The production and characterisation of products of the lipoxygenase enzyme system released by rat peritoneal macrophages

Rat peritoneal monocytes and macrophages when exposed to the ionophore A23187 release products of the lipoxygenase pathway of arachidonic acid metabolism which cause the aggregation and chemokinesis of polymorphonuclear leucocytes suspensions. The major biologically active compound released was leukotriene B which accounted for greater than 80% of the activity. The remaining biological activity was due to the release of a more polar as yet unidentified compound. In addition rat macrophages release 5, 12 and 15-HETE but these mono-HETEs do not significantly contribute to the biological activity.     

Scavenger and antioxidant properties of ten synthetic flavones.

To study the effect of the hydroxyl groups on biological activities of flavones, we synthesized 10 polyhydroxyflavones with varied substitution patterns. The abilities of the 10 compounds to act as radical scavengers were investigated using chemiluminescence in two biological models: the xanthine/xanthine oxidase system and the oxidative burst of rat alveolar macrophages. Stable radical formation was observed by electron spin resonance (ESR) spectroscopy. We found that the presence of the pyrogallol moiety in the B component of flavones gave rise to radical scavenger activity and that C-6 substituted hydroxyl group may also provide the basis for biological activity. Furthermore, compounds with a hydroxyl at C-7 position appeared to be xanthine oxidase inhibitors. One particular compound exhibited radical scavenger activity and xanthine oxidase inhibition. This type of compound should prove to be useful in the treatment of ischemia, for which both properties were required.     

Peptide fragments from the tuftsin containing domain of immunoglobulin G synthesis and biological activity

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [3H-Arg4]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cell's capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsin's receptors.     

Enhancement by double-stranded polyribonucleotides of production by cultured mouse peritoneal macrophages of differentiation-stimulating factor(s) for mouse myeloid leukaemic cells.

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.     

The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.     

In vitro study of gas effects on alveolar macrophages

Summary To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems ave been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO₂ on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.Abbreviations AM alveolar macrophages - CL Chemiluminescence     

Dexamethasone inhibits antitumor potential of activated macrophages by a receptor mediated action

The capacity to inhibit P815 mastocytoma growth was induced in macrophages elicited by trehalose dimycolate by a short in vitro treatment with 10 ng/ml LPS. Activation by LPS was associated with a 3 fold increase in the rate of glucose consumption by macrophages. Incubation of activated macrophages with the glucocorticoid dexamethasone (greater than or equal to 10(-8) M) for several hours (greater than or equal to 5 h) resulted in an inhibition of antitumoral activity and a decrease of glucose consumption. Hydrogen peroxide production is a property expressed by trehalose dimycolate-elicited macrophages independently of the presence of LPS. The capacity to release hydrogen peroxide upon triggering was not affected by a pretreatment of macrophages by dexamethasone. The antiglucocorticoid compound RU 38486, known to bind with a high affinity to glucocorticoid receptors without agonist effect, prevented the inhibitory actions of dexamethasone, indicating that these are receptor-mediated.     

Synthesis of guanidino analogues of PMPDAP and their immunobiological activity

A series of novel 9-, 7- and 3-substituted 2- or 6-guanidinopurines as analogues of potent antiviral and immunobiologically active compound enantiomers of PMPDAP was synthesized and evaluated for their biological activity. Compounds containing the combination of guanidino and amino group at the purine moiety enhanced the interferon-gamma-triggered NO production in murine macrophages and stimulated the secretion of cytokines and chemokines in both murine macrophages and human peripheral blood mononuclear cells. The most active compounds are 27 and 54. None of the compounds tested exhibited any significant cytostatic effect or antiviral effect.     

Macrophage tumor necrosis factor-alpha release is induced by contact with some tumors

