Combinative ligand-receptor interactions: epinephrine depresses RAW264 macrophage antibody-dependent phagocytosis in the absence and presence of met-enkephalin

Macrophages have been shown to possess cell surface receptors for opiates and catecholamines. The abilities of these ligands to affect RAW264 macrophage antibody-dependent effector activity directed against sheep red blood cells were tested. Phagocytosis was measured by the uptake of 51Cr labeled erythrocytes and optical microscopy. Cytolysis was measured by 51Cr-release assays. Met-enkephalin increased specific antibody-dependent phagocytosis in a dose-dependent fashion; the optimal dose was found to be 10(-8) M. Epinephrine diminished phagocytosis in a dose-dependent manner exhibiting a maximal inhibition at 10(-4)-10(-5) M. This inhibition can be blocked by propranolol. The combined effects of simultaneous treatment with met-enkephalin and epinephrine were measured. At the several doses tested, the combined effects of these two ligands on the amount of phagocytosis were equivalent to or more inhibitory than epinephrine alone. Thioglycolate-elicited murine peritoneal macrophages demonstrated similar responses to epinephrine, met-enkephalin, and their combination. Therefore, in vitro models more closely approximating in vivo neuroregulation of macrophage function demonstrate phagocytic inhibition.     

Effects of different durations of exercise on macrophage functions in mice.

The effects of differing durations of daily exercise on macrophage functions in mice were studied. Male ICR mice aged 4 wk were divided into five groups: a nonexercise group (control) and four exercise groups with differing daily exercise durations of 15--120 min (Exr groups). The exercise applied was 5 days/wk treadmill running at 13 m/min for 12 wk. The potentiation of the phagocytosis function of the reticuloendothelial system and the glucose consumption of peritoneal macrophages in the Exr 30, 60, and 120 groups were significantly higher than those in the control group. Superoxide anion production of peritoneal macrophages in both the absence and the presence of phorbol 12-myristate 13-acetate in the Exr 60 and 120 groups was significantly higher than that in the control group. The acid phosphatase and beta-glucuronidase activities of peritoneal macrophages in the Exr 30, 60, and 120 groups were significantly increased. These results suggest that treadmill running exercise for at least 30 min/day (30--120 min) effectively enhances macrophage functions in mice. These data provide preliminary evidence indicating that chronic exercise-induced increases in phagocytic activity exhibit a dose-dependent relationship with exercise duration.     

Macrophage activation to kill Leishmania major: activation of macrophages for intracellular destruction of amastigotes can be induced by both recombinant interferon-gamma and non-interferon lymphokines

Macrophages treated with lymphokine (LK)-rich culture fluids from antigen- or mitogen-stimulated spleen cells or the hybridoma T cell 24/G1, or murine recombinant interferon-gamma (IFN-gamma) from either transfected monkey kidney cells (cos rIFN-gamma) or bacterial (E. coli) DNA (rIFN-gamma) developed the capacity to kill intracellular amastigotes of Leishmania major. Removal of IFN activity from LK by neutralizing fluid phase monoclonal anti-rIFN-gamma antibody, or by solid phase immunoadsorption, left residual macrophage activation factors that induced approximately 50% of the macrophage anti-leishmanial activity of untreated LK. In contrast, rIFN-gamma subjected to the same antibody treatments lost all capacity to induce this macrophage effector function. These results suggest that the intracellular destruction of amastigotes is regulated by several different factors. One of these factors is clearly IFN-gamma, which is pleiotropic in its effects on macrophage functions. The other non-IFN LK factors are immunochemically unrelated to IFN-gamma, and may regulate macrophage microbicidal activities in a more selective manner.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

西藏灵菇胞外多糖组分对小鼠免疫调节作用及机制的研究

摘要：【目的】研究数均分子量为0.1×105~3.0×105（组分1）及1.8×103（组分2）的西藏灵菇胞外多糖组分对正常小鼠免疫功能的影响,并探讨其影响机制。【方法】依据卫生部保健食品功能学评价程序和检验方法,灌胃给药,剂量分别120 mg/kg体重、80 mg/kg体重、40 mg/kg体重,检测脏器／体重比值、半数溶血值（HC50）、自然杀伤细胞（NK）活性、迟发型变态反应（DTH）、腹腔巨噬细胞吞噬功能。采用免疫印迹法,测定小鼠腹腔巨噬细胞中Erk蛋白及COX-2酶的表达量。【结果】组分1能够明     

Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55-65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.     

Enhancement of macrophage spreading and phagocytosis by peroxidative enzymes

Macrophages stimulated by various substances exhibit altered morphology, metabolism, and enhanced phagocytosis. The present studies were done to show if peroxidative enzymes would affect macrophage spreading and phagocytosis. Resident peritoneal macrophages, collected from C57BI mice were exposed to various concentrations of peroxidases and compared with appropriate controls. Results indicated that 0.01 microM myeloperoxidase (MyPO), 0.09 microM horseradish peroxidase (HRP), 0.16 microM lactoperoxidase (LPO) and 70 microM microperoxidase (MPO) significantly enhanced macrophage spreading. It was also noted that peroxidases were able to stimulate phagocytosis by increasing the number of cells with internalized zymosan at least twofold. Stimulation of these functions suggests a possible role of endogenous peroxidases as natural cell activators.     

