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 TNFa 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 µM), TNF (512 U/ml) and NO (71.5 ± 3.2 µM). TNF (256 U/ml) and NO (78.9 ± 9.7 µM) 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 µg/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.

Butyrate induces apoptosis in murine macrophages via caspase-3, but independent of autocrine synthesis of tumor necrosis factor and nitric oxide

We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 µM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 µg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0%), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2%), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0%), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Effect of low extracellular pH on NF-κB activation in macrophages

Objective: Many diseases, including atherosclerosis, involve chronic inflammation. The master transcription factor for inflammation is NF-κB. Inflammatory sites have a low extracellular pH. Our objective was to demonstrate the effect of pH on NF-κB activation and cytokine secretion. Methods: Mouse J774 macrophages or human THP-1 or monocyte-derived macrophages were incubated at pH 7.0–7.4 and inflammatory cytokine secretion and NF-κB activity were measured. Results: A pH of 7.0 greatly decreased pro-inflammatory cytokine secretion (TNF or IL-6) by J774 macrophages, but not THP-1 or human monocyte-derived macrophages. Upon stimulation of mouse macrophages, the levels of IκBα, which inhibits NF-κB, fell but low pH prevented its later increase, which normally restores the baseline activity of NF-κB, even though the levels of mRNA for IκBα were increased. pH 7.0 greatly increased and prolonged NF-κB binding to its consensus promoter sequence, especially the anti-inflammatory p50:p50 homodimers. Human p50 was overexpressed using adenovirus in THP-1 macrophages and monocyte-derived macrophages to see if it would confer pH sensitivity to NF-κB activity in human cells. Overexpression of p50 increased p50:p50 DNA-binding and in THP-1 macrophages inhibited considerably TNF and IL-6 secretion, but there was still no effect of pH on p50:p50 DNA binding or cytokine secretion. Conclusion: A modest decrease in pH can sometimes have marked effects on NF-κB activation and cytokine secretion and might be one reason to explain why mice normally develop less atherosclerosis than do humans.

Endodontic Chelators Induce Nitric Oxide Expression by Murine-cultured Macrophages

Introduction: Endodontic chelators may extrude to apical tissues during instrumentation activating cellular events on periapical tissues. This study assessed in vitro the expression of nitric oxide (NO) concentrations by murine peritoneal macrophages after contact with MTAD (Dentsply/Tulsa, Tulsa, OK), Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy), Smear Clear (Sybron Endo, Orange, CA), and EDTA (Biodinamica, Ibipora, PR, Brazil). Methods: Macrophage cells were obtained from Swiss mice after peritoneal lavage. Chelators were diluted in distilled water obtaining 12 concentrations, and MTT assay identified the concentrations, per group, displaying the highest cell viability (analysis of variance, p < 0.01). Selected concentrations were tested for NO expression using Griess reaction. Culture medium and lipopolysaccharide (LPS) were used as controls. Results: Analysis of variance and Tukey tests showed that all chelators displayed elevated NO concentrations compared with the negative control (p < 0.01). MTAD induced the lowest NO expression, followed by Tetraclean, EDTA, and Smear Clear. No difference was observed between MTAD and Tetraclean (p > 0.01), Tetraclean and EDTA (p > 0.01), and EDTA and Smear Clear (p > 0.01). LPS ranked similar to both EDTA and Smear Clear (p > 0.01). Conclusion: The tested endodontic chelators displayed severe proinflammatory effects on murine-cultured macrophages. Citric acid-based solutions induce lower No release than EDTA-based irrigants. (J Endod 2009;35:824-828)

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Effect of exercise on glutamine metabolism in macrophages of trained rats

This study investigated the effect of exercise on glutamine metabolism in macrophages of trained rats. Rats were divided into three groups: sedentary (SED); moderately trained (MOD) rats that were swim trained 1 h/day, 5 days/week for 6 weeks; and exhaustively trained (EXT) rats that were similarly trained as MOD for 5 weeks and, in the 6th week, trained in three 1-h sessions/day with 150 min of rest between sessions. The animals swam with a load equivalent to 5.5% of their body weight and were killed 1 h after the last exercise session. Cells were collected, and glutamine metabolism in macrophage and function were assayed. Exercise increased phagocytosis in MOD when compared to SED (34.48 +/- 1.79 vs 15.21 +/- 2.91%, P < 0.05); however, H(2)O(2) production was higher in MOD (75.40 +/- 3.48 nmol h x 10(5) cell(-1)) and EXT (79.20 +/- 1.18 nmol h x 10(5) cell(-1)) in relation to SED (32.60 +/- 2.51 nmol h x 10(5) cell(-1), P < 0.05). Glutamine consumption increased in MOD and EXT (26.53 +/- 3.62 and 19.82 +/- 2.62 nmol h x 10(5) cell(-1), respectively) relative to SED (6.72 +/- 0.57 nmol h x 10(5) cell(-1), P < 0.05). Aspartate increased in EXT (9.72 +/- 1.14 nmol h x 10(5) cell(-1)) as compared to SED (1.10 +/- 0.19 nmol h x 10(5) cell(-1), P < 0.05). Glutamine decarboxylation was increased in MOD (12.10 +/- 0.27 nmol h x 10(5) cell(-1)) and EXT (16.40 +/-\ 2.17 nmol h x 10(5) cell(-1)) relative to SED (1.10 +/- 0.06 nmol h x 10(5) cell(-1), P < 0.05). This study suggests an increase in macrophage function post-exercise, which was supported by enhanced glutamine consumption and metabolism, and highlights the importance for glutamine after exercise.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.

Biodritin microencapsulated human islets of langerhans and their potential for type 1 diabetes mellitus therapy

Background. Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. Methods. Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. Results. The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1 beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. Conclusion. This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Effect of the essential oil of Achillea millefolium L. in the production of hydrogen peroxide and tumor necrosis factor-alpha in murine macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endodontic Chelators Induce Nitric Oxide Expression by Murine-cultured Macrophages

Introduction: Endodontic chelators may extrude to apical tissues during instrumentation activating cellular events on periapical tissues. This study assessed in vitro the expression of nitric oxide (NO) concentrations by murine peritoneal macrophages after contact with MTAD (Dentsply/Tulsa, Tulsa, OK), Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy), Smear Clear (Sybron Endo, Orange, CA), and EDTA (Biodinamica, Ibipora, PR, Brazil). Methods: Macrophage cells were obtained from Swiss mice after peritoneal lavage. Chelators were diluted in distilled water obtaining 12 concentrations, and MTT assay identified the concentrations, per group, displaying the highest cell viability (analysis of variance, p < 0.01). Selected concentrations were tested for NO expression using Griess reaction. Culture medium and lipopolysaccharide (LPS) were used as controls. Results: Analysis of variance and Tukey tests showed that all chelators displayed elevated NO concentrations compared with the negative control (p < 0.01). MTAD induced the lowest NO expression, followed by Tetraclean, EDTA, and Smear Clear. No difference was observed between MTAD and Tetraclean (p > 0.01), Tetraclean and EDTA (p > 0.01), and EDTA and Smear Clear (p > 0.01). LPS ranked similar to both EDTA and Smear Clear (p > 0.01). Conclusion: The tested endodontic chelators displayed severe proinflammatory effects on murine-cultured macrophages. Citric acid-based solutions induce lower No release than EDTA-based irrigants. (J Endod 2009;35:824-828)