Opposite effects of bacterial lipopolysaccharide on Fc-receptor-mediated phagocytosis of two bone marrow-derived macrophage cell lines, BDM-1 and BDM-1W3.

We have reported the isolation and characterization of three factor-dependent macrophage cell lines from bone marrow cells of C3H/HeN mice. We have since isolated a subclone, BDM-1W3, from one of these cell lines. We found previously that BDM-1W3 has a different sensitivity to bacterial lipopolysaccharide (LPS) for growth than its parental cell line, BDM-1. In this report, we show that LPS inhibits BDM-1W3 phagocytosis of antibody-coated sheep erythrocytes (Fc-mediated phagocytosis), whereas it enhanced Fc-mediated phagocytosis by BDM-1. It was observed that a loss of Fc-receptor capacity parallels a loss of phagocytic activity in LPS-treated BDM-1W3 cells. LPS stimulated phagocytosis of latex beads by BDM-1 and BDM-1W3, suggesting that Fc-mediated phagocytosis and phagocytosis of latex beads differ in their regulatory mechanisms. When BDM-1 cells were cultured with LPS, they underwent drastic morphological changes, whereas LPS-treated BDM-1W3 cells did not change significantly. Gamma interferon enhanced FC-mediated phagocytosis by BDM-1, while it has no significant effect on that by BDM-1W3. These cell lines should be useful for studying signal transduction mechanisms in LPS-mediated macrophage activation.     

Enhancement of macrophage immune and nonimmune receptor-mediated phagocytosis by a low molecular weight soluble factor from resident thymocytes

Macrophage phagocytic activity is regulated in part by products of activated T lymphocytes. We previously reported that a heat-stable soluble factor derived from resident (nonactivated) thymocytes increases murine peritoneal macrophage Fc-dependent phagocytosis. In the present study, we further investigate the effect of the thymocyte factor on immune and nonimmune receptor-mediated phagocytosis, Fc receptor expression, and its approximate m.w. After 4 days of incubation, cellfree thymocyte supernatant produced a mean (three experiments) 2.10-, 2.08-, and 1.97-fold increase in macrophage phagocytosis of C3-, IgG-, and tannic acid-treated erythrocytes, respectively. Macrophage IL 1 production was not enhanced by a similar concentration of thymocyte supernatant. The thymocyte factor(s) increased the number of IgG2a Fc receptors (FcRI) from 2.4 x 10(5) to 3.8 x 10(5) receptor sites per macrophage. The number of Fc receptors that bind IgG1 and IgG2b (FcRII) was not altered. The soluble factor(s) that increased Fc-mediated phagocytosis passed through both 6000- to 8000-dalton and 2000-dalton cutoff dialysis membranes and eluted from a Sephadex G-25 Fine column over a m.w. range of 200 to 1000 daltons, with a peak activity at 450 daltons. These data suggest that resident thymocytes enhance macrophage phagocytosis of opsonized and nonopsonized particles through the elaboration of a low m.w. substance(s).     

Biomphalaria glabrata: influence of calcium, lectins, and plasma factors on in vitro phagocytic behavior of hemocytes of noninfected or Schistosoma mansoni-infected snails.

In vitro phagocytosis of erythrocytes by hemocytes of B. glabrata, intermediate host of S. mansoni, is strongly influenced by calcium, several lectins, and plasma factors. Our results indicate that two different mechanisms of non-self-recognition in B. glabrata may occur: (1) In the presence of calcium, phagocytosis occurs in noninfected and in infected snails without involvement of any other substances, and hemocytes of schistosome resistant as well as those of susceptible snails are able to recognize and phagocytose the target cells. (2) In the absence of calcium, phagocytosis occurs if bridging molecules (heterologous lectins in our assays) were present for which effector and target cells possess binding sites or if target cells were plasma coated prior to the assays. In suspensions in homologous plasma, hemocytes of both snail strains, infected or noninfected, subsequently showed phagocytic activities of about 70-80%. Preincubation of target cells in homologous plasma resulted in similar high phagocytic activities of hemocytes even in the absence of plasma during the standard assay. In these assays, a significantly higher proportion of hemocytes of resistant snails phagocytosed plasma-opsonized erythrocytes, whereas hemocytes of susceptible snails internalized less erythrocytes per cell and needed 60 min to phagocytose at percentages equivalent to that of resistant hemocytes within 10 min. Preincubation of erythrocytes in resistant plasma significantly increased the subsequent phagocytic activity of susceptible hemocytes, whereas preincubation of erythrocytes in susceptible plasma decreased the phagocytosis level of resistant hemocytes.     

