Phagocytosis In Mouse Macrophages Research Articles

Immune regulatory functions of biologically active proteins from edible fungi.

Proteins from edible mushrooms have a variety of biological activities. Here, thirteen precious edible mushrooms such as Ophiocordyceps sinensis, Ganoderma lucidum, and Morchella esculenta and nine common edible mushrooms such as Flammulina velutipes, Pleurotus ostreatus, and Pleurotus eryngii, etc., from which their proteins were extracted, their composition analyzed and their immunomodulatory activity assessed. Rare mushrooms are a species of edible mushrooms with higher edible value and medicinal value than common edible mushrooms. The results showed that all the different edible mushroom crude proteins increased the proliferation and phagocytosis of mouse macrophages, and we found that these edible mushroom proteins affected the secretion of reactive oxygen species and nitric oxide by mouse macrophages. Further studies on cytokines secreted by mouse macrophages showed a significant increase in pro-inflammatory cytokines, suggesting that edible mushroom proteins promote the polarisation of macrophages into classical M1-type macrophages, further demonstrating that edible mushroom proteins enhance immunity. It was also found that the immunomodulatory activity of the precious edible mushroom proteins was significantly higher than that of the common edible mushroom proteins. These results have important implications for the processing and product development of edible mushroom proteins.

Effects of Rhodotorula mucilaginosa on the Immune Function and Gut Microbiota of Mice.

As a dominant species among marine yeasts, Rhodotorula benthica accounts for ~50% of all marine yeasts. Rhodotorula is rich in a variety of bioactive substances and commonly used in the production of carotenoids by microbial fermentation and is worth developing. Therefore, the present study used a strain of Rhodotorula mucilaginosa isolated from the coastal waters of the South China Sea as the target yeast to investigate its impact on the immune function and gut microbiota of mice. A total of 200 mice were randomly divided into gavage groups and control group and garaged for 30 consecutive days at different concentration. Samples were collected on day 15 and day 30 of gavage administration. The results showed that R. mucilaginosa ZTHY2 could increase the thymus and spleen indices of mice, and its effect on the thymus index was more significant after long-term gavage administration. Short-term (15 days) gavage administration of R. mucilaginosa suspension enhanced delayed hypersensitivity in mice, increased serum IgG, IgA, and IL-2. Long-term (30 days) gavage administration of R. mucilaginosa suspension significantly enhanced the phagocytosis of macrophages in mice and significantly increased serum TNF-α and INF-γ. R. mucilaginosa ZTHY2 altered the structure of the gut microbiota of mice at the phylum and genus levels, leading to an increased relative abundance of Firmicutes and Lactobacillus and a decreased relative abundance of Bacteroidetes. This strain increased the beneficial intestinal bacteria and reduced the harmful intestinal bacteria in mice. This study provides experimental evidence and lays the foundation for the future development and application of this strain as a microecological source of carotenoids.

Impact of Selenium Deficiency on Inflammation, Oxidative Stress, and Phagocytosis in Mouse Macrophages.

Although it has been reported that selenium (Se) deficiency can trigger inflammation, however, there are few reports on the effect of Se on the function of mouse peritoneal macrophages. Herein, we examined the expression of inflammatory factors, oxidative stress levels, and phagocytosis for primary-cultured peritoneal macrophages using control and Se-deficient groups. Our results revealed that Se deficiency induced the accumulation of oxygen free radicals and weakened antioxidant capacity. Se deficiency also significantly increased the expression of inflammation factors including iNOS, IL-1β, IL-12, IL-10, PTGe, and NF-κB. Meanwhile, Se suppression restrained macrophage production of TNF-α. The results of the phagocytosis assay demonstrated that Se deficiency inhibited the phagocytosis of macrophages. In conclusion, Se-deficient macrophages undergo severe inflammation through the NF-κB pathway due to the accumulation of oxygen free radicals and are hindered in their phagocytic capacity.

