Yeast Particles Research Articles

Histamine release from mast cells during phagocytosis and interaction with activated neutrophils.

Histamine release from rat peritoneal mast cells was evaluated during interaction with IgG, C3b-bi and Concanavalin A-opsonized yeast particles and activated neutrophils. In contrast to others we could show no phagocytic uptake of yeast particles attaching to Fc, or Concanavalin A receptors on the mast cell. Attached yeast particles opsonized with C3b/bi were also poorly ingested (less than 10% of the attached particles.) Neither could we detect any significant histamine release. If, however, neutrophils were added to the mast cell-yeast particle complex, histamine release was induced particularly in the presence of Concanavalin A-yeast. We furthermore showed that phorbol myristate acetate-activated neutrophils and eosinophils initiated mast cell degranulation and histamine release. This release is not dependent on myeloperoxidase, but on other oxidative metabolites, since myeloperoxidase-deficient neutrophils also induce histamine release. These experiments show that mast cells exposed to immune complexes and activated neutrophils or eosinophils may augment the inflammatory response.

Effect of interferon on human neutrophilic granulocytes.

The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.

A quantitative microassay for leukocyte chemotaxis, using a microscopic slide system with complement-activating yeast particles as gradient source

A simple quantitative microassay was developed for studying polymorphonuclear leukocyte (PMNL) chemotaxis under conditions where the number of available cells is a limiting factor, e.g., pustules, neutropenia, small children and cerebrospinal fluid. PMNL suspensions are placed on glass slides to which fluorescein-labeled yeast particles have been fixed. After adherence, normal human serum is added to the slides. Owing to complement activation, a chemotactic gradient which attracts the adherent PMNL is formed around the yeast particles. The number of PMNL-associated yeast particles in the presence of normal serum is scored, and compared with cells migrating in the presence of inactivated serum or in the absence of serum. A locomotory index is calculated as the number of yeast particles associated with PMNL divided by the total number of yeast particles.

Bestatin treatment for the correction of granulocyte dysfunction in patients with recurrent furunculosis.

Bestatin, a new immunomodulator which is chemically well-defined, was examined for its capacity to enhance the phagocytic activity of neutrophilic granulocytes from patients with furunculosis. The ability of the granulocytes to ingest fluorescein-labelled yeast particles was significantly decreased in 19 patients with recurrent furunculosis (p less than 0.01). Oral administration of 40 mg bestatin to ten patients increased the phagocytic function of their granulocytes significantly (p less than 0.01).

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP).

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.

C3b Acceptors on macrophages: Inhibition of Fcγ-receptor-mediated phagocytosis by acceptor-bound C3b

The binding of nascent human C3b (i.e. the fragment of C3 just after trypsin cleavage) to mouse peritoneal macrophages was demonstrated by immune adherence. Acceptor-bound C3b could be detected longer than 24 h on the cell membrane. The rosette formation and phagocytosis of SRBC coated with anti-SRBC rat IgG was inhibited by preincubation of the cells with C3 and trypsin (15 min, 37 degrees C). However, the phagocytosis of opsonized yeast particles was not influenced by acceptor-bound C3b, proving that C3b-C3b acceptor interaction did not alter the function of C3b-receptors. Acceptor-bound C3b on the macrophages failed to mediate phagocytosis of human 0,Rh+ red cells having C3b-receptors.

Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay

Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluomemetry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. When extracellular pH was lowered a slight decrease in phagolysosomal pH was detected in macrophages but not in PMN. When measuring total fluorescence, interaction at pH 5.8 for PMN and at pH 4.4 for macrophages is recommended, since the intensity of extra- and intracellular fluorescence are equal under these conditions. Thirty different dyes were tested for dye exclusion and fluorescence quenching of FITC-conjugated yeast particles, and FITC-conjugated IgG. Because of the lysosomotropic effect of basic dyes, acid and direct dyes are preferable as quenching agents. We could not find physical or chemical properties of the dyes that correlated with their quenching effect. Heat aggregated IgG was used as an immune complex analogue in the development of the assay. Trypan blue (0.2 mg/ml) at pH 4.4 was found to be the best quenching agent of extracellular fluorescence when using ingested aggregated IgG. The technique offers a simple method of quantifying ingested protein aggregates and of studying heterogeneity in phagocyte populations.