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG- coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.     

Effect of lipopolysaccharide on the stimulation of macrophage Fc-dependent phagocytosis by splenic B lymphocytes

Macrophage phagocytic activity is regulated by a variety of products derived from activated lymphocytes. It has been reported that nonactivated splenic B and T lymphocytes enhance macrophage glucose metabolism. In addition, the enhancement of macrophage glucose metabolism was further increased by direct effects of bacterial lipopolysaccharide (LPS) on B, but not T, lymphocytes. In the present study, the effect of purified murine splenic B and T lymphocytes on Fc-dependent phagocytosis by thioglycollate-elicited peritoneal macrophages in the presence or absence of LPS has been investigated. Fc-dependent phagocytosis was assayed by measuring the ingestion of 51Cr-tagged sheep erythrocytes. After 3 or 4 days in culture, nonadherent spleen cells (NASC) and B and T lymphocytes from C3H/HeN (LPS-responder) mice produced 92 +/- 27%, 83 +/- 13%, and 147 +/- 33% increases in C3H/HeJ (LPS-hyporesponder) macrophage phagocytic activity, respectively. A similar effect was observed in Balb/c mice. Cell-free supernatant from NASC and B lymphocytes precultured for 2 or 4 days produced a 74 +/- 20% and 157 +/- 42% increase in phagocytosis respectively. At concentrations which have been previously shown to markedly enhance the ability of splenic B lymphocytes to stimulate macrophage glucose metabolism, Escherichia coli K235 LPS (10 micrograms/ml) did not alter the stimulatory effects of any of the splenic lymphocyte populations on macrophage Fc-dependent phagocytosis. These data suggest that B lymphocytes produce a soluble factor(s) which stimulates macrophage phagocytosis. In addition, LPS has different effects on the regulation of macrophage phagocytic activity and metabolism by B lymphocytes.     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Ceramide 1-phosphate, a mediator of phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.     

Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.     

Adenosine receptors are expressed during differentiation of monocytes to macrophages in vitro. Implications for regulation of phagocytosis

Ingestion by phagocytes is known to be markedly enhanced by physiologic signals such as cytokines and extracellular matrix proteins which may be found in inflammatory sites. Little investigation has been made of mechanisms that may depress this increased rate of phagocytosis during resolution of inflammation. We show that adenosine can act as an inhibitor of phagocytosis by macrophages derived from in vitro culture of human peripheral blood monocytes. Adenosine (Ado) is equally effective at inhibiting IgG Fc and complement-mediated phagocytosis. However, Ado has no effect on phagocytosis by freshly isolated monocytes. Inhibition by Ado begins after 2 days in culture and reaches a plateau by 5 days; these kinetics of induction of inhibition of phagocytosis parallel an increase in specific Ado binding to the macrophage plasma membrane. Ado binds to cultured monocytes with a Kd of 6 microM. This affinity and the observation that 2-chloroadenosine and 5'-N-ethylcarboxamidadenosine are the most potent inhibitors of phagocytosis suggest that the Ado receptors expressed during monocyte differentiation are of the A2 type. The inhibition of phagocytosis may be mediated by cAMP, a second messenger coupled to A2 receptors in several cell types. Thus, plasma membrane expression of A2 receptors dramatically increases during monocyte differentiation in vitro. These data show that a potentially physiologic mediator can have very different effects on the function of monocytes and macrophages. This suggests a mechanism whereby phagocytic function at inflammatory sites can be down-regulated if and only if signals for the recruitment of new phagocytes have subsided.     