The purpose of this study was to determine whether macrophages were directly stimulated by tumor cells to release TNF-alpha. We found that several murine and human tumor cell lines and crude cell membrane vesicles prepared from these tumor cells stimulated pyran copolymer-elicited murine peritoneal macrophages (PEM) to release as much as 362 +/- 69 (mean +/- SE) units of TNF activity per 10(6) PEM in vitro. By contrast, several nontransformed cells, including Con A-stimulated splenic leukocytes and CTLL cloned T lymphocytes, failed to stimulate PEM to release TNF. Antibody and complement-mediated depletion of macrophages abrogated the release of TNF; whereas depletion of NK cells and T lymphocytes did not affect tumor-stimulated TNF release, suggesting that tumor cells directly stimulated PEM to release TNF. Tumor-stimulated TNF release was rapid, peaking in 2 to 3 h with subsequent loss of TNF activity from the medium. In the absence of tumor, PEM contained detectable levels of TNF mRNA, but did not release functionally active TNF. The addition of P815 tumor cell membrane vesicles increased both TNF mRNA levels, peaking at 1 to 2 h, and release of high levels of TNF activity. Confounding effects of endotoxin were excluded by the resistance of tumor-stimulated TNF release to neutralization by polymixin B, and by the equivalent responsiveness of PEM from endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive (C3H/HeN) mice to stimulation by tumor cells. Factors which stimulated PEM to release TNF could be extracted from tumor cell membrane, with 77% of the macrophage-stimulating activity recoverable in aqueous phase. In conclusion, we have demonstrated that some tumor cell lines express specific characteristics which can be recognized by macrophages and which stimulate macrophages to release TNF.     

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells.     

Augmentation and Suppression of TNF Release from Macrophages by Inflammatory Polymorphonuclear Leukocytes

It is known that polymorphonuclear leukocytes (PMNs) emerge first in local inflammatory sites, and then they are followed and scavenged by macrophages. We focused on the effect of PMN on tumor necrosis factor (TNF) release activity of macrophages, which is viewed as a possible indicator of the status of macrophage activation. One day after macrophages were cultured with fresh, intact murine PMNs which were induced with sodium casein, the release of TNF triggered by lipopolysaccharide (LPS) was augmented by low concentrations of PMNs, but suppressed by their high concentrations. When the PMN samples were fractionated into soluble and insoluble fractions, the augmenting and suppressing activity was partitioned; the relatively high concentrations of soluble fraction showed the suppressive effect whereas the insoluble fraction in lower concentrations showed augmentation. The suppressive activity was stable at 100 C, but the filtrates of the soluble fraction with membranes having cut-offs of 5,000 or 10,000 were not suppressive at all, suggesting the suppression is not due to low molecular compounds. It was also suggested that the suppressive effect for TNF release was not due to contaminating LPS or transforming growth factor-β. Inflammatory processes may thus be positively and negatively controlled by a quantitative factor of initial PMN populations by regulating the TNF release activity of the subsequent macrophages.     

Induction of tumor-inhibitory macrophages with a novel synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738)

3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.     

Influence of Transglutaminase on the Functions of Mouse Peritoneal Macrophages

Influence of transglutaminase on the production of interleukin-1 (IL-1) and on the release of active oxygen from mouse peritoneal macrophages was examined using cystamine and methylamine, an enzyme inhibitor and a substrate inhibitor, respectively. Casein-elicited or lipopolysaccharide (LPS)-elicited macrophages have higher levels of transglutaminase activity in comparison with resident macrophages, and there exists a definite correlation between endocytosis of erythrocytes and transglutaminase activity in either group of macrophages. The release of IL-1 by resident macrophages stimulated with LPS in vitro was significantly inhibited by the treatment with both transglutaminase inhibitors. However, these inhibitors were not able to inhibit the release of IL-1 from casein-elicited macrophages stimulated with LPS in vitro. The production of active oxygen from LPS-elicited macrophages was inhibited in a dose-dependent manner by the treatment of macrophages with cystamine, but was not by the treatment with methylamine. However, the treatment of LPS-elicited macrophages with cystamine did not inhibit the uptake of glucose into macrophages. These results suggest that transglutaminase activity in mouse peritoneal macrophages is an important factor for macrophage functions.     

Immunomodulatory activity of an anti-HSV-1 synthetic stigmastane analog

Many viral infections are associated with the development of immunopathologies and autoimmune diseases, which are of difficult treatment and for which no vaccines are yet available. Obtaining compounds that conjugate both antiviral and immunomodulatory activities in the same molecule would be very useful for the prevention and/or treatment of these immunopathologies.The compound (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) displays anti-Herpes simplex virus type 1 activity in vitro and reduces the incidence of herpetic stromal keratitis (HSK) in mice, a chronic inflammatory syndrome induced by ocular HSV-1 infection.In the present study, compound 1 showed opposite immunomodulatory properties in vitro. It induced the release of pro-inflammatory cytokines in HSV-1-infected epithelial cells of ocular origin, and significantly reduced the production of these cytokines in LPS-activated macrophages. RNA microarrays revealed various overexpressed and repressed genes in compound 1 treated infected epithelial cells and activated macrophages, many of which are associated with innate immune responses and inflammatory processes. These immunomodulatory properties of compound 1, together with its previously reported antiviral activity, make it a potential drug for the treatment of HSK and many other immunopathologies of viral and non-viral origin.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Effect of Muramyldipeptide,a Synthetic Bacterial Adjuvant,on Enzyme Release from Cultured Mouse Macrophages

