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Previous studies have shown that mannosylated bovine serum albumin (mBSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans and Escherichia coli by resident murine peritoneal macrophages (M φ ). Upregulation of the above M φ functions was associated with the binding of mBSA to the macrophage mannose receptor. The present study was done to determine if certain glyconutrients could stimulate M φ functions in a similar manner. Resident peritoneal murine M φ collected from C57BL/6 mice were exposed to the glyconutrients for 10 and 60 min. The RB was measured using chemiluminescence. Both phagocytosis and killing were measured after incubation with each of the following microorganisms: Candida albicans, Escherichia coli and Staphylococcus aureus . The percent phagocytosis and killing were determined using fluorescence microscopy. Results indicated that certain glyconutrients, caused a dose and time dependent effect on M φ -induced killing of all three microorganisms.

The present investigators have reported previously that macrophages (Mphi) can bind either myeloperoxidase (MPO) or eosinophil peroxidase (EPO) resulting in enhanced cytotoxicity to Candida albicans. Since MPO was shown to be immunomodulatory, the present study was initiated to determine whether either EPO or partially fragmented EPO (fgEPO) also modulated cytokine secretion. Murine peritoneal Mphi; simultaneously stimulated with fgEPO and one of the following, (1) LPS, (2) mannosylated bovine serum albumin (mBSA), (3) interferon-gamma (IFN-gamma), or (4) Poly I:C, demonstrated both dose- and time-dependent decreases in TNF-alpha and IL-6 and a dose-dependent decrease in IFN-alpha/beta. The mRNA levels of Mphi exposed to fgEPO and mBSA demonstrated that fgEPO modulated Mphi cytokine function by decreasing TNF-alpha and IL-6 mRNA transcripts without altering transcription of TGF-beta or GM-CSF. These results demonstrate a possible interaction between the Mphi and eosinophil that could result in reduction of inflammation.

Neutrophils represent one of the host's primary defenses against invading organisms. These cells often arrive at the site of infection prior to macrophages (M phi). Neutrophils release myeloperoxidase (MPO) into the micro-environment during phagocytosis. Previous studies by the present investigators have shown that M phi bactericidal activity is enhanced by exposure to MPO. A recent report suggests that as much as 40% of this protein is enzymatically inactive once it is released into the micro-environment. In the present study, exposure of M phi to an enzymatically inactive form of MPO (iMPO) or another mannosylated protein, mannosylated bovine serum albumin (mBSA), can induce the same enhanced Mø-mediated bacterial cell killing observed with the active form of MPO. Furthermore, this phenomenon is limited as galactosylated BSA (gBSA) did not induce enhancement of bacterial killing. The data suggest that interaction of either enzymatically active or inactive mannosylated proteins with the M phi mannose receptor (MMR), is sufficient to enhance M phi bactericidal activity and further underscores the binding of the MMR and resultant responses as a major host defense mechanism.

The present study describes the inhibition of macrophage-mediated cytotoxicity (MMC) by cocaine and suggests a possible mechanism. Mice (C57BL/6) were injected i.p. with cocaine. At various intervals after exposure to cocaine, peritoneal macrophages (M phi) were removed, cultured in the presence of interferon gamma and LPS, then incubated with 51Cr labeled target cells. A single injection of > or = 10 mg/kg cocaine was sufficient to inhibit cytotoxicity to P815 cells. This inhibition was evident 3 h after exposure to cocaine and could still be demonstrated 24 h later. Since reactive nitrogen intermediates (RNI) have been reported to be one of the major mechanisms by which M phi kill, the amounts of NO2- produced by M phi from cocaine-injected animals were compared with that produced by equivalent controls. Cocaine reduced the level of NO2- in a dose-dependent manner which correlated with MMC. There was a significant reduction in NO2- produced by activated M phi, 3 h after i.p. injection of cocaine but not at 24 h, using > or = 5 mg/kg. At 12 h there were differences between M phi from control animals and animals receiving > or = 10 mg/kg cocaine. By 24 h there were no differences between control and cocaine-injected animals even at the highest dose employed (25 mg/kg). These results suggest that cocaine reduces the killing ability of murine M phi through a temporary reduction of RNI.

Myeloperoxidase is an enzyme which is found in the azurophilic granules of neutrophils and is associated with bactericidal, fungicidal, and tumoricidal activity. The present studies show that human recombinant myeloperoxidase (rec-MyPo) can regulate a number of macrophage (M phi) capacities and functions. Macrophages from mice exposed to rec-MyPo in vitro released reactive oxygen intermediates, tumor necrosis factor alpha (TNF alpha), and interferon alpha/beta (IFN alpha/beta). Enhanced target cell killing was also demonstrated with TNF alpha sensitive but not TNF alpha insensitive cells. Intravenous injection of rec-MyPo induced high titers of systemic TNF alpha and IFN alpha/beta. These results indicate that MyPo can function as an immunomodulator both in vitro and in vivo. Because of these actions, it is apparent that MyPo represents a previously unrecognized endogenous immunomodulator.

