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BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.

Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC-PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC-PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications.

Background: Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions., Methods: We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied., Results: The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Co-culture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1β, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors., Conclusions: We have shown that pMSCs can transition macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair.

Apoptotic cells must be recognized early for phagocytosis to ensure that their toxic contents do not damage neighboring cells. In some cases this is achieved via CD43-capped membrane glycoproteins, the sialylpolylactosaminyl chains of which serve as ligands for phagocytosis by macrophages. However, because many additional changes occur during apoptosis, determining exactly which events are responsible for signaling macrophages to initiate phagocytosis remains a challenge. Here, we examined one clearance mechanism in detail and determined that capping of CD43 alone is sufficient to initiate phagocytosis. We induced macrophage-mediated phagocytosis by using cytochalasin B to artificially cap CD43 on healthy (non-apoptotic) Jurkat cells. Additional experiments confirmed that sialylpolylactosaminyl chains formed through this capping method are a prerequisite for removal, and that nucleolin is the macrophage receptor responsible for their detection. These findings strongly suggest that capping of CD43 presents a sufficient signal for phagocytosis without any additional membrane changes.

This study evaluated the use of isothermal microcalorimetry (ITMC) to detect macrophage-nanoparticle interactions. Four different nanoparticle (NP) formulations were prepared: uncoated poly(isobutyl cyanoacrylate) (PIBCA), polysorbate-80-coated PIBCA, gelatin, and mannosylated gelatin NPs. Changes in NP formulations were aimed to either enhance or decrease macrophage-NP interactions via phagocytosis. Alveolar macrophages were cultured on glass slabs and inserted in the ITMC instrument. Thermal activities of the macrophages alone and after titration of 100 μL of NP suspensions were compared. The relative interactive coefficients of macrophage-NP interactions were calculated using the heat exchange observed after NP titration. Control experiments were performed using cytochalasin B (Cyto B), a known phagocytosis inhibitor. The results of NP titration showed that the total thermal activity produced by macrophages changed according to the NP formulation. Mannosylated gelatin NPs were associated with the highest heat exchange, 75.4 ± 7.5 J, and thus the highest relative interactive coefficient, 9,269 ± 630 M-1. Polysorbate-80-coated NPs were associated with the lowest heat exchange, 15.2 ± 3.4 J, and the lowest interactive coefficient, 890 ± 120 M-1. Cyto B inhibited macrophage response to NPs, indicating a connection between the thermal activity recorded and NP phagocytosis. These results are in agreement with flow cytometry results. ITMC is a valuable tool to monitor the biological responses to nano-sized dosage forms such as NPs. Since the thermal activity of macrophage-NP interactions differed according to the type of NPs used, ITMC may provide a method to better understand phagocytosis and further the development of colloidal dosage forms.

Background: Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2) by ascorbic acid pre-treated quartz (QA) compared to untreated quartz (Q) in the murine macrophage cell line RAW 264.7., Methods: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways., Results: Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-alpha production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself., Conclusion: Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

During pregnancy, trophoblasts are shed into maternal blood from the placenta as they die. Trophoblasts are fetal cells and are therefore immunologically foreign to the maternal immune system, but the effects of shed trophoblasts on the maternal immune system are poorly characterized. We have used an in vitro villous explant model to harvest shed trophoblasts. These shed trophoblasts consist of multinucleated syncytial knots as well as mononuclear cells, and approximately 90% are apoptotic as determined by immunostaining with antibodies recognizing activated caspase-3 and the M30 cytokeratin neoepitope. U937 cells phagocytosed the shed apoptotic trophoblasts and, subsequently, secretion of the anti-inflammatory cytokine IL-10 was increased. In contrast, secretion of the proinflammatory cytokine Il-1beta by U937 cells was decreased after phagocytosis of apoptotic trophoblasts and the changes in both IL-10 and IL-1beta secretion were blocked by co-incubation with the phagocytosis inhibitor cytochalasin B. Shed trophoblasts caused a significant increase also in expression of the, immunosuppressive, tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase. We speculate that the shedding of trophoblasts may not be simply a mechanism the fetus uses to dispose of aged trophoblasts but rather shed apoptotic trophoblasts may provide a chronic source of tolerizing paternally derived antigens to regulate maternal immune responses to the fetus.

Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite--as few as 10 bacteria can cause life-threatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.

Objective: Phagocytosis of apoptotic cells can be facilitated by complement components and short pentraxins, such as serum amyloid P (SAP). In contrast, the long pentraxin PTX3 was shown to inhibit phagocytosis of apoptotic Jurkat cells by dendritic cells and to bind late apoptotic polymorphonuclear leukocytes (PMNs). Recently, levels of the pentraxin PTX3 were shown to parallel disease activity in small-vessel vasculitis, which is often characterized by leukocytoclasia, a persistence of leukocyte remnants in the vessel wall. We undertook this study to test our hypothesis that PTX3 inhibits phagocytosis of late apoptotic PMNs by macrophages, thereby leading to their accumulation in the vessel wall., Methods: Macrophages were allowed to phagocytose late apoptotic or secondary necrotic PMNs that were incubated with or without PTX3 for 30 minutes prior to phagocytosis. Phagocytosis was allowed to occur in the presence of 30% normal human serum with or without SAP and with or without depletion of complement. To discriminate between an inhibitory effect of PTX3 on binding and the internalization of apoptotic PMNs into macrophages, internalization was blocked by cytochalasin B., Results: SAP and complement were both necessary for effective in vitro phagocytosis. In contrast, PTX3 inhibited phagocytosis in a dose-dependent manner, from 11% inhibition at 6.25 microg/ml to almost complete inhibition at 100 microg/ml. Furthermore, PTX3 partly affected binding of apoptotic PMNs to macrophages., Conclusion: PTX3, in contrast to SAP and complement, inhibits phagocytosis of late apoptotic PMNs by monocyte-derived macrophages in a dose-dependent manner. Therefore, PTX3 can play a role in the development of leukocytoclasia by affecting the clearance of apoptotic PMNs, thereby inducing their accumulation in the vessel wall.

