Interferon suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macrophages: related changes in cytoskeletal organization

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

P2Y4 Receptor-Mediated Pinocytosis Contributes to Amyloid Beta-Induced Self-Uptake by Microglia

Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y₁₂ purinergic receptors as triggers for microglial chemotaxis and P2Y₆ receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y₄ receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y₄ receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a “drink me” signal for microglia and P2Y₄ receptors as a potential therapeutic target for the treatment of Alzheimer''s disease.     

Fc gamma R-mediated phagocytosis stimulates localized pinocytosis in human neutrophils

Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcgammaR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcgammaR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca(2+) concentration were prevented. Because stimulation of FcgammaR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcgammaR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca(2+) dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcgammaR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Changes of Endocytotic Activities during the Cell Cycle of Dictyostelium Cells

Changes of endocytotic activities during the cell cycle of the cellular slime mould Dictyostelium discoideum Ax-2 were examined using the temperature-shift method for inducing synchronous growth. The activity of fluid-phase pinocytosis (FPP) was altered Ca²⁺-dependently and stimulated by EGTA. On the other hand, pinocytosis was greatly enhanced by addition of Bacteriological-peptone(BP) to the growth medium for Ax-2 cells, irrespective of the extracellular Ca²⁺-concentration. The maximal pinocytotic activity was attained in the presence of EGTA plus BP, the effects of the two substances being additive. The FPP activity was found to be high in cells in and just after the S phase, when the BP-induced fraction of pinocytosis was rather low. Thus the total activity for pinocytosis in the growth medium remained almost constant throughout the cell cycle, indicating that the rate of nutrient uptake through pinocytosis was not a limiting factor for cell cycle regulation. The change of phagocytotic activity during the cell cycle was somewhat similar to that of the FPP activity. Possible mechanisms of such cell-cycle related changes are discussed in relation to cytoskeletal proteins in the cell cortex. Some properties of BP-induced pinocytosis are also described.     

Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii

Acanthamoeba, with high rates of phagocytosis and pinocytosis of the non-concentrative type, offers favorable experimental material for investigation of similarities and possible differences in these two modes of uptake. Phagocytosis was measured by the rate of uptake of latex beads and pinocytosis by the rate of uptake of radioactive inulin and albumin. The effects of the metabolic inhibitors NaN₃, NaCN, NaF, iodoacetate, 2,4-dinitrophenol and cold were found to be identical on both forms of endocytosis. Both endocytic processes were suppressed by inhibitors of aerobic metabolism and low temperature and were not appreciably affected by inhibitors of glycolysis. The cells recovered capacity to endocytose after exposure to all these compounds except 2,4-dinitrophenol, which was irreversibly toxic. Endocytosis and O₂ consumption were measured as a function of temperature. Below 5 °C both phagocytosis and pinocytosis ceased; between 9 and 15 °C uptake was less than 10% that at 29 °C. From 16 to 29 °C uptake was a linear function of temperature for both pinocytosis and phagocytosis. Curves for O₂ consumption and endocytosis both showed breaks at about 16 °C. Concanavalin A (ConA) inhibited both types of endocytosis more than 50% at concentrations as low as 5 μg/2 × 10⁵ cells/ml. Pinocytosis and phagocytosis were also measured simultaneously in the same cells. Increasing the rate of phagocytosis suppressed pinocytosis, but the combined volume of the two forms of uptake was essentially constant. In contrast, the estimated combined surface intake varied over a two-fold range. These data show no differences between phagocytosis and pinocytosis of the non-concentrative type, and suggest that control of the rate of endocytosis is determined by the volume of an internal compartment. The volume of this compartment, estimated by measuring the volume of latex beads that “saturate” the phagocytic mechanism, amounted to about 500 μm³/cell or roughly 15% of the cell volume.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.     

Calcium requirement for efficient phagocytosis by Dictyostelium discoideum

Extracellular EDTA suppressed in a dose-dependent manner the phagocytosis of yeast particles by Dictyostelium discoideum cells. Activity was restored fully by the addition of Ca(2+), and partially by the addition of Mn(2+)or Zn(2+), but Mg(2+)was ineffective. The pH-sensitive, Ca(2+)-specific chelator EGTA also inhibited phagocytosis at pH 7.5, but not at pH 5, and Ca(2+)restored the inhibited phagocytosis. In contrast, pinocytosis was unaffected by EDTA. Consistent with the idea that Ca(2+)was required for phagocytosis, D. discoideum growth on bacteria was inhibited by EDTA, which was then restored by the addition of Ca(2+). It is concluded that Ca(2+)was needed for efficient phagocytosis by D. discoideum amoebae. A search for Ca(2+)-dependent membrane proteins enriched in phagosomes revealed the presence of p24, a Ca(2+)-dependent cell-cell adhesion molecule-1 (DdCAD-1) that could be the target of the observed EDTA and EGTA inhibition. DdCAD-1-minus cells, however, had normal phagocytic activity. Furthermore, phagocytosis was inhibited by EDTA and rescued by Ca(2+)in the mutant just as in wild type. Thus, DdCAD-1 was not responsible for the observed Ca(2+)-dependence of phagocytosis, indicating that one or more different Ca(2+)-dependent molecule(s) was involved in the process.     

