Macrophage Tumoricidal Activity Research Articles

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro-indirect activation of macrophages by photodynamically killed tumor cells

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-γ, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.

Cytotoxic activity of BCG-activated macrophages against L929 tumor cells is nitric oxide-dependent.

The tumoricidal activity of activated macrophages has been attributed largely to the release of tumor necrosis factor (TNF), or to the production of reactive oxygen or nitrogen intermediates. The L929 tumor cell line (a murine fibroblast-like cell) when treated with actinomycin D (ActD) has been used to measure TNF alpha cytotoxicity. In the present study, we determined the cytotoxic activity of BCG-activated peritoneal macrophages against ActD-untreated L929 tumor cells. Furthermore, we measured the production of hydrogen peroxide (H2O2), nitric oxide (NO) and TNF by macrophages cultured in the presence or absence of L929 cells. As expected, BCG-activated macrophages produced significant amounts of H2O2 (16.0 +/- 3.0 microM), TNF (512 U/ml) and NO (71.5 +/- 3.2 microM). TNF (256 U/ml) and NO (78.9 +/- 9.7 microM) production was unchanged in co-cultures of L929 cells with BCG-activated macrophages but H2O2 production was totally inhibited. The cytotoxic activity was dependent on NO release since L-NAME (2.5, 5.0 and 10 mM), which blocks NO synthase, inhibited the killing of L929 cells. Addition of anti-TNF (20 micrograms/ml) antibodies to the cultures did not affect the tumoricidal activity of macrophages. Our results indicate that macrophage-mediated killing of L929 cells is largely dependent on NO production but independent of H2O2 or TNF release.

Involvement of Ras and MAP kinase (ERK-1) in cisplatin-induced activation of murine bone marrow-derived macrophages.

Cisplatin (cis-diamminedichloroplatinum II), a potent antitumor compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the mechanism by which cisplatin activates these cells is poorly characterised. Our earlier findings indicate that cisplatin treatment stimulates rapid tyrosine phosphorylation in a number of cellular proteins in murine macrophages. This initial tyrosine phosphorylation is an important regulatory mechanism and is followed by activation of several other proteins. In the present study, we report the involvement of other key molecules and the role of tyrosine phosphorylation in their activation in the signaling cascade of cisplatin. We observed the involvement of Ras (a low molecular weight GTP-binding protein) and ERK-1 (a MAP kinase) in this signaling cascade. Cisplatin treatment results in an increase in the expression of both Ras and ERK-1 in a dose-dependent manner, which was dependent upon tyrosine phosphorylation. Genistein a PTK inhibitor inhibited the cisplatin induced expression of Ras and ERK-1. These findings indicate that Ras and ERK-1 are important signaling molecules involved in the tumoricidal activation of macrophages with cisplatin and is dependent on initial tyrosine phosphorylation.

Cisplatin-induced activation of murine bone marrow-derived macrophages require protein tyrosine phosphorylation

The aim of the present study is to evaluate the involvement of tyrosine phosphorylation in the signal transduction mechanism of cisplatin-induced macrophage activation in vitro. Stimulation of bone marrow-derived macrophages (BMDM) with cisplatin (CP) resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 18, 20, 21, 30, 33, 35, 39, 41, 44, 58 and 123 kD, detected by immunoblot using anti-phosphotyrosine antibody. CP-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor genistein. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of CP on murine bone marrow-derived macrophages (BMDM) . Treatment of macrophages with genistein before incubation with CP completely inhibited the CP-induced tumoricidal activation of macrophages as well as production of TNF and NO, whereas pre-treatment of macrophages with phosphatase inhibitor sodium vanadate upregulated macrophage activation in addition to enhanced protein tyrosine phosphorylation. Taken together, these data suggest that tyrosine phosphorylation play a critical regulatory role in the activation of macrophages with CP.

Sensitivity of wild cotton rats (Sigmodon hispidus) to the immunotoxic effects of low-level arsenic exposure.