Purification, characterization and biological activity of a novel polysaccharide from Inonotus obliquus

A novel water-soluble polysaccharide IP3a was successfully isolated and purified from I. obliquus by DEAE-cellulose, Sepharose CL-6B and Sephadex G-200 column chromatography. Chemical characterization and antitumor and immunoregulatory activity of IP3a were investigated. IP3a consisted of rhamnose, arabinose, glucose and galactose in a molar ratio of 2.5:4.6:1.0:2.6 with an average molecular weight of 48,820Da. IP3a exhibited no significant antitumor activities in vitro. However, IP3a could not only inhibit the growth of transplantable Jurkat tumor in mice significantly, but also could enhance the splenocyte proliferation and lymphocyte proliferation induced by ConA and LPS in a dose-dependent manner. At the same time, IP3a could promote cytokine secretion (IL-2, IL-6, IL-12 and TNF-α) and macrophage phagocytosis in mice. In addition, IP3a could increase Bax expression and inhibit Bcl-2 expression significantly. These results suggested that antitumor mechanisms of IP3a might be associated with improving immune response in vivo and inducing apoptosis of tumor cells in vitro. IP3a might be utilized as a potential therapeutic agent against lymphoma cancer with immunomodulatory activity.

Lactobacillus plantarum HY7712 Ameliorates Cyclophosphamide-Induced Immunosuppression in Mice

Lactic acid bacteria (LAB) in fermented foods have attracted considerable attention recently as treatment options for immune diseases, the incidence of which has been increasing worldwide. The ability of 500 strains of LAB, isolated from kimchi, to induce TNF--α production in peritoneal macrophages was investigated. Lactobacillus plantarum HY7712 most strongly induced TNF--α production as well as NF-κB activation. However, HY7712 inhibited NF-κB activation in LPS-stimulated peritoneal macrophages. When HY7712 was orally treated in cyclophosphamide (CP)-immunosuppressed mice for 5 or 15 days, it reversed the body and spleen weights, blood RBC and WBC levels, and splenocyte and bone marrow cells that were reduced by CP. Orally administered HY7712 increased concanavalin A-induced T cell proliferation to 84.5% of the normal group on day 15, although treatment with CP alone markedly reduced it to 53.7% of the normal group. Furthermore, orally administered HY7712 significantly induced the expressions of IL-2 and IFN-γ in ConA-induced splenic cytotoxic T cells of CP-treated mice. Orally administered HY7712 restored the CP-impaired phagocytosis of macrophages in mice. Orally administered HY7712 also restored the cytotoxicity of NK and cytotoxic T cells derived from spleen and bone marrow against YAC-1 in CP-immunosuppressed mice. Based on these findings, orally administered HY7712 may accelerate the recovery of cyclophosphamide-caused immunosuppression, without evident side effects, by immunopotentiating NK and Tc cells, and may provide a mechanistic basis for using HY7712 as an alternative means in lessening chemotherapyinduced immunosuppression in cancer patients.

Encapsulation efficiency of coenzyme Q10‐liposomes in alginate

PurposeThe purpose of this study is to evaluate the effectiveness of alginate as a vehicle to protect coenzyme Q10 in liposomes.Design/methodology/approachEncapsulation efficiency and stability were conducted at varying temperatures (20, 30, 40°C) for 5 d and at exposure to simulated gastric conditions (pH 2) for 2 h. The content of coenzyme Q10 was determined using HPLC (LC/MS). Cytotoxicity and phagocytosis of mouse macrophages (RAW264.7) was determined.FindingsResults showed that thermostability was strongly improved by alginate complex formation with liposomes. Moreover, alginate could maintain coenzyme Q10 at a significantly higher level in simulated gastric pH for at least 2 h (p<0.00).Practical implicationsThis allowed a higher amount of coenzyme Q10 remaining to be absorbed in the small intestine. Alginate not only showed no toxic effect on mouse macrophages but also activated their proliferation and phagocytosis ability.Originality/valueAs a consequence, alginate could be applied as an aid to encapsulation stability and immunostimulating potency.

