IgG-coated Sheep Red Blood Cells Research Articles

An Essential Role for TAGLN2 in Phagocytosis of Lipopolysaccharide-activated Macrophages

Activated macrophages have a greater ability of phagocytosis against pathogens that is mediated by large-scale actin rearrangement. However, molecular machineries that conduct this task have not been fully identified. Here, we demonstrate an unanticipated role of TAGLN2, a 22-kDa actin-binding protein, in Toll-like receptor (TLR)-stimulated phagocytosis. TAGLN2 was greatly induced in macrophages in response to lipopolysaccharide (LPS), a ligand for TLR4, partly via the NF-κB pathway. TAGLN2-deficient macrophages (TAGLN2−/−) showed defective phagocytic functions of IgM- and IgG-coated sheep red blood cells as well as bacteria. Cell signaling pathways involved in actin rearrangement—PI3 kinase/AKT and Ras-ERK—were also down-regulated in LPS-stimulated TAGLN2-deficient macrophages. Moreover, TAGLN2−/− mice showed higher mortality after bacterial infection than wild-type littermates. Thus, our results revealed a novel function of TAGLN2 as a molecular armament required for host defense.

An Essential Role for TAGLN2 in Phagocytosis of Lipopolysaccharide-activated Macrophages

Abstract Activated macrophages have a greater ability of phagocytosis against pathogens that is mediated by large-scale actin rearrangement. However, molecular machineries that conduct this task have not been fully identified. Here, we demonstrate an unanticipated role of TAGLN2, a 22-kDa actin-binding protein, in Toll-like receptor (TLR)-stimulated phagocytosis. TAGLN2 was greatly induced in macrophages in response to lipopolysaccharide (LPS), a ligand for TLR4, partly via the NF-κB pathway. TAGLN2-deficient macrophages (TAGLN2−/−) showed defective phagocytic functions of IgM- and IgG-coated sheep red blood cells as well as bacteria. Cell signaling pathways involved in actin rearrangement—PI3 kinase/AKT and Ras-ERK—were also down-regulated in LPS-stimulated TAGLN2-deficient macrophages. Moreover, TAGLN2−/− mice showed higher mortality after bacterial infection than wild-type littermates. Thus, current results provide a novel function of TAGLN2 as a molecular armament required for host defense.

Transfusion of antibody‐opsonized red blood cells results in a shift in the immune response from the red blood cell to the antibody in a murine model

It is well known that infusion of immunoglobulin (Ig)G-coated cells results in an inhibited antigen-specific humoral immune response compared to the cells themselves, a phenomenon termed antibody-mediated immune suppression (AMIS). Although this AMIS effect has been well described with many different types of cells as well as vaccines and insoluble antigens, the mechanisms behind this effect remain unresolved. To study AMIS in a broad context, three different models of AMIS were studied. In the first, mice were transfused with sheep red blood cells (SRBCs) versus IgG-coated SRBCs. In the second, SRBCs expressing the antigen hen egg white lysozyme (HEL) were studied, and the third model consisted of the diphtheria/tetanus vaccine in the absence versus presence of anti-tetanus IgG. The antibody responses to the SRBCs and HEL-SRBCs, as well as the vaccine, were analyzed for up to 4 weeks after challenge. In these mouse models of immunization, the IgG-coated RBCs or HEL-RBCs induced an antibody response against the IgG, rather than against the RBCs. The decreased response to the RBCs was directly related to the increase of the response against the IgG. The inhibitory AMIS effect using the vaccine strategy again showed an immune response against the IgG, concurrent with a decrease in the immune response against the specific vaccine component targeted. This work demonstrates that, under AMIS conditions, the IgG itself becomes the focus of B cells in the immune system, suggesting a potential mechanism of B-cell regulation.