Arsenic is a ubiquitous contaminant of many toxic waste sites around the country and experimental animal trials have indicated that arsenic may be immunotoxic to laboratory rodents. Because wild rodents such as the herbivorous cotton rat (Sigmodon hispidus) reside on many of these toxic waste sites, we explored the sensitivity of their immune systems to oral exposures of environmentally relevant concentrations of inorganic arsenic. We exposed adult male cotton rats (n = 36) to either 0 (controls), 5 (low dose), or 10 (high dose) ppm sodium arsenite in drinking water for 6 weeks. Daily food intake decreased in a dose-dependent manner, ranging from an average of 10.03 +/- 0.45 in the high-dose group to 11.27 +/- 0.42 (SE) g/animal/day in the control group. Mass of testes in the low-dose group increased significantly compared to controls, but there was no difference between the high-dose and control groups. Masses of liver, kidney, adrenals, popliteal lymph nodes, spleen, epididymides, and seminal vesicles and selected hematological parameters were unaffected by arsenic exposure. In vivo cell-mediated immunity, as measured by a phytohemagglutinin-hypersensitivity response to an intradermal challenge, was suppressed 30% in the low-dose group compared to controls; however, responses of those receiving a high dose of arsenic were similar to controls. Arsenic treatment did not have a measurable impact on lymphoproliferative responses of cultured splenocytes to the mitogens Concanavalin A and Pokeweed mitogen, or to the lymphokine interleukin-2. We also observed no impact of low-level arsenic exposure on macrophage phagocytic activity and tumoricidal activity of lymphokine-activated killer cells in vitro. It is possible that malnutrition caused by decreased food intake may eventually lead to atrophy of lymphoid organs and render animals more susceptible to environmental pathogens. However, direct effects of low-level arsenic exposure on immune function of cotton rats was minimal (a moderate depression in the in vivo cell-mediated immunity assay) and may not be clinically relevant with regard to susceptibility to disease in the wild.

Correlation between apoptosis and tumor-associated macrophages in human invasive ductal breast carcinoma

The relationship between apoptosis and tumor-associated macrophages (TAM) was studied in situ in 60 breast carcinomas (18 G1, 28 G2 and 14 G3 carcinomas) using simultaneous apoptosis (TUNEL method) and macrophage staining (anti-CD68 antibody). Apoptotic tumor cell rate (ATCR), total TAM content (TAM(TOT)) and the proportion of TAM that had either cell-to-cell contact with apoptotic tumor cells or that were phagocytosing them (TAM/APO cells) were quantified. ATCR correlated significantly with TAM(TOT). Within all apoptotic tumor cells, the proportion of TAM/APO cells was lower than 20%. Considering the fact that cell-to-cell contact is essential for macrophage-mediated tumor cell killing, our data suggest that the majority of apoptoses occurring in breast cancer may not be caused by macrophage tumoricidal activity. TAM/APO cells accounted for only 1.7% of all TAM. Thus, tumor cell killing and apoptotic tumor cell phagocytosis seem to be quantitatively less important functions of TAM in human breast cancer in vivo.

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

To explore the mechanisms of immuno-modulatory activities of bleomycin, we investigated interferon gamma (IFN gamma) mRNA expression, tumor necrosis factor alpha (TNF alpha) 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 beta (TGF beta), and these processes in KDH-8 tumor-bearing rats treated with bleomycin. We found that IFN gamma mRNA expression, TNF alpha 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, IFN gamma mRNA expression and TNF alpha production in the KDH-8-bearing rats. In vitro experiments showed that KDH-8-derived TGF beta decreased the IFN gamma mRNA expression and TNF alpha 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 TGF beta.