Commercial naphthenic acids and the organic fraction of oil sands process water induce different effects on pro‐inflammatory gene expression and macrophage phagocytosis in mice

Naphthenic acids (NAs) are believed to be the major toxic component of oil sands process water (OSPW). Different OSPW preparations have distinct NA compositions, and additional organics, that differ from the commercial NAs (C-NAs) often used for toxicology studies. To evaluate whether C-NAs are an adequate model to study OSPW toxicity in complex organisms, we compared the effects of C-NAs and the extractable organic fraction of OSPW (OSPW-OF) on mice immune mechanisms. Mice were orally exposed to different C-NA doses, or OSPW-OF at the same NA dose, for up to 8 weeks, and the expression of pro-inflammatory genes in different organs was determined using quantitative PCR. C-NAs and OSPW-OF altered the expression of pro-inflammatory genes, inducing either expression down-regulation or up-regulation, depending on the organ examined and time after exposure. The time at which gene expression alterations occurred, and the specific sets of genes whose expression was altered, were very different between animals exposed to C-NAs or to OSPW-OF. We evaluated the ability of mouse peritoneal macrophages to phagocytose yeast cell wall, as a measure of the ability of mice to mount a central function of the innate immune response. Phagocytosis was significantly reduced in animals exposed to C-NAs, but enhanced in mice exposed to OSPW-OF. Our results indicate that studies using C-NAs may not necessarily reflect the possible effects induced in animals by process water from tailing ponds.

Studies on the immunomodulatory activity of flavonoidal fraction of Tephrosia purpurea

The flavonoid fraction of Tephrosia purpurea (FFTP) was studied for its effect on cellular and humoral functions and on macrophage phagocytosis in mice. Oral administration of FFTP (10–40 mg/kg) significantly inhibited sheep red blood cells (SRBC)-induced delayed-type hypersensitivity reactions. It also produced a significant, dose-related decrease in sheep erythrocyte-specific haemagglutination antibody titre. However, the fraction failed to show a significant change in the macrophage phagocytic activity. The results obtained indicate the ability of the flavonoidal fraction of T. purpurea to modulate both the cell-mediated and the humoral components of the immune system.

PAG3/Papalpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fcgamma receptor-mediated phagocytosis of macrophages.

The Fcgamma receptor (FcgammaR)-mediated phagocytosis of macrophages is a complex process where remodeling of both the actin-based cytoskeleton and plasma membrane occur coordinately. Several different families of small GTPases are involved. We have isolated a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activity (PAG)3/Papalpha/KIAA0400, from mature monocytes and macrophage-like cells. Mammalian ARFs fall into three classes, and the class III isoform (ARF6) has been shown to be involved in FcgammaR-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocytic cups formed beneath immunoglobulin G-opsonized beads in P388D1 macrophages, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the FcgammaRs were not affected. Other ubiquitously expressed ARFGAPs, G protein-coupled receptor kinase interactors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP activity for ARF1 in COS-7 cells, did not accumulate at the phagocytic cups or inhibit phagocytosis. Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells. We propose that PAG3 acts as a GAP for ARF6 and is hence involved in FcgammaR-mediated phagocytosis in mouse macrophages.

Enhancement of phagocytosis in mouse macrophages by pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides on phagocytosis of fluorescent latex beads by mouse peritoneal macrophages were examined using flow cytometry (FCM). PACAP38, PACAP27 and vasoactive intestinal peptide (VIP) enhanced phagocytosis in a dose-dependent manner. Relative potencies of related peptides at a concentration of 10−6M were PACAP38 > PACAP27 > VIP > secretin > glucagon > (peptide with NH2-terminal histidine and COOH-terminal methionine amide, in short PHM). PACAP(6-38) was as effective as PACAP38. PACAP(6-27) enhanced phagocytosis more effectively than did PACAP27. PACAP(28-38) slightly enhanced phagocytosis. The present result suggest that PACAP38 is one of the mediators that the nervous system uses to modulate the immune system.

Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages.

Fc receptor-mediated phagocytosis in mouse macrophages occurs by a tyrosine kinase-dependent pathway (Greenberg, S., Chang, P., and Silverstein, S.C. (1993) J. Exp. Med. 177, 529-534). To identify proteins that are phosphorylated on tyrosine residues during phagocytosis, we used anti-phosphotyrosine antibodies to perform immunoblotting and immunoprecipitation of lysates derived from Fc receptor-stimulated macrophages. Proteins of 26, 30, 35, 37, 40, 43, 47, 56, 60, 68, 83, 116, and 150 kDa displayed enhanced tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Tyrosine phosphorylation of these proteins was not a consequence of actin polymerization since treatment with cytochalasin D did not alter the pattern of Fc receptor-stimulated protein tyrosine phosphorylation. The 68-kDa tyrosine phosphoprotein was identified as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions (Turner, C.E., Glenney, J.R., and Burridge, K. (1990) J. Cell Biol. 111, 1059-1068). Paxillin colocalized with F-actin beneath nascent phagosomes. In addition to the above proteins detected by anti-phosphotyrosine immunoblotting, the gamma subunit of FcRI and III was shown to undergo tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Of several candidate tyrosine kinases that may be activated during Fc receptor stimulation, p72syk, but not p125FAK, displayed enhanced tyrosine phosphorylation during Fc receptor aggregation. The coordinated tyrosine phosphorylation of the gamma subunit of macrophage Fc receptors, the tyrosine kinase syk, and the cytoskeletal-associated protein, paxillin, may be important steps in integrating signals between Fc receptors and the underlying cytoskeleton.

Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages.

Although Fc receptor-mediated phagocytosis is accompanied by a variety of transmembrane signaling events, not all signaling events are required for particle ingestion. For example, Fc receptor-mediated phagocytosis in mouse inflammatory macrophages (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106:657; Greenberg, S., J. El Khoury, F. Di Virgilio, and S. C. Silverstein. 1991. J. Cell Biol. 113:757) and neutrophils (Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. J. Immunol. 144:1411) occurs in the absence of cytosolic calcium transients. We sought to identify transmembrane signaling events that are essential for phagocytosis. Here we show that tyrosine phosphorylation is an early event after Fc receptor ligation in mouse inflammatory macrophages, and that the formation of tyrosine phosphoproteins coincides temporally with the appearance of F-actin beneath phagocytic cups. The distribution of tyrosine phosphoproteins that accumulated beneath phagocytic cups was punctate and corresponded to areas of high ligand density on the surface of the antibody-coated red blood cells, which provided the phagocytic stimulus. A tyrosine kinase inhibitor, genistein, but not several inhibitors of protein kinase C, blocked the appearance of tyrosine phosphoproteins as assessed by immunofluorescence, the focal accumulation of F-actin beneath immunoglobulin G-opsonized particles, and the ingestion of these particles as well. We suggest that tyrosine phosphorylation is a critical signaling event that underlies Fc receptor-mediated phagocytosis in mouse macrophages, and is necessary for the engulfment per se.

Ca(2+)-independent F-actin assembly and disassembly during Fc receptor-mediated phagocytosis in mouse macrophages.

Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.

Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages.

We have studied the distribution of talin in J774 cells and mouse peritoneal macrophages undergoing Fc receptor-mediated phagocytosis. At early stages of phagocytosis, talin accumulates in the cells' cortical cytoplasm adjacent to the forming phagosome and extends into pseudopods that are encircling the particle. Talin colocalizes with F-actin at these sites. After particle ingestion is completed, F-actin and talin are no longer concentrated adjacent to phagosomes. Thus, talin and F-actin undergo dynamic and coordinate changes in their cytoplasmic location during Fc receptor-mediated phagocytosis.

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages.

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.