Macrophage activation by Lactobacillus casei in mice

Effects of Lactobacillus casei YIT 9018 (LC 9018), which has antitumor activities against allogeneic and syngeneic murine tumors, on macrophage functions were examined. By intraperitoneal (i.p.) injection of LC 9018, acid phosphatase activity and phagocytic activity of peritoneal macrophages were enhanced significantly compared with those of normal peritoneal macrophages. The phagocytic activities showed peaks 2-3 days after the LC 9018-injection. LC 9018 accelerated the phagocytic function of the reticuloendotherial system in ICR mice tested by the carbon clearance test. The cytostatic activity of peritoneal exudate cells (PEC) induced by i.p. injection of LC 9018 into C57BL/6 mice against EL4 cells was also enhanced. On the other hand, PEC induced by L. fermentum YIT 0159, which has no antitumor activity, did not have cytostatic activity. These observations showed that LC 9018 was able to activate macrophages in mice.     

Leukotriene C4 is an essential 5-lipoxygenase intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells

Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately. LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively. Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM. Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM. The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages. Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity. Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4. This implies that besides LTC4, increased cytosolic [Ca2+] is required for A23187 induction of macrophage cytostatic activity.     

Macrophage activation by tuftsin and muramyl-dipeptide

Summary Peritoneal macrophages from tuftsin or MDP-treated mice were tested for their cytostatic activity for tumor cell proliferation. Both substances are able to activate macrophages either after intravenous injection or after incubation in vitro with normal macrophages. But a stimulation as well as an inhibition of tumor cell growth can result from macrophage activation depending on the timing and dose injected. Restoration of the impaired cytostatic capacity of macrophages of mice observed with aging, is obtained by repeated administration of tuftsin. Normal and BCG-stimulated macrophages were examined for their regulatory activity on the proliferation of P815 tumor cells. Low density of macrophages per well determines a stimulation of target cell growth whether the macrophages are normal or activated. When the number of macrophages is increased, under conditions in which normal macrophages are not inhibitory. BCG-stimulated macrophages exert already a strong cytostatic activity. At high macrophage content it appears that normal macrophages can also display an inhibitory activity. Macrophage-tumor cell interactions are highly dependent on the concentration and the state of activation of macrophages.Reprint requests should be addressed to M. Bruley-Rosset     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of 4-methyluracil and carnosine on healing of skin wounds in rats

In experiments in vivo and in vitro the authors studied antioxidative properties of 4-methyluracil and carnosine, their capacity to inhibit sex and accelerate healing of skin wounds. 4-methyluracil and carnosine discover almost the same capacity to decrease in the tissues of the wound and the blood serum in the formation of various intermediate products of free radical oxidation. Data are given on the study of the dynamics of wound healing after a 5-day treatment with equimolar quantities.     

Immunoregulatory role of in vitro differentiated macrophages on human natural killer (NK)-cell activity

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.     

The role of peritoneal macrophages in realizing the growth inhibiting effect of glucocorticoids on reinoculated murine hepatoma 22 cells: the effect of hydrocortisone on the cytotoxic activity and metabolism of phospholipids by macrophages

A single injection of hydrocortisone to rats with ascite hepatoma 22 had practically no effect on tumour growth. Inhibition of tumour growth was observed only after reinoculation of ascite hepatoma to mice that had received no less than 8 daily injections of the hormone. A single injection of hydrocortisone induced inhibition of the cytotoxic activity and decreased phospholipid metabolism in peritoneal macrophages. Contrariwise, long-term administration of the hormone caused marked activation of macrophage cytotoxicity. In this case incorporation of 32P into macrophage phospholipids was restored up to the control level. It is concluded that one of mechanisms underlying the inhibitory effect of glucocorticoids on macrophages is inhibition of phospholipid turnover. Presumably, long-term administration of the hormone promotes the formation of a new population of macrophages insensitive to the inhibitory effect of glucocorticoids and possessing a high cytostatic activity. The appearance of such activated macrophages may account for the enhancement of hydrocortisone effect on tumour cells upon prolonged administration of the hormone.     

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.     

Resistance to a peritoneal lymphoma graft induced by heat-killed S or R Brucella abortus

Summary Mice were injected either with the rough (R) or smooth (S) strains of killed Brucella abortus, after which, at various times, they were given an i.p. injection of a strain-specific lymphoma (EL4). In parallel, samples of peritoneal exudate cells were taken from the Brucella-injected mice, and their in vitro cytostatic activity against EL4 tumour cells was investigated. Protection against the lymphoma graft lasted up to the 7th day with S and the 20th with R. In contrast, cytostatic activity was more intense and lasted longer with S than with R. The parallelism between in vivo resistance to the lymphoma graft and augmentation of macrophage cytostatic activity was satisfactory with R but not with S. The differential effects of S and R on EL4 lymphoma growth could not be simply explained on the basis of a difference in macrophage activation.     

