Latex Phagocytosis Research Articles

Glucose Transporter in Plasma Membranes of Cultured Neural Cells, as Characterized by Cytochalasin B Binding

Identification of hexose transporter sites by cytochalasin B binding was conducted with a centrifugation assay. The determination of KD and Bmax values by LIGAND computer analysis provided binding data that are similar in primary astrocytes (238 nM and 14 pmol/mg protein) and neuroblastoma cells (179 nM and 13.6 pmol/mg protein). In contrast, only an insignificant number of transporter sites was detectable in C6 glioma cells, irrespective of whether membrane fractions were obtained by a two-phase polymer system or by a latex phagocytosis technique yielding inside-out plasma membranes. The latter membrane preparation was utilized to identify and quantitate the transporter molecules at the inner membrane surface of primary astrocytes, i.e., 160 nM (KD) and 5.8 pmol/mg protein (Bmax), respectively.

Mechanism of phagocytosis by Schwann cells

33B rat Schwannoma cell line is known to exhibit phagocytic properties analogous to those of normal Schwann cells. The mechanism of phagocytosis by this cell line was investigated by studying the effect of known modulators of phagocytosis on the uptake of latex particles by these cells. Treatments which block energy production of the host cell, such as incubation at 4°C and treatment with sodium azide, markedly inhibited the phagocytosis of latex particles by these cells. Phagocytosis was dose-dependently, and completely, inhibited by cytochalasin B, demonstrating an important role of microfilaments. Colchicine produced a minor inhibition of phagocytosis only at the highest concentration (10 −3 M) tested, suggesting that intact microtubules are not crucial for latex phagocytosis. Dibutyryl cyclic AMP was without any effect on the phagocytosis. Thus, latex phagocytosis by rat Schwannoma cells is an active, energy-dependent process requiring intact microfilaments with only a minor dependence on microtubules.

Asbestos-induced Modulation of Release of Regulatory Molecules from Alveolar and Peritoneal Macrophages

Asbestos inhalation causes several immunologic abnormali ties in exposed individuals and experimental animals, 1 Pernis B Vigliani EC Selikoff JJ Rheumatoid factor in serum of individuals exposed to asbestos. Ann NY Acad Sci. 1965; 132: 112 Crossref PubMed Scopus (52) Google Scholar , 2 Kagan E Solomon A Cochrane JC Bleissner EI Glukman J Rocks PH et al. Immunological studies of patients with asbestosis. Clin Exp Immunol. 1977; 28: 261 PubMed Google Scholar , 3 Bozelka BE Sestini P Gaumer HR Hammad Y Heather CJ Salvaggio JE A murine model of asbestosis. Am J Pathol. 1983; 112: 326 PubMed Google Scholar and it has been suggested that these phenomena have a role in the induction of pulmonary fibrosis and could be related to alterations of macrophage-mediated regulatory activities. 4 Pernis B Vigliani EC The role of macrophages and immunocytes in the pathogenesis of pulmonary diseases due to mineral dusts. Am J Ind Med. 1982; 3: 133 Crossref Scopus (22) Google Scholar To investigate whether asbestos could affect the release of immunoregulatory molecules from macrophages, we have studied the effect of the exposure in vitro of mouse resident alveolar (AMø) and peritoneal macrophages (PMø) to nontoxic concentrations of asbestos on their suppressive capacity on the mitogen-induced proliferation of syngeneic spleen lymphocytes, on their ability to release arachidonic acid metabolites and superoxide anion (O$$) in response to zymosan and on the spontaneous release of interleukin 1 (Il-1). PMø purified by adherence and AMø obtained by bronchoalveolar lavage from 8-16-week-old C3H/HeN mice were cultivated in flat-bottom wells in the presence of control medium, latex beads, or UICC asbestos amosite, 80 µg/ml, for 21 hours. The medium was then carefully aspirated and the Mø further processed. Suppressive capacity was evaluated as previously described 5 Sestini P Bozelka BE deShazo RD Salvaggio JE Murine alveolar macrophage-mediated lymphocyte cytostasis: kinetics and mechanisms. Cell Immunol. 1982; 30: 108 Google Scholar by adding syngeneic spleen cells stimulated with an optimal dose of Con-A and evaluating the incorporation of tritiated thymidine after 72 hours of culture. The release of O$$ was measured by ferricytochrome c reduction after stimulation with opsonized zymosan. Prostaglandin E2 (PCE2) and F2α, (PGF2α), and leukotriene C4 (LTC4) were evaluated by radioimmunoassay after stimulation with zymosan 200 µg/rnl in medium without serum. Il-1 was evaluated by the C3H/HeJ thymocyte proliferation assay. The presence of interleukin-2 (Il-2) activity in the supernatants was assayed by the ability to support the proliferation of the CTLL cell line. In agreement with previous experiments 6 Bozelka BE, Sestini P, Hammad Y, Salvaggio JE. Effects of asbestos fibers on alveolar macrophage-mediated cytostasis. Environ Res (in press) Google Scholar the exposure of AMø and PMø in vitro to amosite caused a dose-dependent decrease in their suppressive capacity, whereas phagocytosis of latex had no effect. This phenomenon was not due to loss of cell viability as judged by LDH release and trypan blue exclusion, and was paralleled by a marked reduction of the ability to produce Ol. Asbestos exposure caused a small increase of spontaneous PGE2 production from both Mø populations; however, it had different effects on the zymosan-induced release of PGE2 and PGF2α from AMø and PMø. In fact, while PMø showed a reduction of the release of these metabolites after exposure to asbestos compared with untreated cells, AMø showed a marked increase. The release of LTC4, however, was decreased in both Mø populations. Latex beads had minimal effects on these activities. In addition, amosite but not latex beads induced the release of Il-1 in cultures of AMø and PMø. Il-1 activity was present in the supernatants during the first 24 hours of culture, and the release continued during further incubation, even in serum-free medium. No Il-2 activity or dialyzable inhibitors were found in the supernatants.