Cocaine has been demonstrated to have a number of different effects on immune cell functions. We have reported alterations of cellular functions by macrophages (Mφ) exposed to cocaine in vitro, including the inhibition of mouse hepatitis virus replication. Here, we present evidence that cocaine stimulates the secretion of an antiviral product that is neutralized by anti-interferon (anti-IFN). A dose-dependent increase in the secretion of IFN by both Mφ and L929 cells incubated with cocaine, with a concomitant decrease in virus replication, is also reported. The increase in IFN secretion was most pronounced when cells were cultured in the presence of the IFN inducer poly(I·C). The effect of cocaine on IFN production was found to be primarily at the transcript level in both Mφ and L929 cells. These findings further support our previous research demonstrating an antiviral activity of cocaine in vitro. The relevance of this activity to viral infections in general remains to be determined.

Cocaine has been shown to have a number of diverse effects on the immune system. The current investigators have previously demonstrated an inhibitory effect of cocaine on murine hepatitis virus replication in peritoneal macrophages in vitro. The present study was undertaken to examine the effects of cocaine on influenza virus replication and to further characterize that effect in an animal model. Cocaine was capable of inducing a dose-dependent reduction in influenza PR-8 replication using MDCK cells in vitro. Concentrations of 100 μg/ml caused a 50% reduction of virus. To further characterize the effect in vivo, C57Bl/6 mice infected with influenza PR-8 by intranasal instillation were given daily ip injections of 10 mg/kg cocaine just prior to and for 4 days after exposure to influenza. Lungs from mice exposed to cocaine had viral titers that were reduced approximately 50% compared to controls as demonstrated by hemagglutination titers. Additional studies suggest that this reduction appears to be caused by an increase of cocaine-induced interferon.

Tumor necrosis factor-alpha (TNF) is an extremely potent cytokine which is involved in the pathogenesis of a number of diseases. Interruption of its synthesis can result in a reduction of inflammation and subsequent pathology. A new experimental drug pirfenidone (5-methyl-L-phenyl-2-(1H)-pyridone, trade name: Deskar) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. The present study describes the inhibition of TNF in vitro as well as the inhibition of circulating TNF in vivo by pirfenidone. Isolated, thioglycollate-induced peritoneal macrophages (Mphi) from C57BL/6 mice were exposed to either lipopolysaccharide (LPS) or mannosylated bovine serum albumin then incubated with 0.1-0.9 mg/ml of pirfenidone. This substance inhibited the production of TNF in a dose-dependent manner as measured by ELISA. One i.p. injection of either 100 or 200 mg/kg pirfenidone inhibited the induction of circulating TNF following a single i.v. injection of LPS. Endotoxin shock was induced in mice using an i.p. injection of galactosamine and LPS. The higher dose of pirfenidone (200 mg/kg) completely inhibited shock and subsequent mortality. Lower doses of pirfenidone or administration either prior to or post challenge only partially inhibited symptoms. These results indicate that pirfenidone is able to inhibit both TNF induction and subsequent endotoxin shock. Additional studies are warranted to establish this drug as a potential treatment for diseases where TNF plays a major role.

Numerous natural and synthetic quinone compounds possess significant antitumor properties. Various mechanisms have been proposed to account for these properties, including scission and degradation of tumor cell DNA, intracellular "redox cycling" to cogenerate semiquinone free radicals and reactive oxygen intermediates, and the interaction of semiquinone radicals with tumor cell surface flavoenzymes. However, no evidence has been presented to explain adequately the preferential attack on tumor cells by semiquinone radicals, as opposed to normal cells. To address this question, a synergistic interaction was examined. A therapy consisting of a mixture of L-ascorbate and 2,6-dimethoxy-p-benzoquinone, was used for in vivo evaluation. BALB/c mice were depleted of functional T-lymphocytes by xenogeneic monoclonal antibody pretreatment, challenged with Ehrlich ascites tumor, and administered the semiquinone radical-generating therapy. Mice depleted of CD4+ T-lymphocytes responded with rapidly fatal tumor progression, with significantly decreased mean survival times over controls, whereas less severe responses were observed in mice devoid of CD8+ T-lymphocytes. Mice depleted of both T-lymphocyte subpopulations responded with uninhibited tumor growth and rapid mortalities. When tumor challenge occurred after therapy, tumor growth was significantly delayed in mice enriched for CD4+ T-lymphocytes, with demonstrable increases in mean survival time over controls. This reagent combination had no significant effect on T-lymphocyte profiles in secondary lymphoid organs. These data suggest a synergistic phenomenon of semiquinone radical-induced cytostasis coupled with T-lymphocyte helper activity for optimal tumor suppression.

