Protective effects of immunoactive peptide, FK565 against systemic and local infections with herpes simplex virus and murine cytomegalovirus and respiratory tract infection with influenza virus in mice.

The protective effects of FK565 against systemic infections with herpes simplex virus (HSV) and murine cytomegalovirus (MCMV), respiratory tract infection with influenza virus and zosteriform rash with HSV investigated in mice. FK565 showed excellent protective activities against systemic infections with both acyclovir (ACV)-sensitive and -resistant HSV at intravenous and subcutaneous doses of 0.1 and 1 mg/kg and oral dose of 1 mg/kg. FK565 showed superior protective activities at subcutaneous doses of 0.01 and 0.1 mg/kg compared to ACV at subcutaneous dose of 15 mg/kg against MCMV infection. In respiratory tract infection with influenza virus, FK565 showed potent protective effects at intravenous, subcutaneous and oral doses of 0.001 to 1 mg/kg. FK565 markedly inhibited zosteriform spread of HSV on the flank skin at an intravenous dose of 0.1 mg/kg and the mice given FK565 survived longer than the control mice. The peritoneal exudate cells from FK565-treated mice suppressed the growth of HSV in mouse embryo fibroblast more strongly than those from the control mice, although FK565 had no direct antiviral activity against HSV. These findings suggest that FK565 enhanced the host defense ability against viral infections by nonspecific activation of macrophages.     

Immunoactive peptides, FK-156 and FK-565. II. Restoration of host resistance to microbial infection in immunosuppressed mice

The immunoactive peptides, FK-156 and its analogue, FK-565 were evaluated in various models of mice immunosuppressed with cyclophosphamide, hydrocortisone, mitomycin C, carrageenan and tumor cells. Treatment with FK-156 (subcutaneous) and FK-565 (oral) markedly restored host defense ability against microbial infection. The therapeutic effect of ticarcillin or gentamicin alone against pseudomonal infection in cyclophosphamide- and hydrocortisone-treated mice and tumor-bearing mice was much lower than in normal mice. The therapeutic effect of these antibiotics against pseudomonal infection in immunosuppressed mice was enhanced markedly by combined use with FK-156. The killing ability of macrophages and polymorphonuclear leukocytes of the immunosuppressed mice was also markedly enhanced by dosing with FK-156.     

In vivo activation of functional properties in mouse peritoneal macrophages by Nocardia rubra cell wall skeleton

Exudative cells in the peritoneal cavity of mice, particularly polymorphonuclear leukocytes significantly increased 1 day after intraperitoneal injection of Nocardia rubra cell wall skeleton (N-CWS). Macrophages and lymphocytes significantly increased 4 to 7 days after injection. N-CWS also enhanced peritoneal macrophage functions such as phagocytosis of latex particles, production of superoxide anion, production and secretion of lysosomal enzymes such as beta-glucuronidase, lysozyme and acid phosphatase, phagocytosis and intracellular killing of bacteria, and in vitro chemotaxis. The phagocytic function of the reticuloendothelial system was also enhanced. These results indicate that macrophages were activated in vivo by N-CWS.     

In vitro effects of an acyltripeptide, FK565, on antitumor effector activities and on metabolic activities of human monocytes and granulocytes

In vitro effects of an immunostimulatory acyltripeptide, FK565, on antitumor and metabolic activities of human leukocytes were studied. Monocyte cytotoxicity against A375 melanoma targets was significantly increased following pretreatment with FK565 at concentrations of 1 microgram/ml or more. The tripeptide also up-regulated anti-tumor cytostasis by monocytes and showed a strong stimulatory effect on superoxide generation by resting monocytes over a wide range of FK565 concentrations after 18 h preincubation. The monocyte preparations contained an average of 76% LeuM3+HLA-DR+, 12% LeuM3+HLA-DR- and 10% LeuM3-HLA-DR+ cells, and this phenotype distribution was not altered after incubation with FK565. At concentrations above 1 microgram/ml and after 2 h preincubation, FK565 also increased superoxide generation by resting but not stimulated granulocytes. Pre-exposure of cultured bovine endothelial cells to the peptide resulted in a significant inhibition of fMLP-stimulated granulocyte adherence to these cells. These data indicate that in vitro incubation of human monocytes and granulocytes with FK565 (0.1-100 micrograms/ml) had resulted in simultaneous up-regulation of several anti-tumor functions mediated by these cells.     