Effect of intralipid ® on some immunological parameters and leukocyte functions in patients with esophageal and gastric cancer

This study has been undertaken to investigate if the intravenous (i.v.) infusion of fat emulsions may be associated with impairment of some immunological functions thus increasing the risk of septic complications. Fifteen malnourished patients with advanced gastric or esophageal cancer received for 2 weeks preoperatively and 1 week after surgery an isocaloric and isonitrogenous TPN treatment with Intralipid ® (group A: n=8) or glucose alone (group B: n=7) as energy substrate. Cluster analysis of 11 nutritional parameters and some tests of the humoral and cellular immunity (IgG, IgM, C3c, Factor B; polymorphonuclear (PMN) cells, total lymphocytes, T and B lymphocyte counts; ‘in vitro’ PMN chemotaxis, adherence to nylon fibers, phagocytosis of latex particles) were sequentially determined. The incidence and severity of post-operative infections were investigated and a ‘sepsis score’ was calculated for each patient. Pre- and postoperative TPN were not associated with an improvement of the nutritional status. The humoral and cellular immune parameters showed the same behaviour in patients receiving Intralipid ® and in controls. The chemotactic activity of PMN cells was constantly normal, granulocyte adherence fluctuated below the normality range in controls, whereas phagocytosis of latex was similar in both groups. Post-operative infectious episodes were less severe in patients receiving Intralipid. ® Our results do not confirm that Intralipid ® adversely affects some aspects of the humoral and cellular immune response.

Proliferation, kinetics, and fate of monocytes in rat liver during a zymosan-induced inflammation.

The fate and kinetics of monocytes, recruited to the liver by a single zymosan injection, were investigated by light (LM) and electron (EM) microscopy combined with peroxidase cytochemistry and latex phagocytosis. Ultrastructural and cytochemical differences between these cells and resident Kupffer cells persisted during a 7-day period, demonstrating the existence of two types of hepatic mononuclear phagocytes. Both cell types exhibited a pronounced mitotic activity during their numerical increase. Next to peroxidase-positive (POP) Kupffer cells and monocytes, peroxidase-negative (PON) mononuclear phagocytes were observed. These may represent a monocyte subset and/or possibly Kupffer cell precursors.

Cytochemical study of macrophage lysosomal inorganic trimetaphosphatase and acid phosphatase

Cytochemical investigations have associated acid inorganic trimetaphosphatase (TMPase) activity with the lysosomes of certain cell types. We have used the modified staining technique of Berg to show that this enzyme activity is present in normal mononuclear phagocytes and macrophage cell lines. We have found this enzyme activity to be present in murine RAW264 macrophages, in human U937 macrophages, in normal human blood monocytes, and in guinea pig peritoneal macrophages. All of the RAW264 and U937 macrophages showed intense TMPase activity. Many of the human monocytes and most of the guinea pig macrophages were labeled by this method. The reaction product was associated with the lysosomes of these cell types. The lysosomal staining pattern was similar to that of acid phosphatase. Differences with regard to Golgi staining were noted. This indicates that TMPase is a lysosomal enzyme of mammalian macrophages. The distinction between TMPase and acid phosphatase activity has been demonstrated by measuring the pH optimum of each enzyme. Using substrates identical to those of the ultrastructural cytochemistry, we show that the pH optimum of TMPase is 4.0 and that of acid phosphatase is 5.0. The enzymatic activities are therefore ultrastructurally and biochemically distinct. Following phagocytosis of latex, yeast ( Saccharomyces cerevisiae), or Corynebacterium parvum), TMPase has been found to be associated with phagosomes. This enzyme may take part in the degradation of phagocytosed materials, particularly microorganisms which contain inorganic polyphosphates and metaphosphates.