Macrophages (Mphi) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mphi exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mphi-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mphi were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P < or = 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mphi via ROI, and that iMPO which is released at a site of inflammation induces Mphi-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Our previous studies have demonstrated a number of effects of cocaine on macrophage (M phi) functions. The present studies were initiated to ascertain the effects of cocaine on virus production in vitro. C57BL/6 mice were injected intraperitoneally with thioglycollate broth, and the peritoneal M phi collected 4 days later were cultured in 96-well microtiter plates as continuous monolayers. Various concentrations of cocaine were incubated with M phi for up to 5 days. Mouse hepatitis virus (MHV) was added to these cultures, which was followed by a methyl cellulose overlay. Cocaine caused a dose-dependent inhibition of viral plaques after 48 hr of incubation. The inhibitory activity was transferable to fresh cultures of M phi, inhibiting both plaque size and number. Specific polyclonal antibodies to alpha + beta-interferon but not tumor necrosis factor or transforming growth factor-beta partially reversed the inhibition of both plaque size and plaque number. It appears that MHV induced interferon in cultured M phi, an effect that was enhanced by cocaine. Since cocaine has been reported to interfere with calcium mobilization, studies were done using ionomycin, a calcium ionophore, in order to reverse possible effects on intracellular calcium that could affect virus production. The presence of ionomycin completely reversed the inhibition of virus production by cocaine. The antiviral effects of cocaine appear to be caused by modulation of intracellular calcium and, to a lesser extent, by the enhancement of interferon production.

Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (MO). Upregulation of the above MO functions was associated with binding of m-BSA to the MO-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MO in a similar manner. Resident peritoneal MO collected from C57BL/6 mice were exposed to acemannan for 10 min. The RB was measured using chemiluminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MO were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0-5% killing in controls. If MO were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0-5% in the controls. The results of the present study indicate that short term exposure of MO to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.

Candida albicans is an opportunistic pathogen whose resurgence coincides with the rising number of AIDS patients. Neutrophils are known to be involved in the clearance of Candida infections; however, the role of macrophages in host defenses against this organism is not well understood. The present study was undertaken to examine an unrecognized interaction between neutrophils and macrophages resulting in enhanced killing of candidae in vitro. Murine peritoneal macrophages exposed to recombinant myeloperoxidase exhibited enhancement of the respiratory burst, increased phagocytosis, and a dose-dependent increase in intracellular killing of Candida species. Radical scavengers reduced the killing, indicating a role of reactive oxygen intermediates in the candidacidal activity observed. These data suggest that at the site of infection, myeloperoxidase released from neutrophils activates macrophages and induces microbicidal activity.

Cocaine, a naturally occurring alkaloid, is known to affect the immune system. The present authors have demonstrated that peritoneal macrophages, isolated from mice injected with cocaine, have an increased capacity for the production of reactive oxygen intermediates (ROI) and a decreased capacity for the production of reactive nitrogen intermediates (RNI). The present studies were done to determine the effects of certain cocaine metabolites on the induction of ROI and RNI by peritoneal macrophages. C57BL/6 mice were injected i.p. with either saline or 5 mg/kg of one of the following: cocaine, norcocaine, benzoylecgonine, ecgonine methyl ester HCl or ecgonine HCl. The ROI were measured using a chemiluminescence assay and the RNI were measured as nitrite secretion following exposure of isolated M phi to interferon gamma and LPS. Isolated peritoneal M phi from mice injected i.p. with cocaine, norcocaine and benzoylecgonine exhibited an increase in the production of ROI and a concomitant decrease in the production of RNI. However, injections of either ecgonine methylester HCl or ecgonine HCl had no effect on the induction of either ROI or RNI by murine peritoneal M phi. The cocaine metabolites, norcocaine and benzoylecgonine, have been reported to cause hepatic and/or cerebral toxicity. The present study also demonstrated that injection of these metabolites in vivo, also altered M phi functions which were measured in vitro.