Macrophages are critical effectors of antibody therapies for cancer

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.     

Activation of alveolar macrophages after lower respiratory tract infection.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.     

Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.     

Macrophages are critical effectors of antibody therapies for cancer

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.     

Regulation of tyrosine phosphorylation in macrophage phagocytosis and chemotaxis

Macrophages display a large variety of surface receptors that are critical for their normal cellular functions in host defense, including finding sites of infection (chemotaxis) and removing foreign particles (phagocytosis). However, inappropriate regulation of these processes can lead to human diseases. Many of these receptors utilize tyrosine phosphorylation cascades to initiate and terminate signals leading to cell migration and clearance of infection. Actin remodeling dominates these processes and many regulators have been identified. This review focuses on how tyrosine kinases and phosphatases regulate actin dynamics leading to macrophage chemotaxis and phagocytosis.     

Sabotage and exploitation in macrophages parasitized by intracellular protozoans

Macrophages are crucial in immunity to infection. They possess potent antimicrobial function, and efficiently process and present peptide antigens for T-cell activation. Despite this, the intracellular protozoan parasites Toxoplasma gondii, Trypanosoma cruzi and Leishmania spp. target macrophages for infection. Each has adopted unique strategies to subvert macrophage antimicrobial functions. The parasites sabotage killing activities through sophisticated manipulation of intracellular macrophage signaling pathways. These subversive activities are probably dictated by the need to evade microbicidal effector function, as well as to avoid proinflammatory pathology that can destabilize the host-parasite interaction. The molecular details of how intracellular protozoans manipulate macrophage signal transduction pathways for their own ends are beginning to emerge.     

Environmental and chemical dissociation of antibody-dependent phagocytosis from lysis mediated by macrophages: Stimulation of lysis by sulfhydryl-blocking and esterase-inhibiting agents and depression by trypan blue and trypsin

Peritoneal exudate cells and RAW264 macrophage tumor culture line were tested for antibody-dependent effector activity to sheep red blood cells (RBC). Assay in tissue culture dishes showed mostly lysis of targets while tubes promoted phagocytosis. The kinetics suggested that these were independent processes. At concentrations inhibiting ingestion, sulfhydryl-blocking agents iodoacetate, N-ethylmaleimide, and p-chloromercuribenzoate, and esterase inhibitors tosyl-lysine chloromethyl ketone, and diisopropyl fluorophosphate stimulated lysis of RBC. Pretreatment of effector cells but not targets also augmented the lytic reaction. Three other macrophage cell lines with very weak killing activity were stimulated by iodoacetate, but two lymphoid cell lines showed no cytotoxicity with or without the drug. In contrast to these enzyme inhibitors, trypan blue blocked peritoneal exudate and cell line lysis with no effect on phagocytosis. Trypsin pretreatment also inhibited RAW264 but not peritoneal cell cytotoxicity. There was little effect of these agents on macrophage Fc receptors as measured by EA rosettes, or on cell viability or production of lysozyme and β-glucuronidase. We conclude that the two macrophage effector mechanisms are independent, can be inversely modulated by environmental conditions (assay vessel), and are regulated by enzymes or proteins specific for each process.     

PSGL-1 and mTOR regulate translation of ROCK-1 and physiological functions of macrophages

Rho-associated kinases (ROCKs) are critical molecules involved in the physiological functions of macrophages, such as chemotaxis and phagocytosis. We demonstrate that macrophage adherence promotes rapid changes in physiological functions that depend on translational upregulation of preformed ROCK-1 mRNA, but not ROCK-2 mRNA. Before adherence, both ROCK mRNAs were present in the cytoplasm of macrophages, whereas ROCK proteins were undetectable. Macrophage adherence promoted signaling through P-selectin glycoprotein ligand-1 (PSGL-1)/Akt/mTOR that resulted in synthesis of ROCK-1, but not ROCK-2. Following synthesis, ROCK-1 was catalytically active. In addition, there was a rapamycin/sirolimus-sensitive enhanced loading of ribosomes on preformed ROCK-1 mRNAs. Inhibition of mTOR by rapamycin abolished ROCK-1 synthesis in macrophages resulting in an inhibition of chemotaxis and phagocytosis. Macrophages from PSGL-1-deficient mice recapitulated pharmacological inhibitor studies. These results indicate that receptor-mediated regulation at the level of translation is a component of a rapid set of mechanisms required to direct the macrophage phenotype upon adherence and suggest a mechanism for the immunosuppressive and anti-inflammatory effects of rapamycin/sirolimus.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

Peritoneal mast cell degranulation differently affected thioglycollate-induced macrophage phenotype and activity in Dark Agouti and Albino Oxford rats

Aims

Macrophages are heterogeneous population of inflammatory cells and, in response to the microenvironment, become differentially activated. The objective of the study was to explore macrophage effector functions during different inflammatory conditions in two rat strains.