Lymphoid cells in the venous blood of 100 female patients with breast carcinoma. Follow-up studies

The following blood cells were examined in 100 patients with invasive carcinoma of the breast and 100 control patients without malignant disease: leukocytes, lymphocytes, monocytes and different sub-populations of lymphoid cells. Lymphoid cells were characterized by E-, EA- and EAC-rosetting technique, alphanaphtyl acetate esterase activity and by the ability of latex phagocytosis. Both groups showed no significant differences. In 81 patients undergoing postoperative radiation therapy lymphocytes and lymphoid cells were reduced considerably. These changes were still evident after 12 months (6 months after completion of radiotherapy). Mainly the T lymphocytes were effected by this decrease. A negative effect of the radiation induced reduction of immune competent cells must be discussed and examined further by clinical trials.

A latex phagocytosis procedure for the isolation of inside-out plasma membranes from cultured cells of neural origin

A latex phagocytosis procedure for the isolation of inside-out plasma membranes from cultured cells of neural origin

Redistribution of ecto-5'-nucleotidase during phagocytosis by guinea pig polymorphonuclear leukocytes.

We demonstrate that 5'-nucleotidase (5'NT), an ectoenzyme of guinea pig polymorphonuclear leukocytes, is largely excluded from phagosomal membrane, rather than internalized randomly during phagocytosis of heat-killed bacteria, latex microbeads, or zymosan particles. Cells were fixed in 0.25% glutaraldehyde (pH 6.3) at 4 degrees C for 10 min and incubated in a cytochemical medium for the demonstration of 5'NT. In the nonphagocytosing cells, 5'NT was evenly distributed on the external side of the plasma membrane. In cells phagocytosing bacteria, 5'NT appeared to be cleared from the nascent phagosomal membrane; after 5 min of phagocytosis, most of the phagocytic vacuoles containing bacteria, latex, or zymosan particles were devoid of reaction product. When phagosomes containing latex particles were isolated and biochemically assayed, they contained less than 3% of the total cellular 5'NT activity even after 60 min of phagocytosis, and at that time the total cellular 5'NT activity had not declined. When the diazonium salt of sulfanilic acid (DASA), a nonpermeable ectoenzyme inhibitor, was used to determine the distribution of extracellular and intracellular 5'NT activity, no increase in DASA-insensitive intracellular 5'NT was found after phagocytosis of latex or opsonized zymosan. Cytochemical and biochemical evidence led us to conclude that 5'NT is excluded from phagosomal membrane, and that the exclusion is due to redistribution rather than to inactivation by granule enzymes.

Relationship between phagocytosis and immunoglobulin A release from human colostral macrophages.

Macrophages and neutrophils that contain mainly secretory immunoglobulin A (IgA) comprise the majority of cells in human colostrum. These cell populations were separated and analyzed for their ability to release total IgA and secretory IgA when stimulated to phagocytose. Colostral macrophages phagocytosed opsonized bacteria and nonopsonized latex particles; at the same time, IgA was released. Neutrophils poorly phagocytosed opsonized bacteria but actively phagocytosed latex particles. In contrast to the macrophages, the neutrophils did not release IgA, even after active phagocytosis of latex. Consequently, colostral macrophages are the main source of IgA released from colostral leukocytes when these cells are exposed to organisms or particles that are phagocytosed. A function for colostral neutrophils which sequester IgA is proposed.

Monocyte functions in diabetes mellitus.

The aim of this study was to investigate the functions of monocytes obtained from 14 patients with diabetes mellitus (DM) compared with those of monocytes from healthy individuals. It was found that the total number of circulating monocytes in the 14 diabetic patients was lower than that from the healthy individuals. Phagocytosis of Candida albicans was decreased in the monocytes from the patients, whereas pinocytosis of acridine and phagocytosis of latex and sheep red blood cells were normal. The chemotactic response towards casein was enhanced. The possible consequences of these findings for the elucidation of concomitant infections in diabetic patients are discussed.