Acute blood toxicity of the abused inhalant, cyclohexyl nitrite

Cyclohexyl nitrite is an abused nitrite inhalant. This is the first report of toxicity of cyclohexyl nitrite. Mice were exposed to 300–900 ppm cyclohexyl nitrite in an inhalation chamber for 45 min and then bled. Such treatment resulted in a 7–10% reduction in red blood cell counts, haemoglobin and haematocrit levels. Both blood leucocyte counts and spleen cellularity were reduced by 40%. Unlike isobutyl nitrite, subchronic treatment of mice with cyclohexyl nitrite did not impair macrophage tumoricidal activity or production of reactive nitrogen intermediates, but did modulate B and T cell mitogen responses. The data suggest that cyclohexyl nitrite had cytotoxic activity, comparable to that of isobutyl nitrite, which might be related to the anaemia reported in abusers. The immunomodulatory properties of cyclohexyl nitrite differed from those of isobutyl nitrite.

Effects of taxol on TNF-alpha and IL-6 production by human peripheral blood cells.

Taxol, a new drug isolated from the bark of the Pacific yew tree, is being used in treating patients with breast, ovary, lung, neck, and brain cancer.’.* The therapeutic mechanisms of taxol are not yet completely known. Taxol binds to p-tubulin, stabilizes microtubules against dep~lymerization,~ and interferes with microtubules during the cell divisions, blocking the cell through me tapha~e .~ Probably, microtubules are not the only targets of this drug. On murine macrophages, taxol appears to have an action similar to that of bacterial lipopolysaccharide (LPS). There is some evidence that the drug binds to receptors proximal to those of LPS.’ LPS activates antitumor mechanisms such as biosynthesis of tumor necrosis factor alpha (TNF-cY)~,~ and direct macrophage tumoricidal activity.8 Thus, the antitumor activities of taxol may be in part related to its ability to stimulate macrophages. The objective of this study was to determine if cells from the peripheral blood of healthy donors or tumor patients can be stimulated with taxol to produce TNF-a or IL-6 directly or in the presence of “priming” signals, such as those provided by interferon-gamma (IFN-y).

Tolerance to the anti-metastatic effect of lipopolysaccharide against liver metastasis in mice.

We describe the involvement of endotoxin tolerance in the refractoriness of its anti-metastatic effect against murine syngeneic tumors. Three i.v. administrations of LPS at intervals of 4 days after tumor inoculation inhibited liver metastasis of L5178Y-ML25 cells, whereas 3 consecutive i.v. administrations of LPS showed only a slight suppressive effect. Multiple i.v. administrations of LPS, synthetic lipid A, its synthetic derivative DT-5461, Staphylococcus aureus (S. aureus) BioParticles or Staphylococcal enterotoxin B (SEB) on days 1, 5 and 9 after tumor inoculation inhibited liver metastasis of T-lymphoma cells in normal mice. The anti-metastatic effects of LPS, synthetic lipid A or DT-5461 but not S. aureus BioParticles or SEB were diminished in mice injected with LPS at daily intervals for 7 days before tumor inoculation. Mice receiving 3 consecutive i.v. administrations of LPS at daily intervals exhibited suppression of LPS-induced production of endogenous tumor necrosis factor-alpha (TNF-alpha), tumoricidal activity of macrophages, and natural-killer (NK) activity of splenocytes when compared with those of normal mice. Macrophages from mice receiving consecutive daily i.v. administrations of LPS for 3 days showed reduction of LPS-induced tyrosine phosphorylation of several intracellular proteins, including p42(mapk) /ERK2 when compared with that of the cells obtained from normal mice. These data suggest that the LPS-induced anergic state of monocytes/macrophages plays a crucial role in endotoxin tolerance with respect to the metastasis of T lymphoma in the liver.

Inhibition of nitric oxide: effects on interleukin-1 beta-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat.