Polarization of endocytosis and receptor topography on cultured macrophages

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular- weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement- opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.     

AP-1 and ARF1 control endosomal dynamics at sites of FcR mediated phagocytosis

Phagocytosis, the mechanism of ingestion of large material and microorganisms, relies on actin polymerization and on the focal delivery of intracellular endocytic compartments. The molecular mechanisms involved in the formation and delivery of the endocytic vesicles that are recruited at sites of phagocytosis are not well characterized. Here we show that adaptor protein (AP)-1 but not AP-2 clathrin adaptor complexes are recruited early below the sites of particle attachment and are required for efficient receptor-mediated phagocytosis in murine macrophages. Clathrin, however, is not recruited with the AP complexes. We further show that the recruitment of AP-1-positive structures at sites of phagocytosis is regulated by the GTP-binding protein ARF1 but is not sensitive to brefeldin A. Furthermore, AP-1 depletion leads to increased surface levels of TNF-alpha, a cargo known to traffic through the endosomes to the plasma membrane upon stimulation of the macrophages. Together, our results support a clathrin-independent role for AP complexes in endosomal dynamics in macrophages by retaining some cargo proteins, a process important for membrane remodeling during phagocytosis.     

Oxidatively Damaged Erythrocytes Are Recognized by Membrane Proteins of Macrophages

Human erythrocytes incubated with an iron catalyst ADP-chelated Fe³⁺ undergo oxidative damage of the membrane including lipid peroxidation, protein oxidation, and protein aggregation, and become susceptible to recognition by human macrophages. In order to clarify the membrane components of macrophages responsible for the recogrution of the oxidized erythrocytes, binding of the oxidized cells to dot and Western blots of solubilized membrane of macrophages was investigated. The oxidized erythrocytes but not unoxidized cells bound to the dot blots. The binding was effectively inhibited by saccharide chains of band 3, a major glycoprotein of human erythrocytes, and lowered when the saccharide chains of band 3 were removed from the cell surface by pretreatment of the cells with endo-P-galactosidase which specifically cleaves the polylactosaminyl saccharide chains of band 3. The oxidized erythrocytes bound to the membrane proteins of macrophages with molecular mass of about 50, 80, and 120 kDa on Western blots depending on the saccharide chains of band 3 on their surface. The results suggest that the oxidatively damaged erythrocytes are specifically recognized by these proteins of macrophage membrane having saccharide binding ability.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

The inhibitory effect of rifamycin-SV on macrophage mediated erythrocyte lysis.

The semi-synthetic antibiotic rifamycin-SV (10 μg/ml) strongly suppressed macrophage locomotion and extracellular mediated cytotoxicity against syngeneic erythrocytes, while other important macrophage activities, such as pinocytosis and phagocytosis, were unaffected even at 50 μg/ml. Rifamycin was also found to block transferred cytotoxicity mediated by supernates from macrophage cultures under proper conditions to give a soluble macrophage cytolytic factor. Moreover, rifamycin increased target cell resistance against lytic stress caused by agents affecting membrane permeability and structure. It is suggested that rifamycin inhibits the macrophage-mediated cytolysis by a direct protective effect on the target cell itself, and not by acting on the macrophage or its product(s).     

Analysis of macrophage heterogeneity by their phagocytic activity

Heterogeneity of resident peritoneal macrophages was studied as revealed by their phagocytosis and binding of sheep erythrocytes opsonized by specific rabbit IgG. It has been shown that the macrophage heterogeneity is due to the presence of active and inactive cellular subpopulations. A mathematical model, based on the Poisson fitting of experimental histograms, was elaborated for the analysis of subpopulation composition of macrophage pool in the test of rosette formation and phagocytosis. The validity of the model was supported by physicochemical isolation of the macrophage subpopulation which was inactive in erythrocyte binding. The macrophage pool was separated into fractions by absorption--elution at different temperatures. Active and inactive macrophage subpopulations were found in all the fractions but in different ratios.     