Extracellular NAD is a regulator for FcγR-mediated phagocytosis in murine macrophages

NAD is available in the extracellular environment and elicits immune modulation such as T cell apoptosis by being used as the substrate of cell surface ADP-ribosyl transferase. However, it is unclear whether extracellular NAD affects function of macrophages expressing cell surface ADP-ribosyl transferase. Here we show that extracellular NAD enhances Fcγ receptor (FcγR)-mediated phagocytosis in J774A.1 macrophages via the conversion into cyclic ADP-ribose (cADPR), a potent calcium mobilizer, by CD38, an ADP-ribosyl cyclase. Extracellular NAD increased the phagocytosis of IgG-coated sheep red blood cells (IgG-SRBC) in J774A.1 macrophages, which was completely abolished by pretreatment of 8-bromo-cADPR, an antagonist of cADPR, or CD38 knockdown. Extracellular NAD increased basal intracellular Ca 2+ concentration, which also was abolished by pretreatment of 8-bromo-cADPR or CD38 knockdown. Moreover, the chelation of intracellular calcium abolished NAD-induced enhancement of phagocytosis of IgG-SRBC. Our results suggest that extracellular NAD act as a regulator for FcγR-mediated phagocytosis in macrophages.

Role for p40phox in Fcγ-Receptor-Induced NADPH Oxidase Activation.

The phagocyte NADPH oxidase catalyzes the production of superoxide, the precursor to microbicidal oxidants that are essential for normal host defense. The active enzyme complex includes a membrane-bound flavocytochrome b along with p47−phox, p67phox, and Rac-GTP, which translocate from the cytosol to the membrane to activate electron transport through the flavocytochrome upon cellular stimulation with soluble or particulate agonists. Translocation of p67phox is mediated by p47phox, which can be isolated from the cytoplasm prior to oxidase activation as a complex along with a third protein, p40phox. Although p40phox also translocates to the membrane of activated cells, its role in regulation of the NADPH oxidase has been unclear. p40phox is not required for reconstitution of high-level NADPH oxidase activity in cell-free systems or in heterologous whole cell models based in COS7 or K562 cell lines. It has recently been recognized that p40phox binds phosphatidylinositol-3-phosphate (PI(3)P), a product of type III phosphatidylinositol 3-kinase, via an N-terminal PX domain, and that PI(3)P is generated in high levels in the phagosomal membrane, where it recruits proteins important for phagosomal maturation. To examine whether p40phox might play a role in NADPH oxidase activation during phagocytosis, we introduced a cDNA for the human FcγIIA receptor via retroviral-mediated gene transfer into COS7 cells expressing the phagocyte flavocytochrome b, p47phox, and p67phox from stable transgenes. As previously shown by a number of groups for parental COS7 cells, heterologous expression of FcγIIA enabled “COSphox” cells to ingest IgG-coated sheep red blood cells (IgG-SRBC). However, NADPH oxidase activity was not detected, using nitroblue tetrazolium as a probe to detect superoxide production, which is otherwise evident by the accumulation of intraphagosomal formazan deposits in macrophages and neutrophils undergoing phagocytosis. However, numerous NBT-positive (formazan-stained) phagosomes were seen in COSphoxFcγR cells upon transient or stable transfection of an expression plasmid for p40phox. NBT-positive phagosomes were detected within five minutes in a synchronized phagocytosis assay. Upon transient expression of derivatives of p40phox which had point missense mutations in the PX domain predicted to prevent binding of PI(3)P, only rare NBT-positive phagosomes were detected in COSphoxFcγR cells ingesting IgG-SRBC. Transient expression of p40phox derivatives with point mutations in the SH3 domain, which disrupts a potential binding site for p47phox, or in the PC motif, which should disrupt binding to p67phox, resulted in a ~2-fold reduction in the numbers of NBT-positive phagosomes. Introduction of both the SH3 and PC mutations into p40phox resulted in only rare NBT-positive phagosomes following transient transfection of the double mutant and subsequent phagocytosis of IgG-SRBC. Thus, this study identifies a role for p40phox in coupling NADPH oxidase activation to phagocytosis of IgG-coated particles. Results of experiments using mutant derivatives of p40phox suggest that its ability to interact with both PI(3)P and p47phox/p67phox are critical determinants in this process.

