In vivo and in vitro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

Influence of natural antioxidants on in vitro lipoprotein oxidation

Susceptibility of low-density lipoproteins (LDL) to oxidation might be a critical risk factor in the development and progression of atherosclerosis. The oxidation involves the degradation of polyunsaturated fatty acids, the formation of lysolecithin, oxysterols and aldehyde modification of lysine residues on Apo B100. The oxidation products have a number of biological activities such as cytotoxicity, atherogenesis, and carcinogenesis. The aim of this study was to investigate the in vitro antioxidant effects of vitamins E, A, and C on LDL. LDL was isolated from plasma by density gradient high-speed centrifugation and used as 0.1 microM/l isotonic solution. LDL oxidation was triggered by simple incubation with Cu2+ (1, 2, 5, 10, 12, 20 microM/l), in absence or presence of lipid-soluble or water-soluble antioxidants in different concentrations (tocopherols--0.5, 1, 2, and 4 microM/l; cerotenoids--0.1, 0.2, and 0.4 microM/l; ascorbate--2.5, 5, and 10 microM/l). The LDL oxidability was measured by continuous spectrophotometrical monitoring at 234 nm of the increased formation of conjugated diene hydroperoxides. The oxidation curves showed a profile with an inhibition period followed by a propagation period and were quantitatively characterized by two parameters: lag-phase (expressed in minutes), and propagation rate (expressed in changes of absorbance--delta E234nm/min). Lag-phase--the period of inhibition oxidation--was attributed to the ability of LDL (by antioxidants) to resist oxidation in vitro. LDL lag-phase decreased and propagation rate increased with the increasing of copper concentration. In conclusion: 1) susceptibility of LDL to oxidation depends on both the concentration of pro-oxidant stimuli and the entity and concentrations of antioxidants; 2) antioxidants retard the process through which LDL undergo oxidation in vitro when exposed to copper ions; 3) a synergistic effect may also be involved, as water-soluble vitamin C keeps the fat-soluble vitamin E and vitamin A within LDL.     

Deactivation of macrophage oxidative burst in vitro by different strains of Histoplasma capsulatum

It was previously reported that Histoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi, Candida albicans and Cryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

Altered alveolar macrophage function in calorie-restricted rats

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.     

Influence of antioxidants on the peroxidative swelling of mitochondria in vitro

Short courses (6 months) of interferon (IFN) are highly cost-effective in patients with histologically mild chronic hepatitis C. However, IFN treatment lasting 12-24 months is now the standard of care. We utilized a previously published and validated model of the natural history of chronic hepatitis C and the treatment outcomes from two large multicentre treatment trials of IFN-alpha 2b, given for 6 months or 18-24 months, to estimate the incremental cost-effectiveness of prolonged IFN treatment in patients with histologically mild chronic hepatitis C (formerly chronic persistent or mild chronic active hepatitis). In the two treatment trials, pooled analysis of the patients with mild hepatitis showed that 36.4% of them normalized serum alanine aminotransferase and remained virus negative (sustained response) after completing an 18-24 month course of IFN vs 15.3% for a 6-month course and no response in the absence of treatment. The model then estimated that for patients aged 20-50 years, the discounted marginal cost per year of life gained by long-term IFN treatment ranged from US $735 to US $8856, and the gain in life expectancy ranged from 4.35 years to 0.75 years, respectively, compared with an untreated age-matched cohort. Compared with treatment for only 6 months, the incremental marginal cost per life year gained by longer treatment at age 20-50 years ranged from $938 to $9957. The treatment and healthcare costs, sustained response rates and the rate of progression during early disease were identified as significant variables in sensitivity analyses. However, longer treatment always showed a survival benefit compared with 6 months of IFN or no treatment, and the cost of longer treatment is reasonable compared with that for a 6-month course.     

Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production

IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.     