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.

Macrophage cytokine expression significantly affects wound healing. Macrophage secretion of transforming growth factor beta 1 (TGFbeta1) and bone morphogenetic proteins (BMP) may affect osteogenesis at endosseous implant surfaces. The aim of this investigation was to determine the effect of commercially pure titanium (cpTi) substrate topography on adherent macrophage osteogenic and osteoinductive cytokine expression. J774A.1 murine macrophage cell adhesion was examined by scanning electron microscopy, 0-72 h following plating onto polished, machined, and grit-blasted cpTi surfaces. TGFbeta1 and BMP-2 gene expression by adherent macrophages was determined by the reverse transcription polymerase chain reaction. Macrophage adhesion increased with time on all surfaces and spreading increased with increasing surface roughness (polished < machined < grit-blasted). BMP-2 expression was not evident for cells adherent to polished cpTi at 24 h. In contrast, BMP-2 expression occurred at 24 h in cells adherent to machined and grit-blasted cpTi. BMP-2 expression was evident on all surfaces at 72 h and was greatest in grit-blasted titanium adherent cells. Increasing concentrations of cytochalasin B (0-50 microM) inhibited macrophage spreading and reduced BMP-2 mRNA expression, suggesting a relationship between cell shape and BMP-2 expression. This was further characterized using anti-beta1 and anti-beta3 integrin antibodies. The anti-beta1 integrin antibodies inhibited adherent macrophage BMP-2 mRNA expression. Anti-beta3 integrin antibody treatment only modestly reduced BMP-2 mRNA expression. Endosseous implant surface topography induced changes in macrophage shape that were associated with changes in BMP-2 expression in J774A.1 mouse macrophage cell line. This first demonstration of BMP-2 expression by cpTi adherent macrophages suggests that the macrophage may contribute surface-specific osteoinductive signals during bone formation at implanted alloplastic surfaces., (Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 207-216, 2003)

This study was designed to evaluate the anti-inflammatory effect of piperlactam S on chemoattractant-induced migration, functions underlying leukocyte recruitment in vitro. Results showed that RAW264.7 macrophages migrate toward complement 5a, a powerful chemoattractant for macrophages. This phenomenon could be suppressed concentration dependently by piperlactam S (0.3-30 microM). Fluorescence staining demonstrated that piperlactam S and cytochalasin B both effectively reversed complement 5a-induced cell polarization, filopodia extension, as well as the increase in the cell content of F-actin. Functional inhibition by antibodies suggested that Mac-1 (CD11b) integrin plays a central role in complement 5a-induced migration. However, piperlactam S failed to modify Mac-1 expression. Furthermore, complement 5a triggered the activation of Cdc42, a Rho-family protein involved in the regulation of filopodia extension, with a time course that paralleled that of filopodia extension and which was inhibited by piperlactam S. In summary, piperlactam S exerts anti-inflammatory effects possibly by interfering with cell migration, impeding F-actin polymerization, filopodia formation, and/or Cdc42 activation. However, the detailed mechanism by which piperlactam S regulates the above processes needs further study.

Background: Accumulation of immune complexes in the mesangium is a common finding. Since migration of macrophages (Mphi) in the mesangium has been demonstrated to be an important event in the development of glomerular lesions, we studied the role of immune complexes and mesangial cell (MC) interaction in the transmigration (Tm) of Mphi., Methods: To determine the effect of MC and immune complexes (aggregated IgG, IgGAg) on transmigration of Mphi. MC were incubated with or without IgGAg in the lower compartment of a modified Boyden Chamber. To determine the effects of the secretory products (as a result of endocytosis of IgGAg by mesangial cells), MC-IgAg conditioned media was prepared and placed in the lower compartment of the Boyden chamber. We evaluated the effects of MC alone, MC + IgGAg, or MC-IgGAg conditioned media on the transmigration of macrophages across a filter. To determine the effect of free radicals on MC-IgAg endocytosis-induced Mphi migration we evaluated the effect of free radical scavengers such as dimethyl thiourea (DMTU) and tetramethylthiourea (TMTU) in MC-IgAg endocytosis-induced Mphi migration. To determine the role of chemokines in MC-IgAs endocytosis-induced Mphi migration we evaluated the effect of ani-MCP-1 antibodies on MC-IgAg endocytosis-induced Mphi migration, and also studied the effects of IgAg on MC mRNA expression of MCP-1 and RANTES. In addition, we evaluated the role of Fc receptors and actin cytoskeleton of MC in transmigration of Mphi., Results: Mesangial cell endocytosis of IgG aggregates (IgGAg) is associated with enhanced (P < 0.001) transmigration of Mphi (control, 11.2 +/- 0.2 vs. MC + IgGAg, 22.1 +/- 0.9 migrated Mphi/field). IgGAg also induced MC mRNA expression for RANTES and MCP-1 on MC. DMTU and TMTU attenuated (P < 0.001) the MC + IgGAg-induced migration of Mphi as well as IgGAg-induced mRNA expression for RANTES and MCP-1. MC and IgGAg interaction products (MC-IgGAg conditioned media) also increased (P < 0.01) transmigration of Mphi (control, 18.3 +/- 1.7 vs. MC-IgGAg conditioned media, 30.7 +/- 0.6 Mphi/field). This effect of MC-IgGAg conditioned media on the migration of macrophages was dose dependent. Anti-MCP-1 antibody partially inhibited MC-IgGAg-induced migration of macrophages. MC and monomeric IgG (MIgG) interaction (MC-MIgG conditioned media) showed a lower (P < 0.05) migration of Mphi, when compared to the MC-IgGAg conditioned media. MC-IgGAg conditioned media prepared from cytochalasin B pretreated MCs also showed a lower (P < 0.001) migration of Mphi when compared with MC-IgGAg conditioned media-induced migration., Conclusions: These results indicate that MC-IgGAg conditioned media-induced transmigration of macrophages may be mediated through the generation of RANTES and MCP-1 by MC.

