the clinical syndrome. Firstly, an analysis of thegenetic, antigenic and immune characteristics of

Role of Interleukins and Nitric Oxide Secretion by Peritoneal Macrophages in Differential Tumoricidal Effect to Transplantable Melanomas as Regarding their Biological Properties

The secretion of interleukins (IL-18, IL-12) and Nitric Oxide (NO) by peritoneal macrophages from hamsters bearing two lines of transplantable melanoma was estimated. Macrophages of animals with melanoma lines secreted less IL-18 but more IL-12 and NO in comparison with the control macrophages. The distinctly higher cytotoxic activity of macrophages from animals with amelanotic line in comparison with the melanotic line was not accompanied by significant differences in the IL-18 and IL-12 secretion between studied groups of macrophages. Thus, it seems that IL-18 play the role in innate immunity but not in adaptive cellular immunity, whereas IL-12 and NO take part in macrophages tumoricidal activity.

Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production.

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO4 markedly reduced IFN-gamma + LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-gamma + LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Macrophages play a critical role in the anti-tumor activity of Virulizin®

Virulizin, a novel biological response modifier (BRM), has been demonstrated to have a high level of anti-tumor activity against pancreatic cancer and melanoma in many clinical trials and preclinical studies. However, its anti-tumor mechanism has not been fully elucidated. The purpose of this study was to define the mechanism of Virulizin anti-tumor activity in cultures and in a murine xenograft model. The presence of Virulizin stimulated in a dose-dependent manner the cytolytic activity against tumor cells by splenocytes and macrophages, but not by non-adherent splenocytes. The cytotoxic activity of macrophages was significantly increased (approximately 5-fold) in cultures containing 2.5% of Virulizin compared to that of cultures without Virulizin (p<0.001). An increase of 21% in the protease secretion was observed in Virulizin (2.5%)-stimulated macrophages compared to PBS-treated cells (p<0.0001). Moreover, the anti-tumor efficacy of Virulizin observed in CD-1 nude mice was abrogated in mice that were depleted of macrophages, thus stimulation of macrophages may be one mechanism through which Virulizin acts. These results suggest that macrophages may play a critical role in the anti-tumor activity of Virulizin.

Prostaglandin E2 down-regulates viable Bacille Calmette-Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro.

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette-Guérin (BCG)-induced macrophage cytotoxicity in a bladder cancer cell, MBT-2, was studied in vitro. BCG stimulated thioglycollate-elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and PGE2. NS398, a specific COX-2 inhibitor, and indomethacin (IM), a COX-1 and COX-2 inhibitor, enhanced viable BCG-induced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN-gamma and TNF-alpha. Exogenous PGE2 reduced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. These results suggest that PGE2 produced by BCG-activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG-induced antitumour activity of macrophages.

In vitro and in vivo enhancement of canine pulmonary alveolar macrophage cytotoxic activity against canine osteosarcoma cells.

The combination of chemotherapy with immunotherapy may offer an advantage over either therapy alone and provide a greater potential for total tumor eradication. Monocyte/macrophage-mediated tumor cell killing is a major mechanism of the host's defense against primary and/or metastatic neoplasia. We evaluated the tumoricidal activity against canine osteosarcoma cells of canine pulmonary alveolar macrophages (PAM) exposed in vitro to two recombinant canine (rc) cytokines (rcTNF alpha and rcIFN gamma). We also evaluated the in vivo tumoricidal activity of PAM from dogs treated with the macrophage activator, liposome-encapsulated muramyl tripeptide-phosphatidyl-ethanolamine (L-MTP-PE) alone or in combination with doxorubicin (DOX). This study demonstrated that rcTNF alpha and rcIFN gamma significantly enhance in vitro canine PAM cytotoxicity against canine osteosarcoma cells, and that PAM from dogs treated with DOX + L-MTP-PE have enhanced cytotoxic activity against osteosarcoma cells when compared to dogs treated with DOX or L-MTP-PE alone. These findings support the rationale for combining a chemotherapy agent with an immunotherapy agent for the treatment of metastatic disease, and suggest a role for TNF alpha and IFN gamma as agents for stimulating the antitumor activity of macrophages.