The ovulatory process resembles an inflammatory reaction with an infiltration of leukocytes, production of inflammatory mediators such as cytokines, and a general edema and hyperemia. Nitric oxide (NO), a potent vasodilator and the main mediator of macrophage tumoricidal and bacteriocidal activities, is known to participate in inflammatory reactions and has been shown to mediate the interleukin-1 beta (IL-1 beta)-directed tissue-remodeling events within the ovary. The regulation by NO of ovulation rate, leukocyte distribution, and steroid release in the rat ovary was investigated through use of a combination of in vivo and in vitro models of ovulation and a competitive inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), of the NO synthase (NOS) enzyme. Subcutaneous L-NAME (1.5 x 10(-4) mol/kg) administration significantly reduced the in vivo ovulation rate of eCG/hCG-primed rats (L-NAME-treated: 10.6 +/- 1.8 [mean +/- SEM] oocytes per ovary [O/O], 11.0 +/- 1.2 rupture sites per ovary [RS/O]; saline-treated: 18.0 +/- 1.8 O/O, 19.4 +/- 1.1 RS/O; p < 0.01) at 20 h post-hCG. These results were reflected in vitro, where addition of L-NAME (3.5 x 10(-5) mol/L) to LH (0.1 microgram/ml)-perfused ovaries decreased ovulation rate from 8.2 +/- 1.6 to 2.7 +/- 1 ovulations per ovary (p < 0.05) and simultaneously decreased nitrite accumulation at the completion of perfusions from 16.5 +/- 1.9 to 4.1 +/- 0.5 nmol/ml (p < 0.001). The addition of L-NAME to LH+IL-1 beta (4 ng/ml)-perfused ovaries decreased ovulation rate from 15.2 +/- 2.4 to 0.8 +/- 0.8 ovulations per ovary (p < 0.001) and simultaneously decreased nitrite accumulation at 22 h from 22.8 +/- 2.2 to 1.9 +/- 0.6 nmol/ml (p < 0.001). Studies analyzing and manipulating perfusion flow rate indicated that the L-NAME effects on ovulation rate are primarily due to a reduction in flow rate resulting from inhibition of NO, which may be a consequence of the known vasoconstrictor effects of NOS inhibitors. The observed reduction of in vivo ovulation rate by NO inhibition at 20 h post-hCG was associated with a significant reduction in thecal MCA149+ neutrophils at 12 h post-hCG, the expected time of ovulation (L-NAME-treated: 98.4 +/- 9.2 cells per thecal area; saline-treated: 211.5 +/- 11.5 cells per thecal area; p < 0.001), while ED1+ monocytes/macrophages underwent similar but nonsignificant changes. Plasma (20 h post-hCG) and perfusate progesterone were not different with L-NAME treatment, while perfusate estradiol levels were markedly reduced upon addition of L-NAME, suggesting a role for NO in ovulation but not in the process of luteinization. In summary, deprivation of NO by use of the competitive inhibitor, L-NAME, led to fewer ovulations, reduced accumulation of nitrite, a decreased neutrophil count in the theca of preovulatory follicles, and reduced estradiol secretion, while progesterone release remained unaffected. The NO pathway may therefore play an important role in the regulation of ovulation and the mediation of IL-1 beta's pro-ovulatory effects. There are likely to be primarily vascular effects, but also a nonvascular component, to the NO regulation of ovulation, with both components indirectly affecting ovulatory leukocyte distribution and steroid secretion.

The antitumoral effect of the immunomodulator AS101 and paclitaxel (Taxol) in a murine model of lung adenocarcinoma.