Isolated epithelioid cells from disaggregated BCG granulomas - some functional studies.

Isolated epithelial cells from disaggregated rat BCG granulomas show a morphological spectrum from "plasmacytoid" to "vesicular" froms on electron microscopy. Functional studies on these isolated cells showed that all epithelial cells exhibited poor trypsin-resistant glass adherence and poor phagocytosis of latex, zymosan, antibody coated and complement coated and complement coated erythrocytes when compared with macrophages, especially at the vesicular end of the spectrum. A high proportion of all epithelioid cell types was found to have Fc surface receptors but C3 receptors were reduced on vesicular cells compared with the plasmacytoid variety. The findings indicate that the progressive transformation of mononuclear phagocytes into vesicular epithelioid cells through plasmacytoid intermediates is accompanied by a concomitant modification of cell function. The significance of these changes is discussed.

Influence of Various Detachment Procedures on the Functional State of Cultured Murine Macrophages

Macrophages were obtained from seven-day-old bone marrow liquid cultures to which a colony-stimulating factor from L-cell-conditioned medium had been added. It was found that the yield of macrophages from the bone marrow liquid culture was dependent on the type and brand of culture dishes. Highest yields were obtained in teflon membrane bag cultures. The cumulative yield after serial passage of macrophages was 700-fold of the cell input after two months. After mechanical detachment from plastic culture dishes, the survival rate of cells was related to the brand of the plastic dish. Viability also varied greatly after pretreatment of cells with scandicain (28 % ), proteinase K (78 %), or pronase (99 %). After mechanical detachment of macrophages or pretreatment with scandicain, adherence to plastic, latex phagocytosis and chemotaxis was not or only slightly impaired, whereas the same functions after protease treatment were greatly reduced or even abolished and were only recovered after another culture period. Scandicain, proteinase K, and pronase treatment was mitogenic to macrophages in contrast to mechanical detachment. Pronase treatment also induced release of plasminogen activator activity. The results demonstrate that macrophages respond extremely sensitive to environmental conditions and to various insults by changing their functional state.

Latex phagocytosis by polymorphonuclear leukocytes: Role of sialic acid groups

Polystyrene latex particles are rapidly phagocytized by rabbit polymorphonuclear (PMN) leukocytes. The uptake is influenced by macromolecules which have the effect of altering the surface charge of the latex particle. The influence of polylysines of varying chain length on the surface charge of latex particles and of PMN cells was studied by micro-electrophoresis. Charge reversal at the latex surface was found to occur at concentrations considerably below that at which the surface charge of the PMN cells is reversed. Phagocytosis of latex by PMN cells is enhanced in the presence of low concentrations of long-chain polylysines. The enhancement of phagocytosis is strongly reduced if PMN cells are treated with neuraminidase. This suggests participation of siliac acid groups in a stage of particle-cell interaction which precedes engulfment.

Degradation of connective tissue matrices by macrophages. III. Morphological and biochemical studies on extracellular, pericellular, and intracellular events in matrix proteolysis by macrophages in culture.

We have shown that macrophages in culture degrade the glycoproteins and amorphous elastin of insoluble extracellular matrices. Ultrastructural observation of the macrophage-matrix interaction revealed that connective tissue macromolecules were solubilized from the matrix extracellularly. At least part of the matrix breakdown was localized to the immediate vicinity of the cells, as shown by morphological and biochemical studies, although the rate of degradation correlated closely with the secretion of proteinases by various inflammatory stimuli in vivo, by glucocorticoids, prostaglandin E2 or colchicine, or by phagocytosis of latex, zymosan, or cholesterol-albumin complexes in culture was reflected in altered rates of glycoprotein and elastin degradation by the macrophages. Alteration of endocytosis and lysosomal digestion by cytochalasin B, NH4Cl, and proteinase inhibitors did not decrease the overall rate of matrix solubilization, but reduced the processing of the matrix fragments to peptides. Therefore, extracellular, pericellular, and lysosomal events each contribute to degradation of extracellular matrix macromolecules by inflammatory macrophages.