In vitro effects of an acyltripeptide, FK565, on NK-cell activity, LAK-cell generation and cytokine production by human mononuclear cells

In vitro effects of an immunostimulatory acyltripeptide, FK565, on natural killer (NK)-cell activity, lymphokine-activated killer (LAK)-cell generation and cytokine production of normal human peripheral blood mononuclear cells (MNC) were studied. FK565 used at concentrations ranging from 0.1 to 100 micrograms/ml enhanced NK-cell activity only if adherent MNC were removed. The optimal NK-cell enhancing dose was 2 micrograms/ml FK565. At the same range of concentrations, FK565 activated adherent MNC to induce suppression of NK-cell activity in autologous non-adherent MNC preparations. FK565 also potentiated both the generation of LAK-cell activity in the presence of 1000 U/ml of interleukin 2 (IL2) and the effector phase of LAK cells generated at the IL2 concentration of 50 U/ml. The synergistic interaction of IL2 and FK565 on LAK-cell activity was observed for all drug concentrations used. The effects of FK565 on cytotoxic cells could not be attributed to IL2, interferon gamma or tumor necrosis factor-alpha, because FK565 alone had no detectable influence on in vitro production of these cytokines by MNC. The ability of FK56 to modulate effector cells of natural antitumor immunity indicates that it may have promise as a biological response modifier in humans.     

Different effects of bacterial lipopolysaccharide on superoxide anion production by macrophages from normal and tumor-bearing rats

Bacterial endotoxins or lipopolysaccharides (LPS) exhibit a wide range of modulatory activities on immunocompetent cells. Among the numerous effects of LPS on macrophages, an enhancement of superoxide anion (O2-) release has been reported. In previous studies carried out on tumor-bearing rats, it was found that several functions of peritoneal macrophages such as phagocytic, microbicidal and antiviral activities were depressed. In this paper we evaluated the spontaneous or phorbol myristate acetate (PMA)-induced production of superoxide anion by macrophages from tumor-bearing rats with respect to controls. Moreover, the effect of in vitro priming with LPS on O2- production by the same cells was studied. It was found that the pattern of superoxide release by macrophages from tumor-bearing rats is significantly different from controls. Preincubation of macrophages from normal rats with LPS enhanced the spontaneous and PMA-induced production of O2-. In contrast, the same concentrations of LPS did not prime macrophages from tumor-bearing rats.     

Beta-naphthylamine induces anion superoxide production in rat peritoneal macrophages.

Rat peritoneal macrophages were incubated in the presence of beta-naphthylamine (beta-NA), a well known carcinogenic agent, and some parameters of respiratory burst were studied. beta-NA induced a time- and dose-dependent stimulation of superoxide anion (O-2) production, and this enhancement was suppressed by the addition of superoxide dismutase enzyme. Also, no cooperative effect between beta-NA and phorbol 12-myristate 13-acetate was observed. Other observations were as follows: (i) the simultaneous presence of polymyxin B, and staurosporine inhibitors of protein kinase C, inhibited beta-NA-dependent O-2 production; (ii) NADPH-oxidase contained in postnuclear fraction from beta-NA-incubated macrophages showed a greater activity than control fractions; (iii) the stimulation of O-2 production elicited by beta-NA was several-fold enhanced in activated macrophages compared to resident cells. These data suggest that beta-NA produces the activation of NADPH-oxidase through protein kinase C.     

Effects of local anesthetics on rat leukocyte functions]

To determine whether local anesthetics affect functions in macrophages, I examined the effects of 5 local anesthetics, lidocaine HCl, mepivacaine HCl, propitocaine HCl, procaine HCl, and tetracaine HCl, on chemotaxis and production of superoxide anion in rat peripheral macrophages. Rats were intraperitoneally injected with 1% glycogen. Peritoneal exudate cells containing macrophages were obtained from the peritoneal cavity 4 days after the administration. Chemotaxis was evaluated using a 48-well microchemotaxis chamber with a polycarbonate membrane filter. Production of superoxide anion was measured spectrophotometrically by a superoxide dismutase-sensitive reduction of ferricytochrome c. All of the local anesthetics examined at a dose of 1 mg ml inhibited (P < 0.05) chemotaxis and production of superoxide anion in macrophages. Moreover, pretreatment of macrophage suspensions with mepivacaine HCl, propitocaine HCl, procaine HCl, or tetracaine HCl at a dose of 1 mg ml resulted in inhibition of the production of superoxide anion. In contrast, pretreatment with lidocaine HCl at this concentration did not significantly affect the production of superoxide anion. These results suggest that all of the local anesthetics examined at a therapeutic concentration inhibit chemotaxis and production of superoxide anion in rat macrophages.     