The immunomodulator ammonium trichloro(dioxyethylene-0-0')tellurate (AS101) has been shown to possess antitumoral properties in several murine models. In the present study, we demonstrate a synergistic in vivo antitumor effect of AS101 and Taxol against early stage Madison 109 lung adenocarcinoma. Treatment with optimal doses of Taxol (25 and 17 mg/kg) and AS101 (0.5 mg/kg) resulted in 66.6 and 43.3% cures. We propose that the antitumor effect is the result of both a direct and indirect effect of the drugs on tumor cells. AS101 and Taxol directly inhibited clonogenicity of M109 cells in a synergistic dose-dependent manner. Exposure of M109 cells to clinically achievable concentrations of Taxol and AS101 produced a synergistic internucleosomal DNA fragmentation associated with programmed cell death. We suggest that AS101 renders tumor cells more susceptible to chemotherapy in general and to Taxol in particular, partly by increasing the wild-type p53 protein expression that is required for efficient execution of the death program. Moreover, we demonstrate a synergistic effect of AS101 and Taxol in increasing the tumoricidal activity of macrophages. This activity is produced by nitric oxide secretion. The synergistic antitumoral effects of AS101 and Taxol were partly ablated both in vitro and in vivo by inhibition of nitric oxide synthase. These findings indicate that AS101 in combination with Taxol may be a promising antitumor drug, and illustrate the mechanism of action of both drugs when acting synergistically. Phase II clinical trials have been initiated using AS101 in combination with Taxol.

Granulocyte-macrophage colony-stimulating factor inhibits tumor growth during the postoperative period

Granulocyte-macrophage colony-stimulating factor (GM-CSF) may have important antineoplastic properties because it induces macrophage tumoricidal activity in vitro. We examined the inhibitory effect of GM-CSF on tumor growth in a murine carcinoma model and whether this inhibitory effect would persist during the postoperative period. Potential macrophage-mediated mechanisms were studied. The effect of GM-CSF on macrophage function in vitro was assessed by measuring superoxide anion and interleukin-6 production, percentage phagocytosis of Candida albicans, and percentage Ia expression. GM-CSF's effect on tumor volume was assessed first in a murine tumor model and second to examine whether these effects also occurred during the postoperative period in the same model after laparotomy. Macrophage function in the latter study was assessed by measuring superoxide anion, cytotoxicity, and tumor necrosis factor production. GM-CSF treatment was associated with a decrease in tumor volume on day 4 after the initiation of GM-CSF treatment (0.93 +/- 0.08 cm3 for control versus 0.34 +/- 0.08 cm3 for GM-CSF; p < 0.05). This effect was also seen after laparotomy (1.07 +/- 0.2 cm3 for laparotomy+saline versus 0.16 +/- 0.04 cm3 for laparotomy+GM-CSF, p < 0.05). In vivo macrophage function showed increased superoxide anion, cytotoxicity, and tumor necrosis factor-alpha production from macrophages obtained from GM-CSF treated animals compared with saline treated controls. Tumor growth is inhibited by GM-CSF treatment, and this effect also occurs after laparotomy. Thus, GM-CSF may have a therapeutic role in the treatment of the tumor bearing host after operation.

Dietary fish oil modulation of macrophage tumoricidal activity.

Recent studies have shown that macrophages and their functions can be altered by dietary fat. Specifically, diets that are rich in n-3 fatty acids such as fish oils can have significant effects on macrophage cytolytic capacity and the production of select cytokines. The purpose of these studies was to characterize how dietary fish oils altered macrophage tumoricidal activity and the production of tumor necrosis factor-alpha (TNF-alpha). Dietary menhaden fish oil (MFO) significantly decreased the ability of activated macrophages to kill tumor targets compared with macrophages from mice fed safflower oil (SAF), which is high in n-6 fatty acids. Those macrophages from mice fed MFO were hyporesponsive to interferon-gamma. In addition, macrophages from mice fed MFO produced more TNF-alpha after 24 h activation with lipopolysaccharide compared with macrophages from mice fed SAF. That difference in TNF-alpha production was associated with a differential production of and response to prostaglandin E2. Although there are several possible mechanisms by which dietary fat may alter macrophage function and cytokine production, we have investigated signal transduction. Macrophages from MFO-fed mice had a greater increase in intracellular calcium mobilization after treatment with platelet-activating factor (PAF) than macrophages from mice fed SAF. Those differences may be related to an alteration in the PAF signalling pathway by increasing phospholipase C activity. Thus, dietary n-3 fatty acids may significantly alter macrophage tumoricidal activation and TNF-alpha production through the modulation of PGE2 production and signal transduction.