1 Lysophosphatidic acid (LPA) has been widely studied as a naturally occurring and multifunctional phospholipid messenger in diverse tissue and cell types and shown to inhibit adenylyl cyclase (AC) by a G protein-mediated mechanism. 2 In type II AC-expressing mouse RAW 264.7 macrophages, we showed that LPA at 3-50 microM increased cyclic AMP formation in a concentration-dependent manner, the effect being additive with that of forskolin or cholera toxin, and synergistic with that of prostaglandin E1 (PGE1) or isoproterenol. 3 The potentiation effect of LPA was unaffected by the removal of serum or pertussis toxin treatment. 4 Both colchicine and cytochalasin B potentiated the cyclic AMP response to PGE1, the effect being additive to that of LPA. 5 On studying the regulation of type II AC by protein kinase C (PKC), phorbol 12-myristate-13 acetate (PMA) potentiated the PGE1-elicited cyclic AMP response, this effect being non-additive to that of LPA, suggesting that PKC activation was the common mechanism involved in AC potentiation by LPA and PMA. 6 PKC inhibitor Ro 31-8220, but not Go 6976, significantly inhibited the LPA-induced cyclic AMP potentiation. 7 The potentiation effect of LPA was unaffected by long-term treatment with PMA, which resulted in the down-regulation of PKCalpha, betaI, betaII and PKCdelta, but not PKCepsilon, mu, lambda and zeta. 8 By in situ kinase assay, we found a marked increase in atypical PKC activity after LPA treatment. 9 Taken together, we conclude that LPA can elicit a unique signalling cascade in RAW 264.7 macrophages and increase type II AC activity via the activation of atypical PKC.

The foreign body reaction to implanted biomaterials, characterized by the presence of macrophages and foreign body giant cells (FBGC), can result in structural and functional failure of the implant. Recently, we have shown that interleukin-4 and interleukin-13 can independently induce human macrophage fusion to form FBGC via a macrophage mannose receptor (MR) -mediated pathway. The MR is believed to mediate both endocytosis of glycoproteins and phagocytosis of microorganisms, which bear terminal mannose, fucose, N-acetylglucosamine, or glucose residues. Polarization of microfilaments to closely apposed macrophage membranes as observed with fluorescence confocal microscopy led us to ask whether MR-mediated fusion occurred via a filamentous actin-dependent pathway. Cytochalasins B and D and latrunculin-A, agents that disrupt microfilaments, inhibited macrophage fusion in a concentration-dependent manner. The concentrations of cytochalasins D and B that inhibited fusion did not significantly decrease macrophage adhesion, spreading, or motility but did inhibit internalization of Candida albicans during interleukin-13-enhanced, MR-mediated phagocytosis. Very low concentrations of cytochalasin B (< 2 microM) induced a slight enhancement of macrophage fusion. Taken together, the results of this study suggest that cytokine-induced, MR-mediated macrophage fusion requires an intact F-actin cytoskeleton and that the mechanism of fusion is similar to phagocytosis.--DeFife, K. M., Jenney, C. R., Colton, E., Anderson, J. M. Disruption of filamentous actin inhibits human macrophage fusion.

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.

We tested the hypothesis that aggregated lipoprotein(a) [Lp(a)] is avidly taken up by macrophages. Lp(a) was isolated by sequential centrifugations and gel chromatography from a patient with high plasma levels of Lp(a) who was being treated with low density lipoprotein (LDL)-apheresis. Aggregated Lp(a) was prepared by mixing native Lp(a) with 2.5 mmol/L CaCl2, and 54% of the 125I-Lp(a) aggregated after interacting with CaCl2. The binding and degradation of aggregated Lp(a) in macrophages were 4.6- and 4.7-fold higher than those of native Lp(a), respectively. An excess amount of LDL did not inhibit either increase. Cholesterol esterification in macrophages was markedly stimulated by aggregated Lp(a), and macrophages were transformed into foam cells. Cytochalasin B, a phagocytosis inhibitor, strongly inhibited the degradation and cholesterol esterification (78 and 83%, respectively). These findings suggested that aggregation may be partially involved in Lp(a) accumulation, thereby contributing to the acceleration of atherosclerosis.

To determine, whether 558Thr in the carboxyl-terminal domain of moesin is phosphorylated in cells other than platelets, rabbit phosphorylation state-specific antibodies were made to the chemically phosphorylated synthetic hexapeptide KYKpTLR of the moesin sequence, as well as to the unphosphorylated form. The affinity-purified antibody populations were specific for either the phosphorylated or the unmodified peptide conjugated to BSA. Site-specific phosphorylation of moesin is detected in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopodial protrusions. After pretreatment with the phosphatase inhibitor calyculin A, a similar effect to that seen in platelets in found, namely a substantial increase in moesin phosphorylation at 558Thr and redistribution of phospho-moesin together with F-actin into one or more ring-like structures in the cytoplasm, presumably due to binding of phosphorylated moesin to F-actin.