The effect of Intralipid on mononuclear and polymorphonuclear phagocytes

Eight healthy subjects were given Intralipid, a soybean oil emulsion, 20% intravenously for 2 h. During the infusion a significant increase in the nitroblue tetrazolium-reduction of blood monocytes was noted. Preincubation of monocytes in vitro with Intralipid (20 to 100 mg/ml) for 30 min was found to increase the ability of the cells to migrate chemotactically and to phagocytize yeast particles. On the contrary, when neutrophilic granulocytes were preincubated with Intralipid in the same concentrations for 30 min. their nitroblue-tetrazolium-reduction, chemotactic and spontaneous locomotion, as well as their ingestion of yeast particles was depressed.

The stabilization of the C3b molecule in immune deposits by formalin

Formalin-fixed tissue may be used as substrate for immunohistochemistry after enzymatic treatment with proteases. However, whether the antigenicity of C3b is restored is controversial owing to the protease sensitivity of the native molecule. The antigenicity and protease sensitivity of formalin-fixed and non-fixed C3b were tested by using C3b-opsonized yeast particles and the immunofluorescence technique. The results indicate that formalin, probably due to cross-linking, stabilizes C3b molecules and renders them less susceptible to protease treatment. Failures o detect C3b in immune deposits in fixed tissues may be due to factors other than protease.

Modulatory effects of heat-labile serum components on the inhibition of phagocytosis by dexamethasone in peritoneal macrophage cultures

This investigation examined the effects of heat-labile serum substances on the suppression of yeast phagocytosis in dexamethasone-treated cultures of murine resident peritoneal macrophages. When 4–6 day old untreated control cultures were supplemented with either heat-inactivated (56°C, 30 min) or intact (non-heat-inactivated) fetal bovine serum, more than 90% of the macrophage population ingested at least 1 yeast particle during 15 min phagocytosis assays. In cultures treated with 10 −6 M dexamethasone, approximately 30% of the macrophages were phagocytic. In contrast, approximately 70% of the steroid-treated population consisted of phagocytes in cultures supplemented with intact serum. Medium shift experiments demonstrated that the type of serum present during the 15 min yeast phagocytosis assays, but not the 4–6 day incubation periods, determined the size of the phagocytic subpopulations in the treated cultures. Whereas the majority of control phagocytes ingested more than 8 yeast particles, most dexamethasone-treated phagocytes ingested far fewer than 8 particles regardless of the size of the phagocytic subpopulations. In contrast to yeast, the ingestion of latex particles was inhibited to the same extent in dexamethasone-treated cultures that contained either heat-inactivated or intact serum. Thus, dexamethasone action impairs the ability of macrophages to accumulate yeast particles even though the phagocytic subpopulation is larger in treated cultures containing intact serum. This larger subpopulation may result from the activation of the alternative complement pathway by yeast during phagocytosis.

Decrease in the phagocytic activity of peripheral monocytes in patients treated with human interferon-alpha.

The influence of daily intramuscular injections of 3 X 10(6) units of interferon-alpha (IFN-alpha) on the phagocytic activity of peripheral monocytes was studied in 28 tumor patients. One day after initiation of IFN therapy no major change in the capacity of monocytes to ingest yeast particles was observed. After 1 week, 3 months, 6 months, and 9 months of treatment, monocyte phagocytosis had decreased in the majority of the patients tested. In patients where natural killer (NK) cell activity was measured simultaneously with monocyte phagocytosis, a correlation between the degree of enhancement of NK activity and the degree of decrease in monocyte phagocytosis was observed.

Physicochemical and functional changes in human leukemic cell line HL-60.

The recently established human promyelocytic cell line HL-60 was induced to differentiate in the present of DMSO. During this process, physicochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two-phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b-opsonized yeast and phorbol 12-myristate 13-acetate (PMA) did not enhance the CL response conspicuosly until days 3-4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc- and PMA-dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.