Dietary fish oil modulation of macrophagetumoricidal activity

Recent studies have shown that macrophages and their functions can be altered by dietary fat. Specifically, diets that are rich inn-3 fatty acids such as fish oils can have significant effects on macrophage cytolytic capacity and the production of select cytokines. The purpose of these studies was to characterize how dietary fish oils altered macrophage tumoricidal activity and the production of tumor necrosis factor-cc (1NF-α). Dietary menhaden fish oil (NIFO) significantly decreased the ability of activated macrophages to kill tumor targets compared with macrophages from mice fed safflower oil (SAF), which is high in n-6 fatty acids. Those macrophages from mice fed UFO were hyporesponsive to interferon-γ. In addition, macrophages from mice fed MFO produced more TNF-α after 24 h activation with lipopolysaccharide compared with macrophages from mice fed SAF. That difference in TNF-α production was associated with a differential production of and response to prostaglandin E 2. Although there are several possible mechanisms by which dietary fat may alter macrophage function and cytokine production, we have investigated signal transduction. Macrophages from MFO-fed mice had a greater increase in intracellular calcium mobilization after treatment with platelet-activating factor (PAF) than macrophages from mice fed SAF Those differences may be related to an alteration in the PAF signalling pathway by increasing phospholipase C activity. Thus, dietary n-3 fatty acids may significantly alter macrophage tumoricidal activation and TNF-α production through the modulation of PGE 2 production and signal transduction.

Synergistic cooperation between thapsigargin and phorbol ester for induction of nitric oxide synthesis in murine peritoneal macrophages

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucidate the role of intracellular calcium and protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Thapsigargin (TG), Ca 2+ -ATPase inhibitor of endoplasmic reticulum, had modest activity on NO synthesis by itself, whereas phorbol ester, PKC activator, alone had no effect. When TG was used in combination with phorbol ester, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of phorbol ester was shown in the first 6 h after TG treatment. In addition, the ability of TG with phorbol ester on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca 2+ -ATPase, 2,5-di-( t-butyl)-1,4-benzohydroquinone, and Ca 2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca 2+ transient by TG was not affected in the presence or absence of extracellular Ca 2+, indicating that TG must be effective on cytosolic Ca 2+ pool. In addition, chelation of intracellular Ca 2+ by acetoxymethyl ester of 1,2- bis-(2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid (BAPTA/AM), an intracellular Ca 2+ chelating agent, blocked TG- or TG + PMA-induced NO production. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without affecting TG-induced NO production. In addition, when the cells were pretreated with phorbol ester before TG treatment, there was no synergy between TG and phorbol ester, indicating that PKC is not directly involved in the expression of NOS but involved in “triggering” signal. Secretion of NO corresponded with tumor cell killing, but TG plus phorbol ester-activated macrophages failed to kill tumor cell targets in the presence of N G-monomethyl- l-arginine. Collectively, these data illustrate that mobilization of intracellular Ca 2+ provides a “priming” signal for induction of NOS gene expression by itself and it also requires PKC as a “triggering” signal for macrophage tumoricidal activity.

Differential Inhibition of RAW264.7 Macrophage Tumoricidal Activity by 9Tetrahydrocannabinol