Effects of oxysterols upon macrophage and lymphocyte functions in vitro

Oxygenated derivatives of cholesterol (oxysterols), found in high concentrations in atherosclerotic lesions, are potent immunosuppressive agents inhibiting T-cell responses to different stimuli. The action of oxysterols on macrophage functions and macrophage-lymphocyte interaction has been poorly investigated. In this work, the effects of 25-hydroxycholesterol (25-OHCh) and 7-ketocholesterol (7-KCh) upon some functions of murine peritoneal macrophage (PM), such as generation of reactive oxygen intermediates (ROI), secretion of neopterin and interleukin-1 (IL-1)-like activity, Fc-receptor (FcR) activity, and murine and human lymphocyte functions, participating in lymphocyte-macrophage interactions, such as macrophage-activating factor (MAF) and Ia-inducing factor (IaIF) secretion, were studied in vitro. 7-KCh in concentration of 5 micrograms/mL culture medium only, but not 25-OHCh, significantly inhibited ROI generation by zymosan-stimulated PM. Pretreatment of PM for 22 h with 25-OHCh and 7-KCh led to the decrease of IL-1-like activity secretion. 25-OHCh and 7-KCh inhibited both FcR-dependent binding and phagocytosis of sheep red blood cells (SRBC). Oxysterols did not change both spontaneous and lipopolysaccharide-stimulated secretion of neopterin by PM. 25-OHCh dose-dependently and more efficiently than 7-KCh inhibited murine splenocyte secretion of MAF, which activity was determined by the ability of splenocyte-conditioned medium to stimulate ROI generation in PM. Both 25-OHCh and 7-KCh inhibited significantly proliferative activity of human mixed lymphocyte culture (MLC), as well as lymphocyte secretion of IaIF, which stimulates the expression of HLA antigens in cultured human monocytes. Purified Ch did not alter these parameters. These data showed, that some inflammatory functions of macrophages and lymphocytes may be modified by such environmental conditions as the presence of oxysterols.     

Shosaikoto (kampo medicine) protects macrophage function from suppression by hypercholesterolemia

The feeding of cholesterol-enriched diet for 2 weeks was enough to reduce nitric oxide (NO), prostaglandin E(2) (PGE(2) and interleukin-1 (IL-1) productions in thioglycollate-elicited murine macrophages. Although not showing anti-hypercholesterolemic action against ICR mice, Shosaikoto, a Kampo medicine, partially prevented the reduction of NO and IL-1 productions induced by the feeding of cholesterol-enriched diet, and completely released the reduction of PGE(2) production. These data suggest that the malfunction of macrophage induced by hypercholesterolemia may contribute to early atherogenesis and that Shosaikoto retains macrophage function to prevent the development of atherosclerosis, even though serum cholesterol is markedly increased.     

In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.     

Effects of chicken anemia virus on macrophage function in chickens

One-day-old chicks were inoculated intramuscularly with chicken anemia virus (CAV). Four birds were killed at 7, 14, 21, 28, and 42 days postinoculation (PI), and macrophages were recovered from spleen and bone-marrow suspensions. These were tested for interleukin-1 (IL-1) production, Fc receptor expression, phagocytosis, and bactericidal activity. Macrophages recovered from uninoculated chickens at the same sample times, and exposed to CAV in vitro, were also tested. IL-1 production, Fc receptor expression, phagocytosis, and bactericidal activity were significantly lower in all macrophage cultures from CAV-inoculated chickens, and in cultures exposed to the virus in vitro, than in uninfected controls.     

In vitro modulatory effects of interleukin-3 on macrophage activation induced by interleukin-2