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts.

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright's stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45-60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

Methods for studying immuno-effector functions and antigen presenting activity of human macrophages

Macrophages are now recognized as cells that perform a variety of functions making them important cells in both the specific and non-specific immune responses. In the inflammatory response mature monocytes leave the bloodstream to enter macrophages and then migrate to the site of infection. This process is usually co-ordinated by the production of so-called chemotaxins that direct the movements of the macrophages. This can be measured in vitro by a chemotaxis assay that separates macrophages from the chemotactic fluid by a micropore filter. After incubation migrated cells are counted at the other side of the filter and indicate the chemotactic response. At the inflammatory site phagocytosis and killing of invading bacterial micro-organisms is an important feature of macrophages. We describe an assay to measure phagocytosing capacity using IgG-coated sheep red blood cells and a bacterial killing assay. In addition a tumor killing assay is briefly introduced. Finally, the specific immune response is initiated by presentation of antigen to antigen specific T cells by specialized cells such as dendritic cells that are closely related to macrophages. An enrichment procedure for macrophages and dendritic cells is outlined and antigen presentation assays are described.

Suppressive Role of Sialylated N-Glycans in Fc Receptor-Mediated Phagocytosis by Macrophages

By culturing with tunicamycin A 1, an inhibitor of N-glycosylation, or by sialidase digestion, mouse monocytic cells P388D1 were induced to carry out Fc receptor-mediated phagocytosis of IgG-coated sheep red blood cells. There was no significant difference in the numbers of IgG-coated sheep blood cells bound to the cells at 4°C before and after exposure to tunicamycin or sialidase. These results suggest that sialylated N-glycans expressed on the cell surface have a suppressive role in the induction of phagocytosis, and their decreased expression or reduced sialylation results in acquisition of the phagocytic ability of the cells by affecting some processes involved in the ingestion of the particles bound to Fc receptors.

Processing inhibition of N-linked sugar chains associated with induction of Fc receptor-mediated phagocytosis in the mouse monocytoid cells.

The concanavalin A staining of cellular glycoproteins and the direct analysis of their sugar chains released by hydrazinolysis revealed that the processing of N-linked sugar chains of some glycoproteins is suppressed by exposure of mouse monocytoid cells P388D1 to dimethyl sulphoxide, which can induce Fc receptor-mediated phagocytosis. To elucidate the significance of altered glycosylation in inducing phagocytosis, the effects of exposure of the cells to processing inhibitors (swainsonine and castanospermine) were examined and it was found that the cells are induced to acquire an ability to ingest IgG-coated sheep red blood cells, depending on the dose of the inhibitors and incubation time. Analysis of the N-linked sugar chains liberated from cellular glycoproteins by hydrazinolysis confirmed that the processing of the sugar chains is suppressed by the two inhibitors as expected. Since no significant alteration was induced in protein synthesis and DNA synthesis after exposure to the inhibitors, it is suggested that the altered glycosylation of cellular glycoproteins may have some direct role in the induction of Fc receptor-mediated phagocytosis. The inhibitors did not affect the binding of the IgG-coated red blood cells to Fc receptors on the cells, non-specific phagocytosis of latex beads, and the contents of lysosomal enzymes, beta-glucuronidase and acid phosphatase. These results suggest that the glycosylation status of cellular glycoproteins influences some specific processes involved in the ingestion of the ligands bound to Fc receptors.

Inhibitory effect of beta-glucans on zymosan-mediated hydrogen peroxide production by murine peritoneal macrophages in vitro.