Impaired macrophage activation in vitamin D3 deficiency: differential in vitro effects of 1,25-dihydroxyvitamin D3 on mouse peritoneal macrophage functions

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is known to interact in vitro with mononuclear phagocytes. The purpose of this study was to determine the role of the steroid in macrophage activation in vivo. Peritoneal macrophages from normal and vitamin D3-deficient mice were obtained after i.p. injection of activating or eliciting agents. Cells obtained from vitamin D3-deficient mice exhibited defected capabilities to perform anti-tumor activities (cytostasis and cytolysis) and to form oxygen reduction products (H2O2 and O2-). On the other hand, the level of the lysosomal enzyme acid phosphatase was unaffected by vitamin D3 deficiency. In vitro, incubation of macrophages with 1,25(OH)2D3 enhanced their anti-tumor activities, but did not affect the cells' capacity to produce H2O2 and O2-, or acid phosphatase. Our results suggest that 1,25(OH)2D3 is essential for macrophage activation in vivo. However, in vitro, the hormone is only partially capable of affecting the macrophage functions, probably because of the maturation state of the cells.     

Effect of carnosine and 4-methyluracil on the development of experimental hepatitis in rats]

A comparative study of the hepatoprotective effect of carnosine and 4-methyluracil under CCl4-induced acute toxic hepatitis has been carried out. The extent of liver injury and its regeneration were established from morphological data as well as from changes in the activities of alanine aminotransferase (ALT) and histidase and the bilirubin content in blood serum. Hyperlipoperoxidation in the liver and serum was assessed by the amount of TBA-active products. It was found that by day 10 of experimental hepatitis ALT and histidase levels in blood sera of untreated animals exceeded the normal values 1.3- and 3.9-fold, whereas those in the carnosine-treated group approximated the values characteristic of intact animals. The activity of serum ALT in animals treated with vitamin B12 or 4-methyluracil exceeded normal values 1.5 and 1.6 times, whereas that of histidase was 2.5 and 2.7 times as high. Carnosine and 4-methyluracil inhibited (in approximately the same degree) the formation of TBA-active products in the liver. According to morphological dta, cessation of CCl4 injections was accompanied by rapid regeneration of liver tissues in all animal groups. Carnosine enhanced regenerative processes in parenchymatous and connective tissues in a far greater degree in comparison with other drugs. The mitotic index in the carnosine-treated group exceeded more than twofold the corresponding parameters in untreated animals. Possible mechanisms of carnosine action on liver repair are discussed.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Specific lipoxygenase inhibition reverses macrophage cytotasis towards P815 tumor cells in vitro induced by the calcium lonophore A23187

A23187-stimulated cytostatic activity of peritoneal macrophages towards P815 tumor cells served as a model for macrophage activation: a macrophage enriched preparation, separated on the basis of cell size in a discontinuous FCS gradient column, expressed cytostatic activity when stimulated by A23187. This was inhibited dose-dependently, by AA-861 but not by nordihydroguaiaretic acid (NDGA). AA-861 inhibited 5-lipoxygenase specifically, NDGA inhibited both 5-lipoxygenase- and cyclooxygenase activity. The ratio cyclooxygenase/lipoxygenase products increased with AA-861 but not with NDGA. These results show that lipoxygenase products are necessary for expression of cytostatic activity of these arachidonic acid metabolite-producing macrophages and that the ratio cyclooxygenase/lipoxygenase metabolites plays an important role in macrophage activation.     

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34 degrees-36 degrees C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage-like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S-3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater-like structure (about 3-9 microns in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB-treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.     

Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fc gamma receptor-mediated superoxide generation in macrophages

A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.     

The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B₆C₃F₁ mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with ³H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni , which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba -mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba -specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.     

Hormone receptor studies on frog macrophage cells by means of histamine, serotonin and indoleacetic acid.

The phagocytotic activity of frog macrophage cells could be increased by 50% with histamine and by 24% with serotonin. These hormones have a similar effect at the various stages of phylogenetic development, to judge from the respective responses of the unicellular Tetrahymena which showed roughly the same percentual increases of phagocytosis as frog macrophages at roughly the same hormone concentrations. It is concluded that cytoplasmic membrane receptor patterns for a given function do not change in the course of phylogenetic development and the receptors have a capacity for selection, preferring histamine to serotonin, and the latter to the chemically closely related plant hormone indoleacetic acid.     

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.     

Cytostatic effect of gangliosides present in the membrane of macrophages

Stimulated macrophages are known to inhibit the growth of certain tumor cells. Using mouse peritoneal exudates as a source of macrophages and the mastocytoma cell line P815 as the target, the inhibition was found to depend on direct contact between the macrophages and the growing cells. Cytostatic activities were detected in extracts of macrophages as well as in membranes of macrophages bound to substances of low molecular weight. Physical and biochemical characteristics of the cytostatic activity hint toward N-acetylneuraminic acid containing glycosphingolipids (gangliosides). The different macrophage gangliosides were separated by thin-layer chromatography. All types showed cytostatic activity, but the most effective gangliosides were identified as monosialoganglioside GM1 and disialoganglioside GD3.