Effect of in vivo administration of T-2 toxin on peritoneal murine macrophages

We investigated the effect of in vivo administration of T-2 toxin, a 12,13-epoxytrichothecene produced by several Fusarium species, on murine macrophage metabolism. Cytoplasmic and lysosomal enzyme levels, generation and release of superoxide anion, phagocytosis and intracellular killing of Salmonella typhi and murine P815 tumour cell lysis were measured under different experimental conditions. When T-2 toxin was administered to mice at sublethal doses (0.50-1.00 mg/kg/24 hr), the levels of lysosomal and cytoplasmic enzyme activity and the generation of superoxide anion were significantly enhanced as compared to controls. This correlated with increased phagocytosis and intracellular killing of S. typhi. Cytotoxic activity against murine P815 mastocytoma cells exhibited by macrophages isolated from mice treated with T-2 toxin was inhibited in a dose-dependent manner. In vivo administration of T-2 toxin may result in the activation of specific metabolic pathways of peritoneal macrophages, while inhibiting other paths.     

Regulation of macrophage function by the antioxidant N-acetylcysteine in mouse-oxidative stress by endotoxin

Changes in several functions of peritoneal macrophages from mice with oxidative stress caused by intraperitoneal injection of endotoxin (Escherichia coli lipopolysaccharide, LPS) (100 mg/kg), and associated with a high production of reactive oxygen species (ROS), have been observed in our previous studies. Antioxidants such as N-acetylcysteine (NAC) are free radical scavengers that improve and modulate the immune response, especially in oxidative stress situations. Therefore, in the present work, we have studied the effects of the administration of NAC (150 mg/kg i.p.) on different functions of peritoneal macrophages from Swiss mice suffering that oxidative stress, caused by LPS (100 mg/kg). NAC was injected 30 min after LPS injection, and the peritoneal macrophages were obtained at 2, 4, 12, and 24 h after endotoxin injection. The following functions, key stages of the phagocytic process, were studied: adherence to substrate, chemotaxis, ingestion of particles, and production of ROS (reactive oxygen species), as well as tumor necrosis factor (TNFalpha) release. The decrease in chemotaxis and the increase in adherence, ingestion, superoxide anion production, and TNFalpha release shown by macrophages from animals with oxidative stress were counteracted by NAC injection. These data suggest that NAC administration may be useful for the treatment of oxidative stress-linked endotoxic shock, modulating the function of macrophages, specifically in decreasing the production of ROS and of inflammatory cytokines such as TNFalpha.     

Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.

1. Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. 2. We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one. 3. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved. 4. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.     

15-Deoxy-delta(12,14)-prostaglandin J2 is a negative regulator of macrophage functions.

15-Deoxy-delta(12,14)-prostaglandin J2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma). Because PPARgamma is highly expressed in macrophages obtained from stimulant-elicited peritonitis, but not in resident peritoneal macrophages, the effect of dPGJ2 was tested on innate functions of macrophages. dPGJ2 inhibited adhesion and phagocytosis of Escherichia coli. Inhibition of these functions by dPGJ2 was not mediated via the adhesion molecule Mac-1. In addition, dPGJ2 inhibited chemotaxis toward zymosan-activated serum and it also inhibited the production of superoxide anion when macrophages were stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OPZ), but not lipopolysaccharide. Similarly, dPGJ2 inhibited the production of hydrogen peroxide when macrophages were stimulated with either PMA or OPZ. These studies suggest that dPGJ2 may be a negative regulator of macrophage functions.     

Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock

The toxic effects of oxygen radicals produced by immune cells can be controlled to certain degree by endogenous antioxidants because of their scavenger action. This control is specially important in a type of immune cell, i.e., the phagocyte, which produces oxygen-free radicals and uses antioxidants in order to support its functions. Antioxidants, such as ascorbic acid (AA), are free radical scavengers and improve the immune response. In the pathogenesis of endotoxic shock, a disease with high mortality caused by gram-negative bacterial endotoxin, the reactive oxygen species (ROS) produced by phagocytes have been implicated. In a previous study, we observed in peritoneal macrophages from BALB/c mice suffering lethal endotoxic shock caused by intraperitoneal (i.p.) injection of Escherichia coli lipopolysaccharide (LPS; 100 mg/kg) a high production of superoxide anion. Therefore, in the present work, we have studied the in vitro effect of AA, at different concentrations (0.001, 0.01, 0.1, 1 and 2.5 mM), on the various steps of the phagocytic process, i.e., adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production of murine peritoneal macrophages obtained from BALB/c mice with that of endotoxic shock, at 2, 4, 12 and 24 h after LPS injection. The increased adherence, ingestion and superoxide anion production by macrophages from animals with endotoxic shock were lower in the presence of AA, reaching similar values to those of the control animals. The most effective AA concentration in cells from mice with endotoxic shock was 0.01 mM. These data suggest that AA can regulate the phagocytic process in endotoxic shock, principally decreasing free radical production and thus it could reduce endotoxic shock severity.     

Substance P augments nitric oxide production and gene expression in murine macrophages.

We investigated the effects of substance P (SP) on nitric oxide (NO) synthase activity in macrophages by measuring the production of nitrite and the expression of inducible NO synthase (iNOS) mRNA and protein. In LPS-activated macrophages, SP stimulated NO production in time and concentration dependent manners. These SP effects were blocked by a specific NK-1 receptor antagonist. Furthermore, SP stimulation increased the levels of both iNOS mRNA and iNOS protein. These results demonstrate that SP can increase LPS induced NO production in macrophages by augmenting the induction of iNOS expression. We also examined the role of SP on acute-cold stress induced altered production of NO by mouse peritoneal macrophages. SP enhanced the LPS-induced macrophages NO production from stressed mice relative to the non-stressed mice. These results suggest that SP may have an important modulatory role in production of NO by macrophages.     

Hematein inhibits atherosclerosis by inhibition of reactive oxygen generation and NF-kappaB-dependent inflammatory mediators in hyperlipidemic mice

Hematein, a natural compound, is a known anti-inflammatory and antiatherogenic agent in the rabbit model. The authors investigated the effects of this compound on atherogenesis and possible mechanisms of the actions in the hyperlipidemic mice. Low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-cholesterol diet alone for 8 weeks developed the fatty streak lesion in the aortic sinus, whereas this lesion was significantly reduced by hematein treatment without a change in plasma lipid levels compared with control mice. Hematein treatment reduced plasma levels of lipid peroxide and superoxide generation in LPS-stimulated peritoneal macrophage. Hematein treatment inhibited NF-kappaB-DNA binding activity in peritoneal macrophages from Ldlr-/- mice and the activation of NF-kappaB in RAW264.7 macrophages. This compound suppressed plasma nitrite/nitrate levels in Ldlr-/- mice and NO production and iNOS expression in LPS+IFNgamma-stimulated peritoneal macrophages. Hematein treatment also suppressed the activity of iNOS promoters in RAW264.7 macrophages, and reduced the plasma levels of TNF-alpha and IL-1beta and the production of these cytokines in LPS+IFNgamma-stimulated peritoneal macrophages. These results suggest that hematein inhibits atherosclerotic lesion formation, possibly by reducing proinflammatory mediators through a decrease in reactive oxygen species generation and NF-kappaB activation.     

Species differences in impairment and recovery of alveolar macrophage functions following single and repeated ozone exposures.

Effects of single (0.4 ppm for 3, 6, or 12 hr) and repeated (0.4 ppm, 12 hr/day for 3 or 7 days) in vivo ozone exposures on rat and mouse alveolar macrophage functions and cell number were investigated. Single ozone exposure of rats resulted in a small (approximately 15%) decrease in Fc-receptor-mediated phagocytosis and phorbol ester-induced superoxide production by the alveolar macrophages and was followed by recovery above control levels within 12 hr of exposure. Repeated exposures of rats for up to 7 days did not alter alveolar macrophage functions, with the exception of the effects of 3 days of exposure on superoxide production (71 +/- 9% as compared with the controls). In mice, significant changes in alveolar macrophage functions were not observed until 12 hr of exposure (at that timepoint phagocytosis was 74 +/- 2%). Repeated ozone exposures of mice did not cause a further decrease in phagocytosis (at Day 7, 74 +/- 14%). Both after 3 and 7 days of repeated ozone exposure of mice, superoxide production by the alveolar macrophages was inhibited approximately 50%. In rats and mice, repeated ozone exposures led to an increase in the number of alveolar macrophages. In mice, this increase appeared at a later time point (at Day 7 vs Day 3) and was less pronounced (at Day 7, 139 +/- 9% vs 179 +/- 17%) as compared with rats. In summary, our data show that rat and mouse alveolar macrophages have different susceptibilities to both single and repeated in vivo ozone exposures.     