Synthesis and assembly of hepatitis B virus surface antigen particles in yeast.

The surface antigens of hepatitis B virus (HBsAg) has been synthesized in the yeast Saccharomyces cerevisiae by using an expression vector that employs the 5'-flanking region of yeast alcohol dehydrogenase I as a promotor to transcribe surface antigen coding sequences. The protein synthesized in yeast is assembled into particles having properties similar to the 22-nm particles secreted by human cells.

Production of ethanol by solid particles of Saccharomyces cerevisiae in a fluidized bed

Carbon dioxide can be used as the fluid continuous phase for the fermentation of 10 to 40 % aqueous solutions of glucose into ethanol with Saccharomyces cerevisiae using a closed circuit consisting of a fluidized bed of small solid yeast particles, a cooled condenser for the sampling of water and ethanol and a blower. At 18°C, a fermentation of 12 μmoles glucose per min per g dry weight of yeast was achieved.

Modulation of polymorphonuclear leukocyte chemiluminescent response to the chemoattractant f-Met-Leu-Phe.

Upon the interaction between polymorphonuclear leukocytes (PMNL) and soluble or particulate matter, the cells become metabolically activated and produce chemiluminescence. To respond with chemiluminescence to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), the PMNL required conditioning of the cells at 22 degrees C or higher temperature prior to stimulus addition. This was not required when phorbolmyristate acetate or opsonized yeast particles were used as stimulus. When cell samples were obtained from PMNL suspensions during 240 min from preparation, the chemiluminescence signal triggered by f-Met-Leu-Phe progressively increased. Scatchard plot analysis of the chemiluminescence data indicated that storage of the cells resulted in modulation of both the number of functional receptors and the affinity for the receptor. The importance of receptor function control in the inflammatory process is discussed.

Leukocyte adherence inhibition test: Cellular requirements for the elaboration of a polymorph adherence inhibition activity

This paper documents a possible mechanism of the leucocyte adherence inhibition (LAI) test: After interaction with monocytes sensitized by a polyarthritic serum and a normal synovial extract, cultured T cells produced in the supernatant an activity able to modify the adhesiveness of polymorphs (PMN) to glass. After 90 min of contact with sensitized monocytes, T-cell supernatants induced the detachment of three times more PMN than did supernatants from T cells after a contact lasting only a few minutes. The 90-min supernatant caused a dramatic reduction of pseudopod extrusion at the PMN surface, as seen by scanning electron microscopy. The functional effect of the supernatant was able to enable 53% of PMN to engulf yeast particles compared to 30% of control cells. Therefore, it is suggested that the LAI test involves a sequential mechanism leading to the activation of PMN in such a manner that they lose their adherence to glass.

Changes in plasma-membrane-associated filaments during endocytosis and exocytosis in polymorphonuclear leukocytes

We examined the filaments associated with the cytoplasmic surface of the plasma membrane in rabbit exudate PMNs during phagocytosis of particles, or during “frustrated phagocytosis” with exocytosis of storage granules. Cells were plated onto yeast particles glued to coverslips with polylysine or onto coverslips coated with sheets of heat-agglutinated IgG. After periods ranging from 1 to 15 min, we disrupted the cells by a jet of salt solution and exposed their inner membranes. These broken cells were fixed immediately and processed for SEM. Whole cells were also prepared for SEM or TEM. At the site of PMN adherence to an opsonized yeast particle, a network of globular centers and thin, branched filaments appears on the cytoplasmic surface of the plasma membrane, while the outstretching lamellipodia contain a mesh of such filaments but no globular centers. Within 1 to 2 minutes, these structures disappear from the invaginating portion of the developing vacuole, and the cell's storage granules fuse with the barren membrane regions. These activities occur in rapid sequence over the vacuolar membrane after the first contact, until the phagocytosed particle is wholly encircled by a smooth, loose membrane, separated from the cell surface. A comparable filament pattern or complex was seen during “frustrated phagocytosis” on IgG sheets. At times between 1 and 5 min after plating, the cytoplasmic surfaces of these adherent membranes contain denuded central regions and peripheral nets of globular centers with radiating, thin, branched filaments. Granules apparently fuse with the bare areas. Thus we have obtained evidence of filament association with the plasma membrane at sites of adherence (to phagocytosable or nonphagocytosable surfaces) and have traced the subsequent disappearance of the filaments with degranulation.

