Tumoricidal properties of mouse macrophages activated with mediators from rat lymphocytes stimulated with concanavalin A

Macrophage-activating factor (MAF) was obtained from cultures of normal F344 rat lymphocytes incubated with insoluble concanavalin A. The MAF rendered macrophages from normal C57BL/6 mice cytotoxic against the syngeneic B16 melanoma and the allogeneic AC 15091. At the same time, normal syngeneic or allogeneic embryo cells were unharmed, even in the presence of susceptible tumor cells. Optimal MAF levels followed incubation of lymphocytes for 48 hr with Sepharose-bound concanavalin A. A 2-hr incubation of macrophages with MAF was sufficient to initiate activation, providing that 46 hr were allowed to elapse before tumor cells were added. The MAF activity was enhanced after heating the supernatant to 199 degrees. Control experiments largely excluded the possibility that residual unbound concanavalin A caused the observed macrophage-mediated tumoricidal effects.     

Regulation of cytokine-induced neutrophil chemoattractant in rat bone marrow-derived macrophages by inflammatory mediators

During the course of inflammation, macrophages are highly influenced by their local environment and changes in the cytokine milieu. Exposure of macrophages to various factors during different phases of the inflammatory response may have a strong influence on the pattern of gene expression, which a macrophage exhibits. We examined how these mediators affect the regulation of the expression and production of cytokine-induced neutrophil chemoattractant (CINC). Our study demonstrates that CINC can be induced in bone marrow-derived macrophages by lipopolysaccharide, interleukin-1 beta, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma/TNF alpha. These mediators are factors which a macrophage would be expected to encounter early in an inflammatory process. In contrast, transforming growth factor-beta (TGFbeta), which is expressed late in the inflammatory process during mesenchymal cell proliferation and tissue repair, did not induce detectable amounts of CINC and functioned to suppress CINC production stimulated by early inflammatory mediators. Suppression of CINC production occurred whether TGF beta was added simultaneously, 12 or 24 h prior to the stimulus.     

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Cigarette smoking has been shown to increase consequent to the acute administration of methadone. This suggests the possibility that differences in maintenance dose levels might be associated with differential smoking rates. It is of special concern that higher maintenance levels of methadone may lead to more cigarette smoking because of the putative beneficial effects of higher doses on illicit drug use, treatment retention, and the like. Two experiments were conducted to test the hypothesis that higher maintenance doses of methadone are related to more cigarette smoking. Smoking was measured by self-report and expired carbon monoxide, and the amounts were correlated with subjects' methadone dose levels. The results showed smoking rates of 85% and that self-reported smoking significantly correlated (r = -.52) with CO. Maintenance doses, however, were not correlated with smoking levels. This suggests that the acute effects of methadone on smoking are nullified as clients habituate to dose level, and that decisions regarding appropriate methadone dosage can be made on other grounds.     

In vitro alternative pathway activation of complement by the brush border of proximal tubules of normal rat kidney

The purpose of this study was to investigate which structures of the nephron, if any, are capable of directly activating the complement (C) system. To this end, two sets of experiments were performed. First, activation of C was assessed on sections of frozen kidney tissue, using the indirect immunofluorescence technique for the demonstration of C fixation. Second, glomerular or tubular fractions of kidney were incubated with normal fresh serum, and subsequent C consumption was measured. The data obtained support the interpretation that the brush border of proximal tubules activates the alternative pathway of the C system. This phenomenon may have pathogenic significance in conditions of aselective proteinuria.     

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.     

Production of inflammatory mediators by human macrophages obtained from ascites

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.     

In vitro inhibition of carnitine acyltransferase activity in mitochondria from rat and mouse liver by a diethylhexylphthalate metabolite

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.     

