Doris L. Lefkowitz

Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone

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.

Macrophage-neutrophil interaction: A paradigm for chronic inflammation revisited

Macrophages have been described as ‘factories’ of pro‐inflammatory cytokines. Several years ago the present investigators reported that binding of inactive myeloperoxidase (iMPO) to the macrophage‐mannose receptor resulted in the induction of TNF and other cytokines. Also, if endothelial cells were incubated with iMPO, but not enzymatically active myeloperoxidase (MPO), upregulation of cytokine mRNA and cytokines was observed. Taken in their entirety, the data suggest a dichotomy of function for myeloperoxidase; that is, enzymatically active MPO functions primarily in cell killing through the ‘cytotoxic triad’ and iMPO functions as an immunoregulatory molecule through the induction of numerous cytokines. These studies underscore a previously unrecognized interaction among neutrophils, endothelial cells and macrophages, resulting in the induction of TNF and perpetuation of inflammation. The inflammation induced could be relevant in a number of diseases in which neutrophils play a prominent role. The importance of this interaction in the pathogenesis of rheumatoid arthritis is currently under investigation.

Upregulation of phagocytosis and candidicidal activity of macrophages exposed to the immunostimulant, acemannan

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.

Macrophage Activation and Immunomodulation by Myeloperoxidase

Abstract Myeloperoxidase (MyPo) is an enzyme found in neutrophils and monocytes that plays an important role in the microbicidal and cytocidal activities of these cells. The present studies show that this enzyme can also affect both capacities and functions of macrophages. When resident peritoneal macrophages from C57BL/6 mice were exposed to preparations of either human or canine enzyme in vitro, tumor necrosis factor (TNF) was released. The amount of TNF produced was dose dependent and could be neutralized with polyclonal anti-TNF. Low levels of interferon were also produced by these cells. In addition, exposure of murine macrophages in vitro to this enzyme resulted in increased ability to destroy 3T12 target cells. Intravenous injection of mice with myeloperoxidase induced the production of both TNF and interferon, which could be detected in the sera. Possible mechanisms of TNF induction include radical production by myeloperoxidase or ligand-receptor interaction by the binding of this enzyme to the mannosyl-fucosyl receptor. These results, when taken in their entirety, suggest that this enzyme can modulate the immune response through effects on macrophage function.

Regulation of macrophage function by human recombinant myeloperoxidase

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.

[37] Activation of macrophages with oxidative enzymes

Publisher Summary This chapter describes two assay methods used for activating macrophages with peroxidases. The first one employs a target cell cytostasis assay, and the second determines the increase in the “oxidative burst” as measured by the increase in superoxide production, using the chemiluminescence of luminol as the indicator. The microassay for target cell toxicity is different from other cytotoxicity assays as it does not require the presence of a radioactive marker. A fixed number of target cells are exposed to activated macrophages for a period of 48 hours; and at the end of this time, the cells are stained with a dye. The amount of stain present in wells containing target cells and activated macrophages is then compared with the amount of stain present in wells containing target cells with nonactivated macrophages. One of the functions of phagocytic cells is to ingest and kill microorganisms. These cells emit light while ingesting particles. Light arises from the production of high-energy compounds produced during bactericidal activity. This spontaneous luminescence can be used as an assay of phagocytic function or as a sensitive means to quantitate microbicidal metabolic activity.

Exposure of macrophages to an enzymatically inactive macrophage mannose receptor ligand augments killing of Candida albicans.

Abstract Macrophages (Mφ) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mφ 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 Mφ-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mφ 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 ≤ 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 Mφ via ROI, and that iMPO which is released at a site of inflammation induces Mφ-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

MACROPHAGE-MEDIATED CANDIDACIDAL ACTIVITY IS AUGMENTED BY EXPOSURE TO EOSINOPHIL PEROXIDASE: A Paradigm for Eosinophil-Macrophage Interaction

Various disease states are associated with eosinophilia and the release of eosinophil peroxidase (EPO) into the microenvironment. The present study targets the effects of low levels of EPO on macrophage (MØ) phagocytosis and intracellular killing of Candida albicans as well as MØ oxidative activity measured as the luminescence product of luminol dioxygenation. Resident murine peritoneal MØ were exposed to various concentrations of EPO. Chemiluminescence data indicate that nanomolar concentrations of EPO markedly enhanced the dioxygenation activity (respiratory burst) of MØ. In other studies, the exposure of MØ to 0.17 μM EPO for 10 min. enhanced MØ-mediated candidacidal activity 10 fold. The above data indicate that EPO enhances certain MØ functions. Also the results illustrate a previously un-recognized interaction between eosinophils and MØ and implicate yet another possible role for EPO in host defenses against disease.

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.

Cocaine reduces macrophage killing by inhibiting reactive nitrogen intermediates

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.