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.     

The two-step leukocyte migration inhibition factor (LIF) assay. Its use in evaluation of cellular immune function in patients with immunodeficiency diseases.

Administration of interferon (IF) inducers to mice enhances the uptake of antibody-coated erythrocytes (EA) by peritoneal macrophages. To evaluate the role of induced IF in macrophage activation, serum IF titers and phagocytosis of EA by macrophages were determined in recombinant inbred (RI) mice inoculated with Newcastle disease virus (NDV). RI strains carry either a “high” or a “low” response allele for a gene that controls their IF titers induced by NDV. C × BH and C × BK strains, both high responders for IF induction, were also found to be high responders for enhancement of phagocytosis by NDV. Conversely, strains C × BD, C × BI and C × BJ, low responders for IF induction, were also shown to be low responders for phagocytosis of EA by macrophages. In contrast, phagocytosis of EA by macrophages from high responder C × BH mice and low responder C × BD mice was similarly enhanced by the administration of a lipopolysaccharide. When data from all NDV-inoculated mice were analysed, a significant correlation was obtained between serum IF titers and the percentage of macrophages that ingested four or more EA. The results are compatible with two main possibilities: (i) IF induced by NDV enhances phagocytosis of EA by macrophages; or (ii) a macrophage-activating factor different from IF is released together with IF in response to NDV and the activity of this factor correlates with serum IF titers.     

Influence of band 3 protein absence and skeletal structures on amphiphile- and Ca(2+)-induced shape alterations in erythrocytes: a study with lamprey (Lampetra fluviatilis), trout (Onchorhynchus mykiss) and human erythrocytes

Amphiphiles which induce either spiculated (echinocytic) or invaginated (stomatocytic) shapes in human erythrocytes, and ionophore A23187 plus Ca(2+), were studied for their capacity to induce shape alterations, vesiculation and hemolysis in the morphologically and structurally different lamprey and trout erythrocytes. Both qualitative and quantitative differences were found. Amphiphiles induced no gross morphological changes in the non-axisymmetric stomatocyte-like lamprey erythrocyte or in the flat ellipsoidal trout erythrocyte, besides a rounding up at higher amphiphile concentrations. No shapes with large broad spicula were seen. Nevertheless, some of the 'echinocytogenic' amphiphiles induced plasma membrane protrusions in lamprey and trout erythrocytes, from where exovesicles were shed. In trout erythrocytes, occurrence of corrugations at the cell rim preceded protrusion formation. Other 'echinocytogenic' amphiphiles induced invaginations in lamprey erythrocytes. The 'stomatocytogenic' amphiphiles induced invaginations in both lamprey and trout erythrocytes. Surprisingly, in trout erythrocytes, some protrusions also occurred. Some of the amphiphiles hemolyzed lamprey, trout and human erythrocytes at a significantly different concentration/membrane area. Ionophore A23187 plus Ca(2+) induced membrane protrusions and sphering in human and trout erythrocytes; however, the lamprey erythrocyte remained unperturbed. The shape alterations in lamprey erythrocytes, we suggest, are characterized by weak membrane skeleton-lipid bilayer interactions, due to band 3 protein and ankyrin deficiency. In trout erythrocyte, the marginal band of microtubules appears to strongly influence cell shape. Furthermore, the presence of intermediate filaments and nuclei, additionally affecting the cell membrane shear elasticity, apparently influences cell shape changes in lamprey and trout erythrocytes. The different types of shape alterations induced by certain amphiphiles in the cell types indicates that their plasma membrane phospholipid composition differs.     

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

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

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.