In vitro lymphocyte reactivity to rubella antigen following vaccination

In vitro lymphocyte stimulation and several serological tests were used to study the development of immunity after subcutaneous immunization with attenuated rubella virus. Maximal lymphocyte reactivity was obtained 3--4 months after vaccination and a decline after 1.5--2.5 years. Lymphocyte reactivity following natural immunity was of a higher magnitude. No correlation between the degree of lymphocyte stimulation and titers of neutralizing, hemagglutination inhibiting and hemolysis-in-gel antibodies was observed. The influence on the results of some methodological factors and different rubella antigens is discussed.     

In vitro effect of parachlorophenol and camphorated parachlorophenol on macrophages

The purpose of this study was to investigate the "in vitro" effect of parachlorophenol and camphorated parachlorophenol, used in endodontics for the disinfection of root canals, on the substrate adherence capacity of macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. As a test of macrophage phagocytic function, the adherence capacity of macrophages to a plastic surface was determined. Assays were conducted in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that parachlorophenol and camphorated parachlorophenol significantly decreased the substrate adherence capacity of inflammatory macrophages. Taking into account that adhesion is the first step in the phagocytic process of macrophages and in antigen presentation, parachlorophenol and camphorated parachlorophenol could inhibit macrophage function and modulate immune and inflammatory reactions in periapical tissues.     

In vitro induction of aryl hydrocarbon hydroxylase in human pulmonary alveolar macrophages by benzanthracene

Aryl hydrocarbon hydroxylase (AHH) was induced in human pulmonary alveolar macrophages (PAMs) cultured in the presence of benzanthracene. Time and dose--response curves were established for in vitro induction of this enzyme system in PAMs. In addition, a positive correlation was noted between the level of AHH activity in freshly lavaged PAMs and the in vitro inducibility of the enyzme in these cells from either nonsmokers or cigarette smokers. Measurements of the inducibility of AHH in cultured human PAMs provide an experimental system suitable for studying the mechanisms responsible for the initiation of pulmonary carcinogenesis.     

In vitro glucocorticoid receptor binding and transcriptional activation by topically active glucocorticoids

BACKGROUND: To study body composition at the whole-body level in patients with Crohn's disease and a history of intestinal resection compared with healthy controls, we performed a cross-sectional study using dual-energy X-ray absorptiometry (DXA). METHODS: Thirty-one patients, 13 men and 18 women, were included. They had a history of Crohn's disease for a mean period of 20 years (range, 4-45 years). All patients had undergone intestinal resections. The colon had been resected in 24 patients, and the mean length of the resected small intestine was 97 cm (range, 0-305 cm). At the time of investigation the Crohn's disease had been in remission for at least 24 months. Patients presented with significantly increased faecal volume and faecal fat excretion. A group of 69 women and 19 men were investigated with DXA and used as reference group. The fat-free mass (FFM), fat mass (FM), percentage fat mass (FM%), and total body mineral content (TBMC) were measured by DXA, and the results were expressed as a z-score. RESULTS: The mean z-score of the body mass index (BMI) was significantly reduced to -0.35 (P=0.036). The FFM was significantly reduced with a mean z-score of -1.74 (P=0.0001). The FM was unchanged (z-score, 0.12; P=0.42). However, FM expressed as percentage of body weight was significantly increased, with a z-score of 0.88 (P=0.001). The TBMC was significantly decreased, with a mean z-score of -1.42 (P=0.0001). There was positive direct correlation between the BMI and TBMC z-scores. There was no correlation between malabsorption and body composition variables. CONCLUSION: Patients with clinically quiescent Crohn's disease showed significant changes in body composition, with low BMI, significant loss of FFM, and unchanged FM. However, when expressed as percentage of body weight, FM was significantly increased. The TBMC was significantly reduced.     

In vitro exposure of murine macrophages to ultraviolet light induces apoptosis

Ultraviolet light is a known exogenous stimuli with the ability to activate cell death by apoptosis. This study was done to examine the biologic effects of different energy levels of short wavelength UV light on cultured mouse macrophages. Cell proliferation, DNA content, and cellular ultrastructural architecture analysis demonstrated that ultraviolet light induces apoptosis in murine macrophages in culture. Exposure to 0.12J/cm2 evokes progressive cell demise with the classical features associated with apoptosis, whereas exposure to 5.0 J/cm2 results in extensive DNA degradation and crosslinking of cellular proteins. These two phenotypes are qualitatively described.     