Main methods

We have investigated the effects of in vivo treatment with mast cell-degranulating compound 48/80 and/or thioglycollate on peritoneal macrophage phagocytosis and capacity to secrete hydrogen peroxide (H₂O₂), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in Dark Agouti (DA) and Albino Oxford (AO) rat strains. Besides, fresh peritoneal cells were examined for the expression of ED1, ED2 and CD86 molecules.

Key findings

In thioglycollate-elicited macrophages, increased proportion of ED1 + cells was accompanied with elevated phagocytosis of zymosan (DA strain), whereas increased expression level of CD86 molecule on ED2 + macrophages matched elevated secretory capacity for H₂O₂, TNF-α and NO (AO rats). Although mast cell degranulation induced by compound 48/80 increased the percentages of ED2 + macrophages in both rat strains, the proportion of ED2 + cells expressing CD86 molecule was decreased and increased in DA and AO rats, respectively. Furthermore, in DA strain compound 48/80 diminished macrophage secretion of NO, but stimulated all macrophage functions tested in AO strain. If applied concomitantly, the compound 48/80 additively increased macrophage activity induced by thioglycollate in AO rats.

Significance

Macrophages from DA and AO rat strains show different susceptibility to mediators released from mast cells, suggesting that strain-dependant predisposition(s) toward particular activation pattern is decisive for the macrophage efficacy in response to inflammatory agents.     

Characterization of accessory cell costimulation of Th1 cytokine synthesis.

We studied the capacity of macrophage and B cell lines to provide a costimulatory signal that enhances synthesis of IFN-gamma and IL-2 by mouse Th1 clones stimulated with suboptimal doses of immobilized anti-CD3 antibody. The J774 macrophage line and the CH27 B lymphoma line had the greatest costimulatory activity and routinely increased IL-2 production by 10-fold to 100-fold. Other macrophage and B cell lines had less activity and T cell lines were unable to costimulate. The J774 and CH27 lines did not costimulate IL-4 production by a Th2 clone and had only a small effect on IL-2 production by T cell hybridomas. The process of costimulation was fixation-sensitive, contact-dependent and did not involve stable cytokines present in the T cell/accessory cell conditioned media. Neutralizing antibodies for IL-1, IL-6, and TNF failed to inhibit costimulation. Antibodies to the LFA-1/ICAM-1 pair of adhesion molecules also failed to inhibit. Costimulation of IL-2 production by accessory cells was found to have a unidirectional species restriction: mouse accessory cells costimulated mouse and human IL-2-producing T cells, but human U937 cells induced with PMA were effective only for human T cells. The results indicate that accessory cells can significantly regulate Th1 effector function at the level of cytokine production.     

In Vitro Induction of Antibody-Dependent Cytotoxic Macrophages by the Local Anesthetic Lidocaine

Normal macrophages were activated to antibody-dependent cytotoxic effector cells by in vitro treatment with the local anesthetic lidocaine. Experiments on the dose-response and time course of the effect of lidocaine showed that incubation of normal macrophages with 10 mM lidocaine for 10 min at 28 C was enough for induction of antibody-dependent cellular cytotoxicity. The activation by lidocaine was accompanied by enhanced phagocytosis of sheep red blood cells (SRBC) sensitized with anti-SRBC antiserum, but not enhanced ingestion of polystyrene latex particles (PLP). These findings suggest that lidocaine, which has various effects on cell membranes, induces some perturbation of macrophage membranes, resulting in activation of Fc receptor functions in antibody-dependent cytotoxicity and phagocytosis.     

Kojic acid, a secondary metabolite from Aspergillus sp., acts as an inducer of macrophage activation

KA (kojic acid) is a secondary metabolite isolated from Aspergillus fungi that has demonstrated skin whitening, antioxidant and antitumour properties among others. However, limited information is available regarding its effects on macrophages, the major cell involved in cell defence. The aim of the present study was to analyse whether KA affects functional properties related to macrophage activation, such as phagocytosis and spreading ability over a substrate. Treatment of resident macrophages with 50 μg/ml KA for 1 h induced both morphological and physiological alterations in cells. Immunofluorescence microscopy revealed enhanced cell spreading and an increase in cell surface exposure, associated with a rearrangement of microtubules, actin filaments and intermediate filaments. KA also potentiated phagocytosis by macrophages, as demonstrated by the increase in phagocytic activity towards yeast, when compared to untreated cells. KA increased the production of ROS (reactive oxygen species), but not NO (nitric oxide) production. Three tests were used to assess cell viability; MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NR (neutral red) uptake and PI (propidium iodide) exclusion test, which showed that macrophages maintain their viability following KA treatment. Results indicate that KA can modulate macrophage activation through cytoskeleton rearrangement, increase cell surface exposure, enhance the phagocytic process and ROS production. The study demonstrates a new role for KA as a macrophage activator.