Effects of the pretreatment of murine peritoneal macrophages with several polysaccharides on the production of H2O2 induced with unopsonized zymosan were examined. Pretreatment with most of (1-->3)-beta-D-glucans for 6 h at 37 degrees C inhibited the zymosan-mediated H2O2 production by macrophages. The phorbol myristate acetate (PMA)-mediated H2O2 production was not affected by the pretreatment. The pretreatment of macrophages with (1-->3)-beta-D-glucans decreased the ability to ingest unopsonized zymosan, but did not affect the ingestion of IgG-coated sheep red blood cells (IgG-SRBC). These results suggested that the pretreatment with (1-->3)-beta-D-glucans interfered with the interaction of macrophages to zymosan and that the occupation of the receptor for the (1-->3)-beta-D-glucans inhibited zymosan-mediated production of H2O2 by macrophages. Chemical modification by substitution with carboxymethyl groups or hydroxyethyl groups of a (1-->6)-branched (1-->3)-beta-D-glucan reduced the inhibitory effect of pretreatment on zymosan-mediated H2O2 production. The above results indicated the possibility that murine peritoneal macrophages possess certain receptors for beta-anomeric glucans, and one ligand specificity of the receptors is to restrict the intact (1-->3)-beta-D-glucosyl back bone.

Transglutaminase Levels and Immunologic Functions of BCG-Elicited Mouse Peritoneal Macrophages Isolated by Centrifugal Elutriation

BCG-elicited mouse peritoneal macrophages were separated into three subpopulations by counterflow centrifugal elutriation. The three subpopulations were characterized on the basis of the level of a protein cross-linking enzyme, tissue transglutaminase. Subpopulation-3 consisted of large cells (greater than 95% esterase positive and greater than 90% viable) and had at least a fivefold higher transglutaminase activity (35 +/- 6 nmol/hr/mg) as compared to macrophages in subpopulation-1 (6 +/- 2 nmol/hr/mg) and at least a threefold higher enzyme activity as compared to subpopulation-2 (11 +/- 2 nmol/hr/mg). Subpopulation-3 also showed sevenfold higher phagocytosis of IgG-coated sheep red blood cells. The three subpopulations showed no difference in their ability to kill Listeria monocytogenes as determined by [3H]-thymidine release. Subpopulations-2 and -3 caused 90% inhibition of murine adenocarcinoma (EMT-6) tumor cell growth in the presence or absence of lipopolysaccharide. Subpopulation-1 had a poor ability to inhibit EMT-6 cell growth (29 +/- 12%). However, in the presence of lipopolysaccharide, this activity increased by at least threefold (92 +/- 7%). The three subpopulations showed no significant difference in their cytolytic activity against murine mastocytoma (P815) target cells in the presence or absence of lipopolysaccharide. These results suggest that tissue transglutaminase may have no significant role in bactericidal, tumoricidal, or tumoristatic function of macrophages; however, it might have some role in promoting the Fc-receptor-mediated phagocytic function of the macrophages.

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens.

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determined in vitro by a classical rosette assay in 12 postmenopausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, alpha-mannosyl [IgG(Con A)] or beta-galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P less than 0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of arachidonate metabolism on enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages.

We examined whether arachidonate metabolism exerted any influence on the enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages. Enhancement of the intracellular transglutaminase activity was observed on stimulation of macrophages with normal sheep red blood cells (SRBC) or immunoglobulin G (IgG)-coated SRBC, and was inhibited by inhibitors of phospholipase A2 and cyclooxygenase. Moreover, prostaglandin E2 (PGE2), a main product of the cyclooxygenase pathway, leukotriene B4 (LTB4), a product of 5-lipoxygenase, and arachidonic acid also could directly induce high levels of intracellular transglutaminase activity without stimulation of macrophages by SRBC or IgG-coated SRBC, but leukotriene C4, prostaglandin D2, and prostacyclin were unable to induce high activity of the enzyme. Enhancement of transglutaminase activity induced by LTB4 was inhibited by cyclooxygenase inhibitor, but the enzyme activity induce by PGE2 was not inhibited. Furthermore, the quantity of PGE2 released into the culture medium of macrophages stimulated with SRBC or IgG-coated SRBC correlated well with the activity of intracellular transglutaminase in macrophages. Moreover, enhancement of transglutaminase activity by treatment of macrophages with SRBC or IgG-coated SRBC was partially suppressed by sodium benzoate, which is a scavenger of hydroxy radical. These findings suggest that arachidonate metabolism, in particular the cyclooxygenase pathway, plays an important role in the enhancement of intracellular transglutaminase activity.