The wear particles of cobalt chrome alloy and titanium alloy have been implicated as a cause of aseptic loosening of prostheses. It is thought that their ability to induce either cell death or the release of mediators that induce bone resorption contributes to this loosening. This study was designed to test the hypothesis that these adverse biologic effects are due to wear particle corrosion at low pH after they have been phagocytosed by macrophages. Cobalt chrome alloy and titanium alloy particles of similar size and concentration to those found in the tissues surrounding failed prostheses were added to cultured rodent peritoneal macrophages. Treatment of macrophages with drugs that prevent a drop in pH within phagosomes significantly reduced the toxicity of phagocytosed cobalt chrome alloy particles. The same drugs also reduced the levels of prostaglandin E2 and interleukin-6 release induced by phagocytosed titanium alloy particles. When both types of particles were incubated at a low pH, similar to that encountered by phagocytosed particles, soluble products were released that induced the same effects as the particles themselves. These results show that enhanced corrosion of wear particles by phagocytic cells may contribute significantly to the adverse biologic effects of wear particles and identify drug therapies that may be investigated further.

Nitric oxide production by mouse macrophages treated with group B streptococci and gamma interferon was inhibited by cytochalasin B or by antibody neutralization of macrophage-derived tumor necrosis factor alpha. Phagocytosis-induced tumor necrosis factor alpha is responsible for group B streptococcus-induced nitric oxide production in interferon-treated macrophages.

One of the major problems encountered during quantitative studies of phagocytosis is the discrimination without ambiguity between intracellular and extracellular particles. This difficulty is especially acute when zymosan particles are used because of their poor affinity for dyes. We show in this paper that zymosan particles may be stained by a mixture of a basic dye and tannic acid in water (or in an isotonic non saline solution). Crystal violet is one of the most suitable dyes and by combining this staining step with May-Grünwald Giemsa staining, it was possible to observe two populations of macrophage-associated particles. One comprises blue violet particles (BVP), and the second consists of particles with a purple-stained core (PPC). Treating macrophages in culture with various concentrations of cytochalasin B decreases the number of PPC and increases the number of BVP, in a dose dependant manner. Moreover, treatment with alpha-mannan or laminarin decreases the number of cell-associated particles, especially that of PPC. From these observations we concludes that BVP are extracellularly located and PPC are ingested.

In the present study, we investigated the effect of Listeria monocytogenes infection on the cellular level of the transcription factors NF-kappa B, AP-1, and NF-IL6 in the macrophage-like cell line P388D1 by using electrophoretic mobility shift assays. Infection with L. monocytogenes enhanced the formation of two NF-kappa B-like DNA-protein complexes, C1 and C2, whereas the concentration of AP-1 and NF-IL6 complexes remained unaffected. In supershift assays using NF-kappa B-specific antibodies, complex C2 was identified to be a p50 homodimer (KBF1) and complex C1 was identified as a p50/p65 heterodimer. Both complexes were formed within 10 min after addition of the bacteria. Since the synthesis of tumor necrosis factor alpha and interleukin-1 occurs at later times, these cytokines cannot be the mediators of enhanced NF-kappa B formation. Infection experiments with different nonhemolytic mutants of L. monocytogenes and the use of the phagocytosis inhibitor cytochalasin B suggest that events prior to invasion and escape of the bacteria from the phagosome into the cytoplasm enhance the nuclear transport of p50/p65 NF-kappa B components.

Substantial in vivo evidence exists to implicate the macrophage (M phi) in modulating mesangial expansion and glomerulosclerosis (GS) following renal injury. We studied in an in vitro system how M phi activation via Fc-receptor-mediated endocytosis, such as occurs in immune complex-mediated disease states, may influence the effect of M phi secretory products (MSP) on mesangial cell (MC) proliferation and matrix synthesis. MSP from M phi incubated with immunoglobulin G (IgG) complexes caused significantly greater (P < 0.001) enhancement of MC [3H]thymidine incorporation compared with MSP from unstimulated M phi or from M phi activated via nonspecific endocytosis (P < 0.001). MSP from M phi incubated with IgG complexes plus the inhibitor of endocytosis cytochalasin B showed an attenuated effect on MC proliferation (P < 0.02). MSP were also found to enhance MC matrix synthesis (P < 0.001). These data demonstrate that MSP can play a direct role in mesangial expansion by increasing both MC proliferation and matrix synthesis. Surface binding alone of IgG complexes may not be sufficient to activate M phi and enhance their mitogenic effect on MC as endocytosis appears to be required. These findings lend in vitro support to a potential role for the M phi in the process of mesangial expansion and GS following renal injury.

Certain steroids having an oxo group at the C-17 or C-20 position such as pregnenolone and dehydroisoandrosterone inhibit the cholesterol transport from lysosomes to other cellular sites. Taking advantage of the fact that the inhibition is reversed upon removal of the steroids, we studied the factors that control the cholesterol transport from lysosomes to other cellular sites in macrophages. Macrophages that accumulated unesterified cholesterol in their lysosomes were prepared by incubating cells with liposomes containing cholesterol and phosphatidylserine in the presence of a steroid inhibitor. These cells were chased by means of steroid washout, and then the effects of various pharmacological agents on the subsequent metabolism of cholesterol were examined. When the cells were chased in the absence of the agents, some of the cholesterol was converted to cholesteryl esters in the cells, and others were desorbed into the medium as unesterified forms, suggesting recovery of lysosomal cholesterol trafficking. Among the agents tested, bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely blocked both cholesterol esterification and cholesterol desorption at 10 nM. Moreover, agents that neutralize the lysosomal proton gradient, such as ammonium chloride and chloroquine, also reduced both of the processes. Fluorescent microscopic examination of bafilomycin A1-treated cells revealed extensive filipin-cholesterol staining of perinuclear lysosomes. From these data, we conclude that acidic pH is required for the efflux of cholesterol from lysosomes to other cellular sites.