BACKGROUND: The concomitant activation of macrophage-mediated immunosuppressive events might represent one of the most important biologic factors responsible for the decreased efficacy of cancer immunotherapies, including that of interleukin (IL)-2. In previous studies, the authors observed that the increase in the soluble IL-2 receptor (SIL-2R) and neopterin levels was related to the generation of macrophage-mediated immunosuppression and associated with a reduced clinical efficacy during IL-2 cancer immunotherapy. Because both cytokines and neurohormones may influence the macrophage system, the current study was done to evaluate the effects of IL-3 and of the pineal hormone melatonin (MLT) on monocyte response to IL-2 administration. METHODS: Peripheral blood monocytes were incubated with different concentrations of IL-2, IL-3, and MLT, either alone or in association. RESULTS: SIL-2R, neopterin, and tumor necrosis factor-alpha mean concentrations in the medium significantly increased during incubation with IL-2 at a concentration of 100 Cetus units/ml. IL-3 alone, at a dose of 10 ng/ml, also stimulated tumor necrosis factor release; no effect was found on SIL-2R and neopterin. The IL-2-induced neopterin rise was blocked by a concomitant incubation with IL-3 at a dose of 10 ng/ml. Finally, the concomitant incubation with IL-3 and MLT further inhibited neopterin release and significantly decreased IL-2-induced SIL-2R secretion. CONCLUSIONS: These results suggest that IL-3 alone or in association with MLT may modulate macrophage functions during cancer immunotherapy with IL-2 and decrease the IL-2-induced macrophage activation.     

Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis

Genetic and biochemical analysis of phosphofructokinase in the yeast Saccharomyces cerevisiae led to contradictory hypotheses about the function of the subunits of this heterooctameric enzyme. To gain further insight, we exchanged four evolutionary conserved amino acid residues in each of the two yeast subunits affecting presumed catalytic and regulatory functions. In conjunction with a complementary wild-type subunit, each of the mutant subunits led to a loss of a maximum of 50% of phosphofructokinase activity as compared to wild-type cells. Km values for fructose 6-phosphate were increased in most of these mutants. None of the mutant subunits lacking catalytical functions was able to complement the glucose-negative phenotype of a yeast pfk1 pfk2 double mutant when expressed from a single-copy vector. For the beta-subunits, the other mutants did complement, whereas for the alpha-subunits they did not. Concentrations of fructose 1,6-bisphosphate did not drastically change in metabolite determinations in strains carrying one mutant allele, suggesting that the effect of the mutations introduced can be largely compensated by in vivo regulatory mechanisms, as long as one functional subunit is present. The data implicate that each of the yeast phosphofructokinase subunits can serve catalytically as well as regulatory functions.     

Eosinophil chemotaxis induced by several biologically active substances and the effects of apafant on it in vitro

Chemotaxis of guinea pig eosinophils induced by various stimuli in use of a modified Boyden chamber technique in vitro and the effect of a platelet-activating factor (PAF) antagonist, apafant (CAS 105219-56-5, WEB 2086 BS), on it were examined. The eosinophils were obtained by bronchoalveolar lavage from the animals treated by i.v. injection with Sephadex G-200 and purified by Percoll density gradient centrifugation. PAF significantly and potently induced the chemotaxis at a broad range of 10(-17) to 10(-7) mol/l, where no concentration-dependency was observed. Leukotriene B4 also induced the chemotaxis in a concentration-dependent manner at 10(-14) to 10(-12) mol/l and the enhanced migration was not declined until 10(-7) mol/l. Interleukin-5 (IL-5), IL-8 and regulated on activation normal T expressed and secreted (RANTES) only modestly enhanced the chemotaxis in some concentrations at 10(-13) to 10(-7) mol/l with or without significance and with no concentration-dependency while formyl-methionyl-leucyl-phenylalanine (FMLP), a known chemoattractant, increased the migration at 10(-7) to 10(-5) mol/l. Apafant at 10(-8) to 10(-6) mol/l strongly and concentration-dependently inhibited 10(-8) mol/l PAF-induced chemotaxis. However, the drug showed nominal or no influences on their chemotaxis stimulated by the other agonists, at the concentrations of which the enhanced migration was observed. From these results, it is concluded that IL-5, IL-8 and RANTES, different from PAF and LTB4, are not potent stimuli for the eosinophil chemotaxis and that apafant is a selective antagonist of PAF, which is expected to be therapeutically effective for PAF-associated diseases including bronchial asthma.     

In vitro activities of several diaminomethylpyridopyrimidines against Mycobacterium avium complex

Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 microgram/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 microgram/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.     