Human peripheral null lymphocytes II. Producers of type‐1 interferon upon stimulation with tumor cells, Herpes simplex virus and Corynebacterium parvum

Human blood lymphocytes, exposed for 6 to 24 h in vitro to tumor cells (K 562, IGR3, L1210), Herpes simplex virus type 1 (HSV) or Corynebacterium parvum (CP), produced high levels of anti-viral activity which was identified as type-1 interferon (IF). In mixed lymphocyte tumor cell cultures (MLTC), the generated type-1 IF was definitely shown to originate from the lymphocytes and not from the tumor cells. Supplementation of leukocyte cultures with 10% fetal calf serum instead 10% human AB serum had little influence on tumor cell-induced IF production, but strongly reduced CP-induced IF production. Lymphocyte fractionation procedures involving iron/plastic treatment, nylon wool columns, Ig-anti-Ig columns and rosette (E, EA) separation led to the identification of null cells as highly efficient producers of type-1 IF. T cells obtained by different ways (E-rosette sedimentation, passage through 1 nylon and 2 Ig-anti-Ig columns, or thoracic duct lymphocytes) were poor IF producers in response to tumor cells, HSV and CP, but secreted anti-viral activity when stimulated with phytohemagglutinin. In MLTC, the level of generated type-1 IF roughly stimulated with phytohemagglutinin. In MLTC, the level of generated type-1 IF roughly paralleled nautral killer (NK) cell activity. Evidence is presented that type-1 IF can be produced by an Fc receptor-negative null cell subset, whereas NK activity requires Fc receptor-positive cells. It is suggested that production of type-1 IF represents one of the earliest functions in the differentiation process of mononuclear phagocytes and is likely to develop before the appearance of Fc receptors, diffuse esterase staining and latex phagocytosis.

Implications in the burn neutrophil of serum and cellular zinc levels

A parabolic correlation between leukocyte zinc content and phagocytic capacity apparent in clinical studies has been tested in a rat model. Dietary zinc restriction decreased average serum zinc levels from control values of 1.65 ± 0.41 to 0.70 ± 0.28 μg/ml during the first 13 weeks of restriction ( P < 0.0005) and to 0.56 ± 0.23 μg/ml during the second 13 weeks ( P < 0.0005). A parabolic variation in latex phagocytosis was observed in rat neutrophils during 33 weeks of zinc restriction. During study Weeks 12 through 21 a trend toward improved neutrophil antibacterial activity corresponded with a period of increased phagocytic activity in zinc-deficient rats. After Week 27 an opposite trend of impaired antibacterial activity corresponded to significantly decreased phagocytosis relative to controls ( P < 0.0005). This study suggests that acquired neutrophil functional defects may be corrected by replacement of specific nutritional components.

A functional assessment of macrophages from osteopetrotic mice.

Macrophages from osteopetrotic mice caused less bone resorption than normal sib macrophages in culture. The bone-resorbing activity lay in the culture supernatant, and reduced activity in cultures of macrophages from osteopetrotic mice correlated with reduced glass spreading and reduced latex phagocytosis. We suggest that macrophages from osteopetrotic mice show defective phagocytic recognition of glass, latex, and bone and that this defective recognition may reflect the cause of the failure of resorption which results in osteopetrosis.

Ultrastructural Alterations and Phagocytic Function of Cryopreserved Platelets

Fresh human platelets and platelets cryopreserved in 4% dimethylsulfoxide were examined ultrastructurally before and after incubation in a suspension of latex particles. Cryopreserved platelets had fewer discoid forms than fresh platelets. The cryopreserved platelets had many sphered platelets containing an increased number of vacuoles; the sphered platelets were more electron-lucent margination and pallor of organelles. Phagocytosis of latex into vacuoles was markedly impaired in the cryopreserved platelets. The morphologic alterations suggested that the freezing, thawing, and washing procedures reduced the functional activity of platelets.

Spontaneous Cell-Mediated Cytotoxicity (SCMC) Associated with Lymphocytes Negative for Acid α-Naphthyl Acetate Esterase (ANAE) Activity

A modified histochemical procedure for nonspecific acid α-naphthyl acetate esterase (ANAE) activity in human lymphocytes was used to identify a subpopulation of E-rosette forming cells. Performing a one hour reaction at pH 6.5 the distinct dot-like staining pattern was almost exclusively observed on high affinity E-rosettes which sedimented readily in a regular Ficoll-Metrizoate gradient. By combining latex phagocytosis with staining for ANAE activity, a clear-cut distinction between mononuclear phagocytes and lymphocytes could be made. An attempt was undertaken to relate the ANAE marker on human lymphocytes to their functional capacity in spontaneous cell-mediated cytotoxicity (SCMC) reactions. Using as targets two allogeneic (K562,IGR3) and a xenogeneic cell line (L1210) it could be clearly demonstrated that high SCMC activity is mediated by ANAE negative mononuclear cells, whereas enrichment for ANAE positive lymphocytes resulted in a loss of SCMC.