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.

Antigens in extracellular fluids can be processed and presented with major histocompatibility complex (MHC) class I molecules by a subset of antigen presenting cells (APCs). Chicken egg ovalbumin (Ova) linked to beads was presented with MHC class I molecules by these cells up to 10(4)-fold more efficiently than soluble Ova. This enhanced presentation was observed with covalently or noncovalently linked Ova and with beads of different compositions. A key parameter in the activity of these conjugates was the size of the beads. The APC that is responsible for this form of presentation is a macrophage. These cells internalize the antigen constructs through phagocytosis, since cytochalasin B inhibited presentation. Processing of the antigen and association with MHC class I molecules appears to occur intracellularly as presentation was observed under conditions where there was no detectable release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). Similar results were obtained in BALB/c mice immunized with beta-galactosidase-beads. The implications of these findings for development of nonliving vaccines that stimulate CTL immunity are discussed.

Aggregation studies have become a useful criterion for analyzing leukocyte motility and activation in vitro. The T-cell-derived lymphokine human leukocyte inhibitory factor (LIF) is a modulator of many important polymorphonuclear (PMN) functions in addition to aggregation such as chemotaxis, lysosomal degranulation, phagocytosis, bactericidal killing, augmented antibody-dependent cellular cytotoxicity (ADCC), induction of neutrophil Fc-gamma, complement type-1 and FMLP receptors, and production of superoxide and H2O2. Our investigations focused on the ability of LIF to modulate the aggregation of macrophages (MO) induced by calcium ionophore A23187. The ionophore A23187 directly induced potent aggregation of macrophages, which was markedly enhanced when the cells were pretreated with LIF. However, the addition of LIF in the absence of other costimuli did not directly induce MO aggregation. LIF was shown to enhance PMN aggregation induced by N-formyl-Met-Leu-Phe (FMLP), but did not augment the aggregation of FMLP-stimulated macrophages, indicating a cellular specificity of aggregation-inducing costimuli following LIF priming. Additional cytokines examined for possibly inducing MO aggregation were interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), and interleukin-6 (IL-6); all proved to be incapable of inducing aggregation directly, nor did they enhance the effect of A23187 ionophore on macrophage aggregation. Additionally, we found that LIF can directly stimulate MO to activate specific pathways of the arachidonic acid cascade, inducing the synthesis and release of thromboxanes and leukotriene B4. LIF did not augment the potent ability of A23187 to induce increased production of LTB4 or TxA2 by human MO. These new results coupled with our previously published data indicate that LIF can enhance the activation of both MO and PMN leukocytes when exposed to either A23187 or FMLP, respectively. Moreover, these data suggest that LIF can contribute directly to monocyte-macrophage leukocyte activation, in addition to PMN activation, during inflammatory responses, resulting in greater cell aggregation, activation, and specific proinflammatory arachidonic acid product release.

Autoclaved yeasts are stained light pink by May-Grünwald Giemsa (MGG). If treated with tannic acid solution just before MGG staining, they display a deep violet color. It seemed possible that these properties could be used to discriminate between extra- and intracellular yeasts in a phagocytosis test, extracellular yeasts being violet and intracellular yeasts being pink. To validate this protocol, quantitative studies of phagocytosis by MALU cells (a murine macrophage cell line) were performed in the presence or absence of drugs known to interfere with phagocytosis. After treatment of cells with cytochalasin B, the mean number of pink yeasts per cell decreased in a dose-dependent manner, the mean number of violet yeasts increased in a dose-dependent manner, whereas the total number of cell-associated yeasts remained almost unchanged whatever the dose used. After treatment with alpha-mannans or chloroquine, the mean numbers of both violet and pink yeasts decreased in a dose-dependent manner. These results confirmed that (i) violet yeasts are extracellular, (ii) autoclaved yeasts recognize lectin receptors, and (iii) unstained (pink) yeasts are intracellular. We show that this simple method can be used for quantitative light microscopic analysis of both the attachment and internalization steps in the phagocytosis of yeasts.

Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.

A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 micrograms/ml). Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.

Low density lipoprotein (LDL) and beta-very low density lipoprotein (beta-VLDL) are internalized by the same receptor in mouse peritoneal macrophages and yet their endocytic patterns differ; beta-VLDL is targeted to both widely distributed and perinuclear vesicles, whereas LDL is targeted almost entirely to perinuclear lysosomes. This endocytic divergence may have important metabolic consequences since beta-VLDL is catabolized slower than LDL and is a more potent stimulator of acyl-CoA/cholesterol acyl transferase (ACAT) than LDL. The goal of this study was to explore the determinants of beta-VLDL responsible for its pattern of endocytic targeting. Fluorescence microscopy experiments revealed that large, intestinally derived, apoprotein (Apo) E-rich beta-VLDL was targeted mostly to widely distributed vesicles, whereas small, hepatically derived beta-VLDL was targeted more centrally (like LDL). Furthermore, the large beta-VLDL had a higher ACAT-stimulatory potential than the smaller beta-VLDL. The basis for these differences was not due to fundamental differences in the means of uptake; both large and small beta-VLDL were internalized by receptor-mediated endocytosis (i.e., not phagocytosis) involving the interaction of Apo E of the beta-VLDL with the macrophage LDL receptor. However, large beta-VLDL was much more resistant to acid-mediated release from LDL receptors than small beta-VLDL. Furthermore, partial neutralization of the multiple Apo Es on these particles by immunotitration resulted in a more perinuclear endocytic pattern, a lower ACAT-stimulatory potential, and an increased sensitivity to acid-mediated receptor release. These data are consistent with the hypothesis that the interaction of the multivalent Apo Es of large beta-VLDL with multiple macrophage LDL receptors leads to a diminished or retarded release of the beta-VLDL from its receptor in the acidic sorting endosome which, in turn, may lead to the widely distributed endocytic pattern of large beta-VLDL. These findings may represent a physiologically relevant example of a previously described laboratory phenomenon whereby receptor cross-linking by multivalent ligands leads to a change in receptor targeting.