Effects of filtrates from enterotoxinogenic Escherichia coli on some functions of human granulocytes and monocytes

The effects of filtrates from enterotoxinogenicEscherichia coli B609C on some functions of human granulocytes and monocytes have been studiedin vitro. A considerable increase in the nitroblue tetrazolium (NBT)-reduction was observed in granulocytes exposed for a short time (30 min) to subtoxic concentrations of the filtrate. The spontaneous and chemotactic motility of such cells was decreased, while the ability to ingest fluorescein-labelled yeast particles remained unchanged. A stimulation of the NBT-reduction was also observed in monocytes, but it was lower than that of granulocytes, and higher concentrations of the filtrates were needed. Contrary to the observation made for granulocytes, the spontaneous and chemotactic motility of monocytes increased after exposure to the active filtrates. The ingestion of yeast particles by monocytes was then increased as well. Heat-labile enterotoxin was at least partially responsible for the effects observed, since the stimulation was decreased by heat-inactivation of the filtrate or by neutralization with antiserum against enterotoxin.

Distribution of actin-binding protein and myosin in polymorphonuclear leukocytes during locomotion and phagocytosis

Polymorphonuclear (PMN) leukocytes, highly motile ameboid cells vital for mammalian defense against infection, acquire a distinct polarized morphology during locomotion and phagocytosis. An organelle-excluding pseudopod extends in the direction of movement and surrounds objects during phagocytosis. The anterior pseudopod contains a three-dimensional network of actin filaments. Actin-binding protein (ABP) and myosin cause the crosslinking and contraction, respectively, of actin filaments in vitro. We used indirect immunofluorescence to study the redistribution of myosin and ABP molecules in rabbit PMN leukocytes during locomotion and phagocytosis. In unpolarized PMN leukocytes, ABP and myosin had a diffuse distribution with some predilection for the cortex. In polarized PMN leukocytes crawling toward yeast particles, myosin and ABP staining concentrated in the anterior pseudopod. In PMN leukocytes fixed during phagocytosis of the yeast particles, antimyosin and anti-ABP staining concentrated strikingly in the distal portions of the pseudopod embracing the yeasts. Staining for catalase, a cytoplasmic protein in PMN leukocytes, for lactoferrin, a protein of specific granules, and for myeloperoxidase, a protein of azurophilic granules, was not concentrated in pseudopods. Taken together with available morphologic and biochemical information, these findings are consistent with a mechanism wherein interactions of actin, ABP and myosin redistribute cortical cytoplasm into pseudopods involved in locomotion and phagocytosis.

Alpha-1-antitrypsin-induced inhibition of complement-dependent phagocytosis

In a previous investigation, inhibition of complement-dependent rosette formation by alpha 1-antitrypsin (α 1-AT) was observed, and it was demonstrated that α 1-AT interacts through its carbohydrate portion with C3 and its fragments. In the present study, the effect of α 1-AT on the complement-receptor-mediated phagocytosis by human peripheral blood monocytes was examined. Purified α 1-AT inhibited in a dose-dependent manner phagocytosis of C3-carrying yeast particles. Inhibition was selective, concerned only C3-receptor-mediated phagocytosis, neither Fc-receptor-mediated phagocytosis nor uptake of untreated yeast particles was blocked by α 1-AT. It was demonstrated that α 1-AT exerted its inhibitory effect through binding to C3-carrying particles. The activity of α 1-AT towards C3 and fragments of C3 was not mediated by its antiprotease effect, but by its carbohydrate moiety. This finding suggests that α 1-AT may have an impact on various immune functions involving complement receptors.