The effects of cocaine on the respiratory burst (RB) and the phagocytic activity of murine macrophages (M phi) were studied. C57BL/6 mice were injected intraperitoneally with various concentrations of cocaine or appropriate vehicle. Peritoneal and alveolar M phi were isolated from mice and cultured in vitro. Both alveolar and peritoneal M phi from cocaine-exposed mice exhibited an increase in the RB when compared with M phi from saline-injected controls. This increase in the RB was apparent 60 min after injection and persisted for at least 48 hr. Injection of the cocaine metabolites, ecgonine methyl ester hydrochloride and ecgonine hydrochloride, did not affect the RB. The increase in the RB was correlated with an increase in phagocytosis of opsonized zymosan in vitro. However, an in vivo phagocytic assay using mice injected ip with cocaine, which was subsequently followed by sheep erythrocytes, demonstrated the opposite effect. Fewer peritoneal M phi from cocaine-injected mice were observed with ingested erythrocytes. These data underscore the complex effects of cocaine on M phi functions.

SummaryMarihuana and its principal active ingredient Δ9-tetrahydrocannabinol (THC) has been shown to be immunosuppressive in mice. The present study was conducted in order to determine if the immunosuppression was regulated by the maturity of the animal. Splenic lymphocytes from 4-week-old mice exposed to THC displayed a reduced capacity for mitogenic stimulation following in vitro exposure to phytohemagglutinin. Rosette formation, IgG plaque formation, and synthesis of circulating antibody to sheep erythrocytes (SRBC) were also inhibited after exposure of young mice to THC. Cell-mediated immunity and rosette formation in 14-week-old mice was not significantly altered by treatment with THC as evidenced by the equivalent capacity of splenic lymphocytes from both drug and control animals to respond to mitogenic stimulation and to bind SRBC. Older mice were also found to be less responsive to immunosuppression by THC in relation to IgG plaque formation and hemagglutination titer.

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.

The effects of ethanol alone or combined with pyrazole or disulfiram were studied in reference to interferon production. Saline, ethanol (2 g/kg), pyrazole (2 mg/kg), or disulfiram (2 mg/kg) were injected, intraperitoneally, 3 hr after poly I:C, an interferon inducer. Drugs were administered either by a single injection, or twice daily for 7 days, and the mice were sacrificed 6 hr after poly I:C. Sera from mice were pooled, dialyzed, and assayed for interferon by the microplaque reduction method. Administration of ethanol alone reduced interferon production 76% and 66% after the single and the multiple injections, respectively. A similar inhibition of interferon occurred after either pyrazole or disulfiram, with a greater potency for pyrazole. Treatment with pyrazole prior to ethanol reduced interferon production further, but only after the 7-day schedule. All drugs tested affected cellular immunity as measured by interferon production. Moreover, reduced interferon production by these agents was associated with a loss of splenic lymphoid tissue, suggesting that ethanol, pyrazole, and disulfiram could affect susceptibility to viral infection.

Studies were conducted comparing interferon production of various mouse strains. Methods used included the production of interferon in vivo and in vitro using spleen cell cultures. Strain differences observed in vivo were generally consistent with the results obtained using cell culture methods. In general, high-producing strains in vivo were also high producers using in vitro methods. Strain differences in vivo were greatest using BCG with old Tuberculin (OT) as a challenge. In general, BDF1 and C57BL/6J mice were high producers while BALB/cByJ and NZB/BLNJ were consistently low responders to both type I and type II interferon inducers. C3H/He and HA/ICR mice could be classified as intermediate interferon producers.

The ability of ascorbic acid, sodium salicylate, and caffeine to alter the circulating serum level of interferon was investigated in mice. The animals were singly injected subcutaneously with one of the compounds, 4-8 h later again singly injected intraperitoneally with poly I:C, and bled 6-8 h afterward. The sera from the mice were assayed for interferon titer by the use of the plaque inhibition method utilizing vesicular stomatitis virus. Ascorbic acid, sodium salicylate, and caffeine increased the serum level of interferon; however, the increase produced by sodium salicylate was dose-dependent, i.e. low doses increased interferon titers, high doses decreased the titers. Caffeine produced minimal increases in the interferon titer. These observations suggest that a potential prophylactic result may occur in virus infections from administration of the three compounds either singly or in combination at the proper concentration.

SummaryMorphine, Dilaudid, and methadone were injected into mice in order to assess their effect on interferon production. It was found that all three narcotic analgesics significantly reduced the level of serum interferon induced by either poly I:C or endotoxin. The level of inhibition was directly related to drug dosage. The inhibitory effect of morphine can persist at least 9 days following a single injection. Interferon levels of certain tissues particularly spleen were also depressed in morphine–treated animals. This effect on the level of interferon does not appear to be mediated through an effect on body temperature nor can it be eliminated by the morphine antagonist naloxone. These drugs were also tested on cells in vitro using poly I:C as an inducer and do not appear to have a major effect on either the induction or the action of interferon in vitro.The authors thank Dr. John J. Dallman, Department of Community Medicine, Medical College of Georgia for his assistance in the statistical treatment o...