Our previous studies have shown that a phagocytic challenge with IgG-coated erythrocytes (EIgG) depressed macrophage triggered H2O2 production in vitro, and in vivo there was a decrease in the survival rate following bacteremia. The phagocytosis of an equal number of IgG-coated erythrocyte ghosts had none of these effects, indicating that the contents of the erythrocytes are important for these effects. The present study evaluated the role of the scavengers of reactive oxygen intermediates within erythrocytes in the depression of H2O2 production triggered with phorbol myristate acetate following a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages (PM) were challenged with EIgG prepared from normal E or E with inactivated catalase, depleted glutathione, hemoglobin converted to methemoglobin, or fixed with formaldehyde. The depression of triggered H2O2 production was similar when equal numbers of normal EIgG and EIgG with inactivated scavengers were phagocytized. When the phagocytic challenge with normal EIgG was carried out in the presence of cytochalasin B, no depression of triggered H2O2 production was observed. Cytochalasin B partially blocked the phagocytosis of EIgG, so that with larger doses of EIgG there was sufficient ingestion of EIgG to depress H2O2 production in untreated PM. These results indicate that the scavengers of reactive oxygen intermediates present in erythrocytes are neither required nor sufficient to depress H2O2 production by macrophages.

Dexamethasone decreases 2-D-deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages in vitro in a concentration- and time-dependent manner (Ki for 1 microM-dexamethasone after a 2 h exposure = 0.71 +/- 0.21 microM; Ki for 0.1 microM-dexamethasone after exposure for 4 h = 0.10 +/- 0.06 microM). The inhibition of 2-dGlc uptake is consistent with a decrease in the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is: (1) the Km for zero-trans 2-dGlc uptake in quiescent macrophages was increased by dexamethasone, but there was no significant effect on the Vmax.; (2) dexamethasone increased the rate of exit of sugar from cells preloaded with 2-dGlc; (3). the free sugar accumulation within the cytosol of the cells above the external solution concentration was significantly decreased by dexamethasone. These effects of dexamethasone on 2-dGlc transport were antagonized by simultaneous exposure to the steroid RU 38486 (Ki = 0.04 +/- 0.01 microM; 4 h incubation). Although dexamethasone inhibited zero-trans uptake, the maximum rate of infinite-trans exchange uptake of 2-dGlc into cells preloaded with 3-O-methyl-D-glucose (40 mM) was unaltered by dexamethasone or RU 38486, indicating that the dexamethasone-dependent decrease in zero-trans uptake was not due to a change in the number of transporters in the plasma membrane. Dexamethasone also inhibited the phorbol myristate acetate-induced stimulation of hexose monophosphate shunt (HMPS) activity, and this was reversed by RU 38486. Cytochalasin B, the potent sugar-transport inhibitor, inhibited HMPS activity and 2-d[2,6-3H]Glc uptake equally, indicating a single site of action. By contrast, dexamethasone showed differential inhibition of HMPS activity and 2-d[2,6-3H]Glc uptake, suggesting that it not only acts by decreasing the coupling between hexokinase and sugar transport, but also at one or more additional points.

2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.

When exposed to hen ovalbumin (OA)-complexed IgG antibodies, guinea-pig macrophages undergo O2- generation and a rapid rise in the intracellular concentration of free Ca2+ ((Ca2+]i). These responses were found to depend on the IgG isotype of antibodies used; OA-complexed IgG2 antibody (OA gamma 2) induced these responses 3-5 times more intensively than did OA-complexed IgG1 antibody (OA gamma 1). The inhibitory effects of monoclonal antibody to Fc gamma receptor for IgG2 alone (Fc gamma 2R) and that to Fc gamma receptor for both IgG1 and IgG2 (Fc gamma 1/gamma 2R) showed that Fc gamma 2R triggered both an increase in [Ca2+]i and activation of the respiratory burst NADPH oxidase more effectively than did Fc gamma 1/gamma 2R. As the number of Fc gamma 2R molecules per macrophage is about one-half that of Fc gamma 1/gamma 2R molecules, the ability of Fc gamma 2R to trigger these responses may be much higher than that of Fc gamma 1/gamma 2R. This difference between their abilities was further demonstrated by measuring the responses induced by cross-linking of Fc gamma 2R or Fc gamma 1/gamma 2R molecules. In addition, the O2- generation with OA gamma 1 was found to be enhanced with cytochalasin B, and to be lowered by depletion of the intracellular Ca2+ of macrophages with Ionomycin and EGTA, though cytochalasin B and the Ca2+ depletion did not affect the O2- generation with OA gamma 2. These results suggest that the mechanisms of Fc gamma 2- and Fc gamma 1/gamma 2 R-mediated signal transmission leading to activation of the NADPH oxidase also differ from each other.