Effect of anticancer drugs on macrophage-mediated antibody-dependent cytotoxicity and secretion of reactive oxygen intermediates

The in vitro effects of methotrexate, vincristine, dexamethasone and adriamycin to modulate the antibody dependent cellular cytotoxicity (ADCC) reaction and secretion of superoxide (O 2 −) and hydrogen peroxide (H 2O 2) by phorbol myristate acetate (PMA) stimulated rat peritoneal macrophages were studied. Macrophage-mediated ADCC, as measured by the lysis of 51Cr-labeled IgG-coated sheep red blood cells in an 18 h assay, was significantly enhanced in the presence of 100 ng/ml of PMA. Treatment of macrophage monolayers for 24 h with vincristine (10 −5–10 −7 M), dexamethasone (10 −5–10 −9 M) and adriamycin (10 −6M) inhibited ADCC by PMA stimulated macrophages. Vincristine, dexamethasone and adriamycin also inhibited the secretion of O 2 − and H 2O 2 by PMA stimulated macrophages at similar concentrations that reduced the ADCC reaction. Methotrexate (10 −3–10 −7 M) did not inhibit either ADCC or O 2 − and H 2O 2 secretion by PMA stimulated macrophages. These results suggest that select anticancer drugs can inhibit macrophage mediated cytotoxicity of antibody coated target cells by suppressing the release of reactive oxygen intermediates by activated macrophages.

Peritoneal Macrophages in Short Dwell Time Effluent Show Diminished Phagocytosis

We investigated the effect of different i.p. dwell times of dialysis fluid on the composition and phagocytic capacity of the peritoneal cells. We examined dialysate after 1.5 and 15 h dwell time in 10 continuous cyclic peritoneal dialysis (CCPD) patients and after 8 h dwell time in 14 continuous ambulatory peritoneal dialysis (CAPD) patients. There was no significant difference in viability, composition, and Fc receptor positive cells among the peritoneal cells in the studied effluents of CCPD and CAPD patients. However, the yield of peritoneal cells and the phagocytic capacity of IgG-coated sheep red blood cells ([lgG]SRBC) by peritoneal macro phages in 1.5 h dwell time dialysate was significantly decreased when compared with 15 h dwell time dialysates. In addition, the total phagocytic capacity of peritoneal cells in 1.5 h dwell time dialysate is dramatically decreased when compared with the 15 h dwell time dialysate and 8 h dwell time dialysate of CAPD patients. These findings must be taken in consideration by performing three rapid exchanges after the diagnosis of a peritonitis.

Alteration of rat Kupffer cell function following mitomycin-C administration.

Cancer chemotherapeutic agents modify the human immune system in diverse ways including both immunosuppression and immunostimulation. We evaluated the effects of mitomycin-C on rat Kupffer cell phagocytosis, C3b receptor binding, and lysosomal enzyme activity. Kupffer cell cultures were greater than 95% pure. Phagocytosis of IgG-coated sheep red blood cells was demonstrated by 84% of control cells but by only 25% of cells isolated two weeks following mitomycin-C administration (p less than 0.0005). This depression in phagocytic ability had returned to control levels by four weeks posttreatment. Similarly, C3b receptor binding of IgM and complement coated sheep red blood cells was observed in 88% of control Kupffer cells, but declined to 47% at two weeks after drug administration (p less than 0.005) and returned to normal after four weeks. Lysosomal enzyme activity was not impaired by mitomycin-C. Histologically severe ulceration of the colon of treated animals was seen one and two weeks after drug administration, but healed by four weeks post-mitomycin-C treatment. Depression of macrophage function as a consequence of cancer chemotherapy may have important clinical consequences in host defense against bacteria and tumor metastases.