Monolayer cultures of macrophages obtained by peritoneal lavage of normal or thioglycollate-stimulated mice spontaneously secreted lysosomal enzymes into the culture medium. When the elicited macrophages were cultured in the presence of muramyldipeptide (MDP), a 20–30% increase in the release of β-glucuro-nidase was consistently observed and the intracellular activity decreased to about 45% of that of control cells after 6–8 days' culture. A stimulatory effect of MDP on lysozyme secretion, though less profound, was also observed. In contrast, release of neither enzyme was stimulated in resident macrophages by the addition of MDP. A neutral α-glucosidase, which has recently been found to localize also in granules of macrophages, remained inside the cells and neither its activity nor its release was affected by the addition of MDP to either type of macrophages. A large amount of lactic dehydrogenase was released only when the resident, not the elicited, macrophages were cultured for 3–4 days and then phagocytosed zymosan.     

Apoprotein E mediates the interaction of beta-VLDL with macrophages

beta-Very low density lipoproteins (beta-VLDL) isolated from cholesterol-fed rhesus monkeys stimulated cholesteryl ester synthesis and accumulation in mouse peritoneal macrophages. The apoprotein specificity and requirement for the cell surface uptake of beta-VLDL was investigated by treating the beta-VLDL with trypsin (beta-VLDL (T], incubating the beta-VLDL (T) with other lipoproteins or apoproteins, reisolating the beta-VLDL (T) and measuring its biological activity which, for this study, is defined as the ability of the lipoprotein to stimulate cholesterol esterification in the macrophages. Trypsin treatment of beta-VLDL abolished its biological activity. Apoprotein analysis of the beta-VLDL (T) demonstrated the absence of intact apoproteins B-100, B-48, and E. The J774 macrophage-like cell line and mouse peritoneal macrophages responded similarly with respect to cholesterol esterification following incubation with inactive and treated beta-VLDL. The J774 macrophage-like cell line was used to establish the conditions necessary for the restoration of biologic activity to the trypsinized beta-VLDL. The loss of biological activity of beta-VLDL (T) could be reversed by restoring apoprotein E-containing LDL from hyperlipemic monkeys or purified apoprotein E. Apoprotein A-I had no such effect. The restored biological activity of the beta-VLDL (T) was proportional to the amount of apoprotein E acquired by the lipoprotein. beta-VLDL particles composed of apoprotein E and either intact or degraded apoprotein B-100 had comparable biological activity. Thus, intact apoprotein E, without intact apoprotein B, is a sufficient mediator for the biological activity and metabolism of beta-VLDL by macrophages and plays a major role in receptor-lipoprotein interaction.     

Effects of acute cigarette smoke exposure on macrophage kinetics and release of tumour necrosis factor alpha in rats

Some biological parameters before and after an acute episode of cigarette smoking in rats have been evaluated. The carboxyhaemoglobin levels depended either on the number of cigarettes, or on the time of exposure to cigarette smoke and returned to pre-smoking values in about 2 h. The evaluation of the kinetics of alveolar and peritoneal macrophages in rats after a smoking session of three cigarettes within an hour, indicated that alveolar macrophages in the bronchoalveolar lavage fluid significantly increased 8 h after the smoking, whereas the number of peritoneal macrophages remained practically constant. The incubation of these cells for various times at 37( degrees )C in a humidified atmosphere, resulted in a spontaneous release, 24 h thereafter, of variable amounts of tumour necrosis factor alpha (TNFalpha), which remained practically constant during the following days. Neither alveolar macrophages of control rats, nor peritoneal macrophages of both control and smoking rats were able to release TNFalpha. Moreover, after lipopolysaccharide induction of alveolar macrophages of both control and smoking rats, an increased release of TNFalpha was observed, indicating that these cells were in an active state.     

Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages.

Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood. The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages. The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days. Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr. By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells. Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli. Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively. Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity. Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF. HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight. We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF. This process may contribute to the host response to some neoplastic diseases.