The human alveolar macrophage product, enzyme-releasing peptide (ERP), has a molecular mass of 8,000 Da, and releases azurophilic and specific granule constituents from neutrophils. A murine monoclonal anti-ERP antibody (12E10H), previously used to show a lack of antigenic identity between ERP and C5a, interleukin 1, tumor necrosis factor, and gamma-interferon, showed no cross-reactivity with interleukin 8. 12E10H and a fluorescein-labeled second antibody were used to visualize ERP on the macrophage surface. ERP was removed from alveolar macrophages by a 3-min incubation with 5 X 10(-7) M bovine pancreatic trypsin at 37 degrees C. The washed trypsinized cells could readhere to plastic and exclude trypan blue. Dilution of the trypsin-derived ERP released myeloperoxidase from cytochalasin-B-treated neutrophils dose dependently. The enzyme-releasing ability of the trypsin-derived material was removed by immunoprecipitation using antibody 12E10H bound to Staphylococcal protein A Sepharose 4B. The estimated molecular mass of the trypsin-derived ERP (by molecular sieve chromatography on HPLC) was approximately 8,500 Da. Other proteases (plasmin, thrombin, and cathepsin G) also released ERP from the cell surface, but the ERP was not an active secretagogue for neutrophils. However, macrophages cultured with protease inhibitors did not show decreased ERP accumulation in the medium. Our data indicate that ERP exists on the surface of human alveolar macrophages and can be released by proteases found within the lung environment in some disease states.

We present a rather simple cytofluorometric technique for the study of exocytosis of lysosomal contents from individual cultured cells. It is based on the use of the lysosomotropic weak base acridine orange (AO) which, in its stacked form, as it occurs within lysosomes, emits red fluorescence when excited by blue light. Mouse peritoneal macrophages were cultured for 48 h and, after 2 h in serum-free medium, stained with AO. The cells were then exposed to F10-medium with or without newborn calf serum (NCS), zymosan A (Z) or cytochalasin B (CB) for different times at 20 or 37 degrees C. After staining, the macrophages showed no change in red fluorescence intensity, if stored at room temperature in the dark. If, however, the cells were kept in the incubator at 37 degrees C, the cells showed slightly decreasing red fluorescence intensity with time. This decrease was markedly potentiated by the presence of NCS, Z or CB, which are known to induce secretion of lysosomal enzymes from macrophages in vitro. Selective lysosomal enzyme release was confirmed biochemically during treatment with zymosan A. The technique presented here may be of value in further studies on the stimulation of, and the mechanisms behind, lysosomal exocytosis in cultured cells.

Activated macrophages kill several types of tumor cells in vitro, whereas non-activated macrophages lack this capacity. We, however, observed that non-activated macrophages efficiently kill F9 teratocarcinoma as well as other teratocarcinoma cell lines. Dexamethasone, a glucocorticoid known to prevent macrophage activation, did not perturb the killing of F9 teratocarcinoma cells. Neither tumor necrosis factor alpha, nor the reactive oxygen intermediates, i.e. hydrogen peroxide, superoxide anion, and hydroxyl radical, nor serine proteases participated in this killing, shown by employing various agents which interfere with their production, secretion, or function. Using acridine orange/ethidium bromide vitality staining, the F9 teratocarcinoma cells were shown to be phagocytized alive by macrophages and subsequently killed intracellularly. Intact lysosomal function is required for the killing of F9 cells, as the lysosomotropic drugs chloroquine and ammonium chloride markedly inhibited this killing without perturbing their engulfment. The signal transduction pathway induced in the macrophages upon interaction with F9 teratocarcinoma cells seems to differ from that induced by macrophage activation. Neither the protein kinase C inhibitors polymyxin B and H-7 [1-(5-isoquinolinylsulfonyl)-2-methyl piperazine] nor the protein kinase C activator phorbol 12-myristate-13-acetate affected the killing of F9 cells. However, chlorpromazine (a powerful inhibitor of calmodulin), dibutyryl cAMP (a cAMP analog), and prostaglandin E2 inhibited the macrophage-mediated killing of F9 cells. In vivo studies indicate that an increased number of macrophages at the F9 tumor inoculation site (the peritoneal cavity) as a result of elicitation by thioglycollate prevents F9 tumor development. Our findings indicate that non-activated macrophages kill teratocarcinoma cells using a mechanism which differs from that employed by activated macrophages in the killing of other tumor cells.

The effect of calmodulin antagonists on the amounts of free fatty acids produced by rabbit alveolar macrophages was determined by fluorometric high-performance liquid chromatography. Opsonized zymosan-induced arachidonic acid production was dramatically suppressed in the presence of W-7 and trifluoperazine without an effect on the production of other fatty acids. Calmodulin antagonists inhibited phospholipase A and abolished the release of arachidonic acid from phospholipids. The present results suggest that a zymosan-sensitive pool of 20:4, which is different from that of other fatty acids, is present in macrophages and that calmodulin antagonists selectively inhibit phospholipase A, which preferentially degrades phospholipids with 20:4.

Resident alveolar macrophages and lung lavage fluids were obtained from normal rabbit lungs. Superoxide production by alveolar macrophages exposed to lung lavage fluids and cytochalasin B was measured by superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction. Glycogen-elicited peritoneal macrophages were used as a control. Cytochalasin B, as well as lung lavage fluids, enhanced superoxide production by resting alveolar macrophages. The cytochalasin B-induced superoxide production was associated with enhanced attachment to glass and with remarkable alterations of the cell surface morphology, probably relating to interference with the microfilament functions of cells. On the other hand, glycogen-elicited peritoneal macrophages showed only a slight production of superoxide when exposed to the same stimulants.

