Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

Latent lymphokines: isolation of guinea pig latent lymphocyte-derived chemotactic factor for monocytes. Its activation by trypsin and a soluble factor from macrophages.

Guinea pig lymphocytes when depleted of macrophages and stimulated by the T cell mitogen phytohemagglutinin produce a latent form of lymphocyte-derived chemotactic factor for monocytes (LDCF-M). Latent LDCF-M is also produced when immune lymphocytes are stimulated in vitro with specific antigen, horseradish peroxidase. Latent LDCF-M from both sources can be activated and converted to "classical" LDCF-M by trypsin and by a soluble factor obtained from sonicated macrophages. These observations suggest that macrophages may modulate lymphokine activities in vivo by releasing soluble factors that convert inactive latent lymphokines to biologically active substances.     

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.     

Stimulation of guinea pig macrophage pinocytosis by lipopolysaccharides (LPS): Evidence that LPS acts directly on the macrophage

Bacterial lipopolysaccharide can enhance the pinocytosis of horseradish peroxidase in guinea pig macrophages. This increased uptake can be seen as early as 6 hr after incubation in a spinner suspension culture. An equivalent amount of enhanced pinocytosis can be seen if plated, washed peritoneal exudate macrophages are used. Plated guinea pigs PECs, washed five times, were examined for the presence of B cells, and none were found. Thus, LPS appears to stimulate the macrophage directly and does not require a lymphocyte intermediary. The active stimulatory moiety appears to be lipid A, which can be blocked by preincubation of LPS with polymyxin B.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

A quantitative assay for mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors using plasma clots

Cytochemical procedures were used to identify and quantitate granulocyte and macrophage precursors from mouse bone marrow cells in plasma clot cultures. Excellent clonal morphology and cellular enzyme activity were obtained when using plasma clots as the support matrix and buffered formalin acetone as the fixative. For cytochemical identification, naphthol AS acetate esterase staining was used for macrophages and peroxidase for granulocytes. These enzyme properties were confirmed by inactivation studies with a variety of inhibitors, group specific chemical modifications, and pinocytotic affinity for horseradish peroxidase. When mouse bone marrow cells (3 X 10(4) cells/dish) were cultured in plasma clots with human placental or L-cell-conditioned medium, 70 to 110 colonies were produced. Both pure granulocyte (CFU-g) and pure macrophage colonies (CFU-m) were observed, but approximately 5% of the total colony number was composed of mixed granulocyte/macrophage colonies (CFU-gm). The number of plated cells correlated strongly with the colony number (0.990 less than r less than 0.999).     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine.

Although nicotine is thought to be one of the major immunomodulatory components of cigarette smoking, how nicotine alters the host defense of the lung and, in particular, immune responses of alveolar macrophages, which are critical effector cells in the lung defense to infection, is poorly understood. Nicotinic acetylcholine receptors (nAChRs) are the receptor for nicotine and may be involved in the modulation of macrophage function by nicotine. In this study, therefore, nicotine-induced suppression of antimicrobial activity and cytokine responses of alveolar macrophages mediated by nAChRs to Legionella pneumophila, a causative agent for pneumonia, were examined. The murine MH-S alveolar macrophage cell line cells expressed the messages for alpha4 and beta2 subunits of nAChRs, but not alpha7 subunits, determined by RT-PCR. The nicotine treatment of MH-S alveolar macrophages after infection with L. pneumophila significantly enhanced the replication of bacteria in the macrophages and selectively down-regulated the production of IL-6, IL-12, and TNF-alpha, but not IL-10, induced by infection. These effects were completely blocked by a nonselective antagonist, d-tubocurarine, for nAChRs, but not by a selective antagonist, alpha-bungarotoxin, for alpha7-nAChRs. Furthermore, the stimulation of nAChRs with another agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, showed the same effects, which were blocked by the antagonist d-tubocurarine, on the bacterial replication and cytokine regulation with that of nicotine. Thus, the results revealed that nAChRs, the major exogenous ligands of which are nicotine, are involved in the regulation of macrophage immune function by nicotine and may contribute to the cigarette-induced risk factors for respiratory infections in smokers.     

High concentrations of bacterial lipopolysaccharide, but not microbial infection-induced inflammation, activate macrophage C3 receptors for phagocytosis

Macrophage C3 receptors are normally immobilized in the plane of the cells' plasma membrane and are unable to promote phagocytosis even though they promote avid particle binding. We have previously identified a lymphokine that activates macrophage C3 receptors for phagocytosis both in vitro and in vivo, and others have found that certain types of nonimmunologically mediated inflammation are also able to activate mononuclear phagocyte C3 receptors. These findings raised the possibility that macrophage C3 receptor activation is a universal consequence of inflammation. We sought in the present experiments to determine whether or not inflammation induced by microbial infection in a nonimmune host resulted in activation of macrophage C3 receptors. We injected mice i.p. with either viable microorganisms, microbe-containing immune complexes, or bacterial LPS. Macrophages were harvested by peritoneal lavage 4 days later; nearly all lavage fluids grew the microorganism with which the mouse had been injected, indicating that an infection had been established. Monolayers of macrophages were established and their interaction with sheep E coated with C3 (EIgMC) was determined. All macrophages bound EIgMC, but only macrophages from mice injected with either very high concentrations of LPS or microbe-containing immune complexes ingested them. C3 receptors of macrophages that ingested EIgMC were mobile; others were not. Thus, inflammation induced by microbial infection does not commonly, if at all, activate macrophage C3 receptors; microbe-containing immune complexes and high concentrations of LPS do. The mechanism of receptor activation in each case is C3 receptor mobilization, which is probably mediated by a lymphokine.     