In vitro oxidation of pyrazinamide and allopurinol by rat liver aldehyde oxidase

Aldehyde oxidase was purified about 120-fold from rat liver cytosol by sequential column chromatography using diethylaminoethyl (DEAE) cellulose, Benzamidine-Sepharose 6B and gel filtration. The purified enzyme was shown as a single band with M(r) of 2.7 x 10(5) on polyacrylamide gel electrophoresis (PAGE) and M(r) of 1.35 x 10(5) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified enzyme, in vitro conversion of allopurinol, pyrazinamide and pyrazinoic acid was investigated. Allopurinol and pyrazinamide were oxidized to oxypurinol and 5-hydroxy-pyrazinamide, respectively, while pyrazinoic acid, the microsomal deamidation product of pyrazinamide, was not oxidized to 5-hydroxypyrazinoic acid. The apparent Km value of the enzyme for pyrazinamide was 160 microM and that for allopurinol was 1.1 mM. On PAGE, allopurinol- or pyrazinamide-stained band was coincident with Coomassie Brilliant Blue R 250-stained band, respectively. These results suggest that aldehyde oxidase may play a role in the oxidation of allopurinol to oxypurinol and that of pyrazinamide to 5-hydroxypyrazinamide with xanthine dehydrogenase which can oxidize both allopurinol and pyrazinamide in vivo. The aldehyde oxidase may also play a major role in the oxidation of allopurinol and pyrazinamide in the subgroup of xanthinuria patients (xanthine oxidase deficiency) who can oxidize both allopurinol and pyrazinamide.     

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.     

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.     

In vitro suppression of the normal mitogenic T lymphocyte response by steady state sickle cell disease sera

This study is part of a long term evaluation of sickle cell disease (SCD) as a paradigm for immunosuppression. Serum was obtained from 43 SCD patients during the steady (healthy) state. Peripheral blood mononuclear cells (PBMC), separated by density gradient were obtained from 8 normal healthy donors. PBMC were utilized in assays directly or as a source for obtaining, total T (CD3) and helper T (CD4) cell populations separated by specific T cell columns. Standard in vitro phytohemagglutinin (PHA) stimulation of lymphocyte cultures was done with culture media containing 10% SCD serum, as compared to normal pooled O, Rh+ (O+) serum. Mitogenic responses were expressed as mean counts per minute (cpm) and stimulation index of triplicate cultures. Results revealed PHA responses were positive in all experiments when a standard stimulation index of 10 or greater was used as a test parameter for comparison. Positive results were demonstrated in 43/43 (100%) of triplicate cultures regardless of serum type in all experiments. Conversely, by using mean cpm as the test criterion, suppression of PHA response was shown in SCD serum supplemented cells as follow; 36/43 (84%) of PBMC, 35/43 (81%) of CD3 and 37/43 (86%) of CD4 cultures. The degree of suppression ranged from > 10% to 98% in individual experiments, as compared to O+ serum. Inhibitors of normal T lymphocyte in vitro PHA response appear to be present in a significant percentage of SCD sera even during the healthy state of disease. Type 2 cytokines which suppress cell mediated immunity would seem to be the most likely inhibitory agents.     

The in vitro effect of hydrocortisone on endocytosis in cultured mouse peritoneal macrophages

Three juvenile patients with cerebellar astrocytomas which have seeded the spinal subarachnoid space are presented. Histologic verification of the similarity between the posterior fossa tumor and its spinal implant was obtained in two of the three patients. The cerebellar tumors in all cases have been benign (grade I),and the behavior, other than their seeding has also been indolent. Review of pertinent literature discloses no similar experience with cerebellar astrocytomas. Aggressive therapy is advocated for the rare patient with subarachnoid seeding from this benign lesion.