Echinococcus multilocularis: inhibition of murine neutrophil and macrophage chemotaxis

Resident peritoneal neutrophils and macrophages from mice infected with 50 +/- 5 cysts of Echinococcus multilocularis were collected at 2, 4, 6, 8, 10, and 16 weeks postinfection. Their ability to respond and migrate to purified parasite larval antigens or endotoxin-activated mouse serum (EAMS) in comparison to normal peritoneal cells from uninfected mice was tested in vitro using Boyden chambers. Early in the infection, both cell types responded to the specific and nonspecific chemoattractants as the control group. However, at 8 and 10 weeks postinfection, the neutrophils and macrophages lost their response to parasite antigens but retained their ability to migrate to EAMS. Toward the 12th and 16th week postinfection, both cell types lost their ability to migrate to the specific as well as the nonspecific factors. The data presented suggest that the cellular mechanisms of recognition and chemotaxis in mice infected with alveolar hydatid cysts are impaired.     

Leishmania mexicana mexicana: attachment and uptake of promastigotes to and by macrophages in vitro

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania mexicana mexicana: Attachment and Uptake of Promastigotes to and by Macrophages In Vitro1

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and glutaraldehyde sensitive. The promastigote ligand is proteolytic enzyme and glutaraldehyde insensitive. Uptake follows attachment and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages. The first sub-population to be taken up survived better in macrophages over 24 h than subsequently engulfed sub-populations.     

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

Factors regulating the metabolism of low-density lipoprotein-proteoglycan complex in macrophages

We studied the factors regulating the metabolism of low-density lipoprotein (LDL)-proteoglycan complex, LDL and acetyl-LDL in mouse peritoneal macrophages. Macrophage conditioned medium stimulated the degradation of LDL-proteoglycan complex and acetyl-LDL in a dose-dependent manner and enhanced cholesteryl ester synthesis mediated by these ligands. The conditioned medium had no such effect in a cell-free system. The conditioned medium enhanced the degradation of both the LDL and proteoglycan components of the complex. The degradation of LDL was not affected by the conditioned medium. The active factor in the conditioned medium was labile to boiling, suggesting that it may be protein in nature. The conditioned medium also lost its stimulatory activity after dialysis through a membrane with an exclusion limit of 25,000 daltons, suggesting the involvement of cytokines and/or other growth factors. Macrophage activation was accompanied by a 2-3-fold increase in the degradation of LDL-proteoglycan complex and acetyl-LDL as compared to the degradation of these ligands in resident macrophages; however, this had no effect on LDL degradation. The degradation of all three ligands increased markedly with decreasing cell density. Preincubation of macrophages for 48 h with increasing concentrations of fetal bovine serum produced a substantial increase in the subsequent degradation of LDL-proteoglycan complex and acetyl-LDL, while it had very little effect on the degradation of LDL. The active factor in serum was destroyed by boiling, suggesting that it may be a protein. These results show that the scavenger receptor, mediating the uptake and degradation of LDL-proteoglycan complex and acetyl-LDL and LDL receptor are regulated differently in mouse peritoneal macrophages.     

Macrophage binding and the uptake of oxidized low density lipoprotein are regulated by intracellular protein phosphorylation

The involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosin light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.     

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.     

Complement is an essential component of the immune response to adeno-associated virus vectors

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-κB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1β (IL-1β), IL-8, and MIP-1β. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.     

Induction of tissue transglutaminase in mouse peritoneal macrophages

Tissue transglutaminase accumulates rapidly and to very high levels (1-2% of cellular protein) in mouse peritoneal macrophages cultured in mouse serum. The induction is due to accelerated synthesis of the enzyme (150-fold increase) that occurs within 90 min of exposure of the cells to a heat-labile constituent of serum or plasma. The induction is reversible and is not reproduced by known activators of macrophage function such as lipopolysaccharide, muramyl dipeptide, and tuftsin. In animals, elevated levels of tissue transglutaminase are also found in inflammatory macrophages elicited by thioglycolate broth.     

Human receptor for measles virus (CD46) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon

Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-gamma). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-alpha/beta blocked NO production. Antibodies against IFN-alpha/beta also inhibited the augmenting effect of MV on IFN-gamma-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-gamma alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-alpha/beta. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-alpha/beta upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-alpha/beta production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-alpha/beta abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-alpha/beta in response to MV infection, and IFN-alpha/beta synergizes with IFN-gamma to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

Interaction of high density lipoproteins with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages

Morphological and biochemical experiments were carried out to investigate the interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages. It is demonstrated that resident mouse peritoneal macrophages express HDL receptors. Subsequent to receptor-mediated binding, HDL are internalized and intracellularly transported into endosomes. These endosomes do not fuse with the lysosomal compartment but interact with the margin of intracellular plasma lipid droplets. Macrophages do not degrade, but rather resecrete internalized HDL particles as described for the transferrin-receptor pathway. HDL binding to freshly isolated macrophages is saturable at a concentration of approximately 320 ng HDL-protein/mg cell protein and a Scatchard plot indicates the presence of some 130 000-190 000 receptors/cell with a Kd of approximately 9 X 10(-7) M. Binding of HDL on the macrophage surface is significantly enhanced in cholesterol-laden macrophages, whereas the increase in the rate of uptake and secretion is less pronounced. Within the HDL fraction the HDL2 subclass showed higher binding, uptake and secretion activity as compared with HDL3. From these experimental data we postulate that cholesterol uptake from macrophages is mediated by HDL particles which interact with these cells via a receptor-mediated retroendocytosis pathway.     

Tumoricidal activation of murine alveolar macrophages by muramyldipeptide substituted mannosylated serum albumin

Rat and mouse alveolar macrophages have almost no spontaneous tumoricidal activity and are only slightly activated by muramyldipeptide (MDP). When MDP was carried by serum albumin, the activation was higher than with free MDP but only at high concentration. When MDP was bound to a neoglycoprotein (mannosylated serum albumin) - which binds to the sugar binding receptor at the macrophage cell surface and is actively endocytosed - the activation of rat or mouse alveolar macrophages is dramatically enhanced even at very low concentration of neoglycoprotein -bound MDP. Furthermore, neoglycoprotein -bound MDP injected i.v. or i.p. was found to be able to activate alveolar macrophages, the activity of which was maximal after 48 hours in mice and 72 hours in rats. Such conjugates have so potential values as new immunostimulant agents in cancer and parasite therapy.     

Enhanced interleukin 1 (IL-1) production mediated by mouse serum amyloid P component

Purified serum amyloid P component (SAP), the major acute-phase reactant of mice, induces enhanced interleukin 1 (IL-1) production by elicited monocytes/macrophages in vitro. SAP also enhanced IL-1 elaboration by macrophages from lipopolysaccharide (LPS)-low responder mice and in the presence of polymyxin B, indicating that the small amounts of LPS present in the SAP preparation did not augment IL-1 production. Concentrations of SAP of 0.1 to 10.0 micrograms/ml enhanced IL-1 production by elicited and bacillus Calmette-Guerin (BCG)-activated peritoneal macrophages, but not by resident peritoneal macrophages. The inflammation-induced monocyte/macrophage population displayed selective binding of SAP. The mouse macrophage line P388D1, also could bind SAP and display enhanced IL-1 production in response to SAP. SAP did not bind to the macrophage cell line RAW264.7 nor did it enhance IL-1 secretion by this line. The results suggest that this acute-phase reactant has the potential to enhance inflammatory and immunological events mediated by IL-1.     

Biological activities of mouse haptoglobin elicited by administration of an antitumor polysaccharide,PSK

The concentration of mouse haptoglobin in serum was increased by administration of an antitumor polysaccharide, PSK. The administration of the purified mouse haptoglobin inhibited the growth of Sarcoma-180 cells implanted in ICR mice. Furthermore, this glycoprotein enhanced macrophage activitiesin vitro, as judged from the cytostatic and cytolytic activities, glycose consumption, O₂-production, and interleukin-1 production of macrophages. In addition, mouse haptoglobin enhanced the cytolytic effect of cytotoxic T-lymphocytes. These results suggested that haptoglobin has a role in restoring or enhancing the resistance of the host against tumors.Abbreviations FCS fetal calf serum - LPS lipopolysaccharide - CTL cytotoxic T-lymphocytes - IL-1 interleukin 1 Part of this work has been presented at the 14th International Congress of Chemotherapy, Kyoto, Japan, June, 1985.     

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Selectivity in endocytosis of serum and cytosol proteins by macrophages in culture

It was found that cultured mouse macrophages preferentially endocytose and degrade short half-life serum and cytosol proteins. With increasing duration of endocytosis, the components of longer half-life are increasingly endocytosed and degraded. The relevance of these observations to the selectivity of protein turnover is discussed.     

Measurement of cholesterol bidirectional flux between cells and lipoproteins

We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.     

Role of a serum factor in enhancement of in vitro interactions between Plasmodium berghei sporozoites and hamster peritoneal macrophages

Interactions between Plasmodium berghei sporozoites and hamster peritoneal macrophages were studied. Hamster serum was shown to enhance the percentage of sporozoites tha attached to macrophages, thus confirming previous studies by other workers using mouse macrophages and mouse serum. The enhancement factor within hamster serum was concentrated by a fractionation procedure consisting of ammonium sulfate precipitation followed by Concanavalin A-Sepharose affinity chromatography. This serum fraction has been shown previously to contain component(s) that bind to P. berghei sporozoites.