Angiotensin II (At II) in the concentration range of 10(-5) to 5 x 10(-7) M inhibited active and passive erythrocyte-antibody (EA) rosette formation on monolayers of rat peritoneal macrophages (PM) but stimulated it in the range of 10(-7) -10(08) M. Cytochalasin B (CB) and vinblastin (Vb) inhibited the dose-dependent biphasic effect of At II on the Fc receptor (FcR) expression of macrophages. The hormone acts on the last step of rosette formation, i.e. on the binding of sheep red blood cells (SRBC) but does not interfere with the binding of 125I-Ig to FcR. The influence of At II on macrophage rosette formation can be modified by changes in the density of FcR-Ig complexes on the surface of target cells, by the nature of the immunoglobulin (sub)classes involved in rosette formation, and by the biological properties of rosette-forming corpuscular antigen. Based upon the above results we suggest that At II exerts its effect on the contractile elements associated with the cytoskeleton of the macrophage.

Rat peritoneal macrophages released high amounts of prostaglandin E (PGE) when treated in vitro with 10(-7) to 10(-4) M colchicine. PGE production occurred after a lag period of 4 hr and proceeded at a constant rate for more than 24 hr. Lymphocytes could not be stimulated to PGE release by colchicine. Disaggregation of microtubules appeared to be an essential event, since lumicolchicine was inactive and addition of heavy water (D2O) abolished colchicine-induced PGE formation. Cytochalasin B (5 microgram/ ml) did not interfere with PGE production by colchicine during the initial 12 hr, but thereafter it gave rise to an activity capable of degrading or converting newly synthesized PGE. Although details of the mechanisms by which colchicine in association with disrupted microtubules may induce PGE release remain unclear, these observations suggest that components of the cytoskeleton may efficiently influence the biosynthesis of prostaglandins.

Mouse peritoneal macrophages and polymorphonuclear leucocytes were examined for their ability to kill Mycoplasma pulmonis in vitro. Killing of mycoplasmas was shown to require specific antibody, suggesting that antibody-dependent phagocytosis may be involved in resistance to infection. Convalescent mouse serum appeared to be less effective than rabbit antiserum in promoting the killing of mycoplasmas by phagocytic cells. Moreover, mycoplasmas recovered directly from the respiratory tract of infected mice were more resistant to killing by macrophage cultures than organisms grown in vitro. The possibility that these observations may contribute to an understanding of the persistent nature of M. pulmonis infections in mice is discussed.

The three-dimensional visualization of Triton X-100 resistant cytoskeletons has been used to demonstrate that an absorbed dose of 120 Gy from X rays causes a distinctive and reproducible alteration of the cytoskeleton of intact rat alveolar macrophages in vitro. The alteration has also been shown to be rapidly and completely "repaired" and to be apparently similar to alterations caused by colchicine but dissimilar to those caused by cytochalasin B. From these observations and those of other workers who have studied the irradiation of extracted microtubular proteins in vitro, we think it likely that microtubules rather than microfilaments are the radiosensitive component of the macrophage cytoskeleton.

Pulmonary macrophages defend lung surfaces by ingesting deposited particles. We investigated to what extent this uptake of particles can be modulated in vivo by two drugs, colchicine and cytochalasin B (CytB). 198Au colloidal gold, in isotonic saline carrier fluid, was intratracheally instilled into male Syrian golden hamsters. The uptake of these particles by pulmonary macrophages was measured when the carrier fluid contained only colloidal gold and when this test particle was combined with graded doses of either drug. We found that macrophage uptake of the particles 1 h after instillation was depressed 37% when colchicine was added to the instillate (150 micrograms/100 g BW). Depression of particle uptake was also seen with CytB at 15 micrograms/100 g BW. Experiments with tritiated colchicine and CytB showed that both drugs were rapidly cleared from the lungs early in the 1 h phagocytic period. The effect of intravenous colchicine and CytB on the clearance of intravenously injected gold colloid by the liver and spleen reticuloendothelial system was negligible. The results of these experiments, in conjunction with in vitro effects of colchicine and CytB, provide insight into the components of cell function active in particle uptake in situ.

The phagocytic capacity of peritoneal macrophages from resistant C3Hf mice and sensitive C57Bl/6 mice was studied in vitro using a virulent and an avirulent strain of Salmonella typhimurium. Virulent and avirulent 3H-labelled bacteria opsonized with normal mouse serum were killed to an equal extent (about 40%) by macrophages from C3Hf mice and C57Bl/6 mice within 5 min after contact. Killing of both bacterial strains by macrophages from C3Hf mice continued at a lower rate for the next 30 min until about 40% of the remaining bacteria were killed. In this later phase macrophages from C57Bl/6 mice killed avirulent S. typhimurium to an extent comparable with the killing by macrophages from C3Hf mice, whereas macrophages from C57Bl/6 mice were unable to kill virulent S. typhimurium. Cytochalasin B did not inhibit the rapid initial killing of bacteria opsonized with normal mouse serum, but completely inhibited the slower phase of killing. From these results it is concluded that the resistance of the mice to infection with S. typhimurium correlates with the bactericidal activity of their peritoneal macrophages, and that killing of the bacteria occurs in an early extracellular phase followed by an intracellular phase. It is only the latter phase which reflects the animal's resistance to infection. The chemiluminescence response to macrophages to opsonized live S. typhimurium was independent of the susceptibility of the mice from which the macrophages were taken. Cytochalasin B and 2-deoxy-D-glucose reduced the chemiluminescence generated by opsonized or non-opsonized S. typhimurium. Comparison of the kinetics as well as inhibition, by cytochalasin B and 2-deoxy-D-glucose, of chemiluminescence and killing of S. typhimurium showed that the killing reaction of the peritoneal macrophages was not related to their chemiluminescence response.