delta 9tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has been shown to inhibit macrophage cell contact-dependent cytolysis of tumor cells. The purpose of this study was to determine whether THC inhibited macrophage cytolytic function by targeting selectively tumor necrosis factor (TNF)-dependent pathways versus L-arginine-dependent reactive nitrogen intermediates. An in vitro system employing RAW264.7 macrophage-like cells as effectors and TNF-sensitive mouse L929 fibroblasts or nitric oxide (NO.)-sensitive P815 mastocytoma cells as targets, was employed to assess the effect of THC on cytolysis. Macrophages were pretreated with THC or vehicle for 48 hr, subjected to multistep activation with 10 U/ml recombinant mouse gamma-interferon (IFN-gamma) plus 100 ng/ml LPS or to direct activation with 1 microgram/ml LPS, and co-cultured with tumor cells in the presence or absence of THC. THC inhibited TNF-dependent killing by macrophages subjected to either multistep or direct activation. Decreased amounts of TNF-alpha were detected in medium of macrophage cultures treated with THC. In contrast, THC inhibited NO.-dependent cell contact killing only for macrophages subjected to direct activation. Decreased levels of NO2-, a stable degradation product of the short-lived and highly toxic effector molecule NO., were produced by these macrophages. In addition, the effect of the enantiomeric pairs (-)CP55,940/(+)CP56,667 or (-)HU-210/(+)HU-211 on macrophage cell contact-dependent killing was assessed. Inhibition of macrophage tumoricidal activity against TNF-sensitive L929 cells was effected by both isomers of THC analogs. In contrast, both of the enantiomeric pairs had an effect on killing of NO.-sensitive P815 mastocytoma cells only for macrophages subjected to direct activation. These data suggest that cannabinoids inhibit macrophage cell contact-dependent killing of tumor cells by a noncannabinoid receptor-mediated mechanism. However, specific cytolytic pathways are inhibited differentially by cannabinoids depending on the activation stimuli to which macrophages are exposed.

Stimulation of macrophages with IFNγ or TNFα shuts off the suppressive effect played by PGE 2

PGE 2 has been shown to be able to interfere with various lymphocyte and macrophage functions, but its effects on macrophage activation are still unclear. In this study, carried out on peritoneal macrophages obtained from healthy, tumour-bearing and Corynebacterium parvum-treated mice, we demonstrated that PGE 2 is involved in the down-regulation of macrophage activation, but it cannot exert its inhibiting effect when macrophages are further stimulated with activating cytokines, such as IFNγ and TNFα. Our findings provide new insight into how macrophage tumoricidal activity may be induced and maintained even in presence of significant levels of PGE 2.

The Induction and Augmentation of Macrophage Tumoricidal Responses by Platelet-Activating Factor

Platelet-activating factor (PAF) has previously been shown to stimulate intracellular signal transductional events similar to the macrophage tumoricidal activators interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Macrophages stimulated with IFN-γ and LPS utilize various cytolytic mediators in order to kill tumor cells. However, PAF has been shown to only induce and enhance tumor necrosis factor-α (TNFα)-mediated cytolysis by macrophages. Therefore, the purpose of this study was to determine whether PAF, in comparison with IFN-γ and LPS, could stimulate macrophages for both TNFγ- and non-TNFγ-mediated tumoricidal activity. For this, the activation of macrophages for a cytolytic response was characterized by the production of TNFγ and nitric oxide (NO-2), as well as, their ability to kill select tumor cells. PAF together with IFN-γ stimulated macrophage secretion of NO-2. In addition, PAF enhanced IFN-γ- and LPS-stimulated NO-2 production. PAF, together with IFN-γ, also activated macrophages for tumoricidal activity against TNFγ-resistant tumor cells. In assays to determine the temporal sequence of activation, increased tumor cell cytolysis was observed only when macrophages were first treated with IFN-γ. Moreover, PAF enhanced macrophage tumoricidal activity when added with LPS and IFN-γ. With respect to TNFγ production, macrophages activated with high concentrations of PAF stimulated significant levels of TNFγ compared to macrophages without PAF. A similar level was observed following multiple additions of a lower concentration of PAF, Also, PAF induced macrophage cytolytic activity against a TNFγ-sensitive tumor cell. In addition, PAF significantly enhanced LPS-induced TNFα production. Thus, PAF can play a modulatory role in the activation for non-TNFγ-mediated tumoricidal activity of macrophages.

Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte‐derived macrophages

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.