The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function

We hypothesized that a previously observed exercise-induced suppression of alveolar macrophage antiviral resistance results from increases in corticosterone and/or epinephrine. Mice (CD-1) were run to fatigue on a treadmill (exercise), or placed in Plexiglas lanes above the treadmill (control). The role of corticosterone was assessed by further dividing mice into groups receiving one of the following treatments; sham surgery, adrenalectomy, or adrenalectomy plus corticosterone replacement. Macrophage antiviral function was suppressed in the exercised mice compared to the control mice. However, macrophage antiviral function was not suppressed in the exercised mice that underwent adrenalectomy or adrenalectomy plus corticosterone replacement. We tested whether another adrenal factor (epinephrine) may be involved by dividing mice into exercise and control groups treated with either saline or propranolol. Macrophage antiviral function was again suppressed in the saline-treated exercised mice compared to saline-treated control mice, but no differences were found between the exercised mice receiving propranolol, control mice receiving propranolol, or saline-treated control mice. Isoproterenol, when added to alveolar macrophages in culture, also suppressed antiviral resistance. These findings suggest that decreased macrophage antiviral function following exercise may be due to increased release of adrenal catecholamines.     

The beneficial effect of natural antioxidants on the endothelial function of regenerated endothelium

HYPOTHESIS: Lipid peroxidation and LDL oxidation have been implicated in intimal hyperplasia and endothelial dysfunction following direct arterial trauma. OBJECTIVES: The aim of this study was to evaluate the effects of megadose of natural antioxidant vitamin E (VE) combined to vitamin C (VC) on the regenerated endothelial dysfunction. METHOD: A first group of rats (VE and C) (n = 10) was pretreated with VE (500 IU/kg/day) and VC (1200 mg/kg/day) for 4 weeks before aortic (thoracic) endothelial denudation with a Fogarty catheter. Rats were then fed with the same vitamin supplemented diet for 2 months. A second group (n = 10) was similarly denuded and treated with soya oil (SO), VE vehicle, for the same period; a third group (n = 10) was denuded only (DN); and a fourth group was maintained on a regular diet (CL) without denudation. Endothelial-dependent and independent relaxation was assessed in organ chambers. RESULTS: Vascular relaxation to nitric oxide analogue sodium nitroprusside was not affected either by the regenerative process or the vitamin supplementation. However, endothelial-dependent relaxation to acetylcholine, was significantly preserved in VE and C (p < 0.01) treated animals compared to DN group. CONCLUSION: These results suggest that, in this model, dietary megadose of antioxidants vitamin initiated 4 weeks before denudation can improve the post-regenerative endothelial dysfunction and could contribute to the prevention of the atherosclerotic process.     

Marijuana and cocaine impair alveolar macrophage function and cytokine production

Use of marijuana and cocaine is on the rise in the United States. Although pulmonary toxicity from these drugs has occasionally been reported, little is known about their effects on the lung microenvironment. We evaluated the function of alveolar macrophages (AMs) recovered from the lungs of nonsmokers and habitual smokers of either tobacco, marijuana, or crack cocaine. AMs recovered from marijuana smokers were deficient in their ability to phagocytose Staphylococcus aureus (p < 0.01). AMs from marijuana smokers and from cocaine users were also severely limited in their ability to kill both bacteria and tumor cells (p < 0.01). Studies using NG-monomethyl-L-arginine monoacetate, an inhibitor of nitric oxide synthase, suggest that AMs from nonsmokers and tobacco smokers were able to use nitric oxide as an antibacterial effector molecule, while AMs from smokers of marijuana and cocaine were not. Finally, AMs from marijuana smokers, but not from smokers of tobacco or cocaine, produced less than normal amounts of tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-6 when stimulated in culture with lipopolysaccharide. In contrast, the production of transforming growth factor-beta, an immunosuppressive cytokine, was similar in all groups. These findings indicate that habitual exposure of the lung to either marijuana or cocaine impairs the function and/or cytokine production of AMs. The ultimate outcome of these effects may be an enhanced susceptibility to infectious disease, cancer, and AIDS.