A low molecular weight, heat-labile factor enhances neutrophil Fc receptor-mediated lysosomal enzyme release and phagocytosis.

When exposed to solid-phase immune complexes, polymorphonuclear neutrophils (PMN) degranulate and release proteases capable of degrading the major structural macromolecules of the joint. Evidence indicates that the PMN response to such activators may be modified by factors present at the sites of inflammation. We have evaluated the effects of a low molecular weight factor present in synovial fluid from rheumatoid arthritis (RA) patients on Fc receptor-mediated PMN degranulation and phagocytosis. Synovial fluid samples from 11 RA patients were studied; 10 of them contained factor(s) which augmented phagocytosis and degranulation mediated through the Fc receptor. This factor(s) alone, however, did not initiate neutrophil degranulation: its MW is less than 10,000 daltons and it is unstable when heated to 56 degrees C. We also examined supernatants that were produced by coculturing adherent human mononuclear cells stimulated by IgG-coated sheep red blood cells with autologous nonadherent mononuclear cells. A factor(s) with properties similar to those found in the synovial fluids (i.e., heat-labile at 56 degrees C and capable of augmenting Fc receptor-mediated degranulation and phagocytosis) was found in the stimulated human mononuclear cell culture supernatants. Filtration studies indicated that the MW of this factor(s) is between 2,000 and 10,000 daltons. No such activity was detected in culture supernatants of unstimulated mononuclear cells. Production of the cytokine was blocked by cycloheximide, indicating that protein synthesis was necessary. This factor(s), by enhancing PMN degranulation, may augment PMN-mediated tissue destruction in diseases such as RA.

Effects of ethanol on human monocyte IgG Fc receptors.

The IgG Fc receptor function of human monocytes (Mo) exposed to ethanol in vitro was assessed by a rosette assay in which IgG-coated sheep red blood cells were used as test particles. Pre-incubation of Mo in autologous serum with ethanol (55 mM and above) for 15 min at 37 degrees C, caused a significantly lower percentage of Mo-forming rosettes. This reduction by ethanol was not observed when Mo were preincubated with ethanol in fetal calf serum or in serum-free conditions, but was present when Mo were pre-incubated with ethanol in serum-free media to which IgG (10-150 micrograms/ml) had been added. Donor-dependent differences were observed in the reduction of Mo-forming rosettes in the presence of autologous serum and ethanol. Mo allowed to form rosettes without ethanol were exposed to ethanol during a subsequent phagocytosis process. A dose-dependent inhibition of the internalization of test particles was found in Mo from all donors.

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice.

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.

Effects of retinoic acid on the differentiation of THP-1 cell lines containing aneuploid or diploid chromosomes.

The effects of retinoic acid on the differentiation of human monocytic leukemia cell lines containing aneuploid (THP-1-Cs5) or diploid chromosomes (THP-1-R) were studied and compared. The induction of cell adhesion to a substratum, phagocytosis of sheep red blood cells (SRBC) or IgG-coated SRBC, pinocytosis of dextran sulfate, and NBT dye reduction by the cells were examined. The occurrence of these processes was much greater in RA-treated THP-1-Cs5 cells than in RA-treated THP-1-R cells. Of all these functional activities, the most remarkable differences between the two cell types were seen for cell adhesion and phagocytosis of SRBC. Morphological changes in RA-treated THP-1-Cs5 cells were observed by light and electron microscopy. RA-treated THP-1-Cs5 cells had a moderately-developed Golgi apparatus, and abundant lysosomes, mitochondria and lipid droplets in the cytoplasm. Among various retinoids examined, RA was the strongest inducer of the differentiation of the THP-1-Cs5 cells into mature cells. These findings suggest that THP-1-Cs5 cells which contain aneuploid chromosomes are more efficiently functionally differentiated by RA than are THP-1-R cells.