Cytotoxicity and antigenicity of antimicrobial synthesized peptides derived from the beetle Allomyrina dichotoma defensin in mice

Synthetic peptides, peptides A (Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-NH(2)) and B (Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH(2)), derived from the beetle Allomyrina dichotoma defensin, have antimicrobial activities. Immunotoxicological effect of these peptides was evaluated by cytotoxicity of mouse peritoneal macrophages. In addition, antigenicity of these peptides was studied by evaluating antibody responses in mice immunized with these peptides. The toxicity of peptide A toward mouse peritoneal cells was less than that of polymyxin B, when morphologically evaluated in a cytotoxicity test. Almost all of mice injected intraperitoneally (i.p.) with either peptide A or B at 50-150 mg/kg survived, whereas all mice injected i.p. with polymyxin B at the doses of more than 25 mg/kg died within 24 h. Interestingly, almost all of mice injected intravenously with these peptides at the doses of 10 and 25 mg/kg also survived. Furthermore, mice immunized with these peptides conjugated with keyhole limpet hemocyanin (KLH) showed little or negligible anti-peptide A or B antibody production, although anti-KLH antibody was significantly produced. The results indicated that peptides A and B were less cytotoxic than polymyxin B and also had poor antigenicity to produce specific antibody in mice.     

In vitro effect of mercury and vanadium on superoxide anion production and plasminogen activator activity of mouse peritoneal macrophages

The in vitro effects of mercuric chloride and vanadate were examined on two functions of mouse peritoneal macrophages, i.e., the superoxide anion production and the plasminogen activator (PA) activity. Vanadate, at concentrations which do not affect the viability of the cells, does not seriously alter any of these functions. High concentrations of mercury depress the respiratory burst; this effect results from loss of the reducing properties of cellular NADPH. Low concentrations of mercury stimulate the effect of phorbol 12-myristate 13-acetate on PA activity. The mechanism of this stimulation does not involve the protein kinase C system. It is hypothesized that mercury could enhance the synthesis of PA, its translocation to the cell surface, or its binding to the membrane receptors.     

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages

1. Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon-gamma (IFN-gamma) induced apoptotic death through a nitric oxide-dependent pathway. 2. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation-dependent apoptosis. 3. These drugs decreased the up-regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl-2 than in LPS/IFN-gamma treated cells. 4. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. 5. Incubation of macrophages with LPS/IFN-gamma barely affected caspase-1 but promoted an important activation of caspase-3. Both CsA and FK506 inhibited pathways leading to caspase-3 activation. Moreover, the cleavage of poly(ADP-ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. 6. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase-3 in cells treated with nitric oxide donors. 7. These results indicate that CsA and FK506 protect macrophages from nitric oxide-dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.     

Alleviation of picryl chloride-induced delayed type hypersensitivity reaction by saponin fraction of Gleditsia sinensis

The objective of the present study was to investigate the effects of saponin fraction from anomalous fruits of Gleditsia sinensis (SFGS) on picryl chloride-induced delayed type hypersensitivity (PC-DTH) and functions of T lymphocytes and macrophages in mice. SFGS (100, 200 mg/kg), orally administered during either sensitization stage or effector stage, produced remarkable inhibition of PC-DTH. In vitro, SFGS (1, 2, 4 microg/ml) concentration-dependently attenuated concanavalin A (Con A)-elicited mouse splenocyte proliferation and interleukin 2 (IL-2) production. At concentrations of 10 and 20 microg/ml, SFGS inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and interleukin 1beta (IL-1beta) of mouse peritoneal macrophages. The findings indicate that SFGS attenuates PC-DTH in mice, which is probably mediated by preventing proliferation and differentiation of T cells during the sensitization stage and suppressing activation of macrophages during the effector stage.