Photoperiod alters macrophage responsiveness, but not expression of Toll-like receptors in Siberian hamsters

Defense against pathogens is a critical component of comparative and ecological biology. However, pathogen recognition, a process necessary for the facilitation of systemic immune response, remains understudied in a comparative context, yet could provide insight into how the immune system interacts with pathogens in variable environments. We examined pathogen recognition by macrophages in relation to an ecological variable, day length, in Siberian hamsters (Phodopus sungorus). Because peritoneal macrophages collected in long, summer-like day lengths are more responsive to a lipopolysaccharide (LPS) challenge compared to macrophages collected during short, winter-like day lengths, we hypothesized that these functional differences are mediated by variation in pathogen recognition, which occurs through binding to Toll-like receptors (TLRs). We predicted that expression of TLR2 and 4, the receptors that bind and respond specifically to LPS, would be upregulated in long vs. short days, and that expression of these receptors would reflect macrophage responsiveness to LPS. Macrophages collected during long days were again more responsive to LPS challenge compared to short-day macrophages; however, TLR2 and TLR4 expression was similar between photoperiods and were unrelated to our measure of macrophage responsiveness suggesting that other downstream intracellular mechanisms may be responsible for photoperiod-based variation in macrophage responsiveness in this species.     

A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells

The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.     

A macrophage receptor for (mannose/glucosamine)-glycoproteins of potential importance in phagocytic activity

Lung macrophages, in the absence of serum factors in vitro, strongly bound and ingested yeast cells ($\text{Candida}\text{krusei}$ and zymosan). Binding was temperature-and calcium-dependent, and was inhibited by the presence of D-mannose, D-glucosamine, horseradish peroxidase and beta-glucuronidase. Pretrypsinization of the macrophages also prevented binding of yeast cells. Binding was not affected by D-mannitol, D-glucose, D-galactose nor L-fucose. I suggest that macrophage binding of yeast cells is mediated by a mannose/glucosamine receptor on the cell membrane. This receptor may be responsible for opsosin-independent phagocytosis of activators of the alternative complement pathway and, as well, the phagocyte-dependent clearance of certain lysosomal enzymes.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

活性氧对巨噬细胞呼吸爆发影响及云芝多糖的保护作用

用化学发光法观察到叔丁基氢过氧化物对培养的小鼠腹腔巨噬细胞呼吸爆发有强烈的抑制作用。云芝多糖经腹腔注射后,能增强巨噬细胞呼吸爆发功能对叔丁基氢过氧化物损伤的抵抗力。云芝多糖处理的巨噬细胞谷胱甘肽过氧化物酶基础活力显著提高,在叔丁基氢过氧化物作用下,云芝多糖处理的巨噬细胞仍有较高的谷胱甘肽过氧化物酶活力。说明巨噬细胞的免疫功能与谷胱甘肽过氧化物酶活力有关,非特异性免疫多糖可提高细胞抗氧化能力,减轻活性氧损伤作用。     

An avidin-biotin-peroxidase method for Fc receptors on macrophages isolated from and in sections of rat lung

Receptors for the Fc region of immunoglobulin G (Fc receptors) were detected on pulmonary macrophages by adapting an avidin-biotin-peroxidase technique to isolated cells and sections of rat lung. After incubation with soluble rabbit immunoglobulin G (IgG), surface bound IgG was identified consistently and reproducibly on glass-adherent pulmonary macrophages and on macrophages in tissue sections made from incubated lung slices. Control experiments indicated that binding was specifically mediated by surface Fc receptors. This method may be useful for identifying macrophages in intact tissues.     

Galactose-specific receptors on liver cells. I. Hepatocyte and liver macrophage receptors differ in their membrane anchorage

Colloidal gold particles coated with asialoglycoproteins are bound by hepatocytes as well as by liver macrophages. Binding by both cell types is inhibited by N-acetylgalactosamine and related saccharides and is dependent on the presence of Ca2+. We have now performed an electron microscopic study on receptor anchorage in the plasma membranes. Cells with prebound ligand were treated with 20 mM EDTA at 4 degrees C, washed free of chelator and tested for residual galactose-specific receptor activity. Whereas hepatocytes preserve binding activity (73% of untreated control), liver macrophages lose galactose-specific receptor activity (12% of untreated control). Liver macrophages regain binding activity after a 2 min incubation at 37 degrees C allowing for receptor recycling. If the macrophages were fixed with low glutaraldehyde concentration prior to EDTA treatment they fully retained their receptor activity (74% of control). Ligands were also removed from both cell types by incubation with 80 mM N-acetylgalactosamine. After washing the cells free of the competing monosaccharide, both the hepatocytes as well as the macrophages show full binding activity (120% and 85% of untreated controls). Therefore, membrane anchorage sites of the macrophage receptors are not identical to ligand-binding sites. These results suggest a Ca2+-Mg2+-dependent receptor anchorage on the macrophage plasma membrane. As shown in the accompanying paper (Roos, P.H., Hartmann, H.J., Schlepper-Sch?fer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847, 115-121), EDTA-induced dissociation from the membrane can be used for isolation of the galactose-specific receptors of liver macrophages.