Effect of arsenic on cell growth of the cellular slime mould, Dictyostelium discoideum

The growing D. discoideum cells were killed in a dose-dependent manner when exposed to 100 and 140 ppm of arsenic (As2O3) at mid-log phase for 20 min. Reduced plaque sizes and changed cell and colony morphologies were observed in the treated cells. Endocytotic functions (both phagocytosis and pinocytosis) were also inhibited in the treated cells. Arsenic treated cell showed a lower DNA and protein synthetic activities. These findings are discussed in relation to known mechanism of action of the heavy metal on growth-related cellular functions.     

Effects of induction of endocytosis on adhesiveness ofAmoeba proteus

Summary The adhesive behaviour ofAmoeba proteus during induction of endocytosis has been studied with interference reflection microscopy. Although phagocytosis and pinocytosis, jointly termed endocytosis, are closely related phenomena, it was found that the way they affect adhesiveness of amoebae differs substantially. Phagocytosis is accompanied by an increase in area of cell surface contacting the substratum, whereas during pinocytosis a sharp decrease of contact area is observed. The resistance to detachment increases in prey-stimulated and phagocytosing amoebae but declines as the pinocytosis is being induced. These results suggest that the influence of the induction of phagocytic activity on the cell periphery differs from that of induction of pinocytosis.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Inhibition of endocytotic functions in Dictyostelium discoideum treated with a carbamate pesticide

Administration of a carbamate pesticide carbaryl (1-Naphthyl-N-methyl carbamate) at a concentration of 60 and 100 ppm greatly inhibits the endocytotic functions during growth of the cellular slime mold D. discoideum. The ingestion of fluorescien isothiocynate (FITC) labeled E. coli is reduced between 30 and 40% in the treated cells as compared to controls. Similarly, the uptake of FITC-labeled dextran, which has been used as fluid-phase marker for pinocytosis also show 40-50% inhibition in the treated cells. 3H-leucine uptake and incorporation are also inhibited in the treated cells. SDS-PAGE analysis of cytoskeletal proteins shows a higher actin association with the membrane of treated cells. The results demonstrate the detrimental effects of Carbamate on the soil microbe even at a very low concentration and the efficacy of the slime mold cells as a biosensor for the carbamate-induced cytotoxicity.     

Pinocytosis in Dictyostelium discoideum cells : A possible implication of cytoskeletal actin for pinocytotic activity

Pinocytosis in Dictyostelium discoideum axenic strain (Ax-2) cells in the growth phase is progressively inhibited at higher Ca2+ concentrations, the activity being maximal at submicromolar Ca2+ concentrations. The cytoskeletal actin content is also markedly reduced in the presence of 10 mM EGTA. This was confirmed by electronmicroscopy using intact cells and Triton X-100-insoluble cell cortices. Interestingly, the pinocytotic activity seemed to be somewhat increased in response to cytochalasin B (CB). Aggregation-competent Ax-2 cells which are usually devoid of pinocytotic activity can resume their activity considerably following treatment with 10 mM EGTA. Under these conditions, cytoskeletal actin declines markedly, as also was the case for growing Ax-2 cells. Our findings indicate a correlation between the pinocytotic activity and presence of cytoskeletal actin: reduced amounts of actin in the cell cortex seem to favour pinocytosis. Conceivably, membrane-associated actin filaments may function as a powerful anchor, restricting the flexibility of the cell membrane and thereby inhibiting the pinosome formation. Other properties of pinocytosis like a developmental change as well as the effects of pH and temperature are also described and were compared with the properties of wild-type strain, NC-4.     

AMPKα promotes basal autophagy induction in Dictyostelium discoideum

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5′-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα⁻ cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα⁻ cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα⁻ cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK–ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, β-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα⁻ cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.     

Effect of a mushroom toxin, orellanine, on the cellular slime mold Dictyostelium discoideum and the bacterium Escherichia coli

Abstract Orellanine, the toxic principle of Cortinarius orellanus efficiently inhibited the growth of the amoebae Dictyostelium discoideum . No significant effect on phagocytosis or pinocytosis was observed. The growth of the bacterium Escherichia coli was inhibited with a sensitivity similar to that of D. discoideum .     

Histology of digestion in nymphs of Rhipicephalus appendiculatus fed on rabbits and cattle naive and resistant to the ticks

Nymphs feeding on ears of four rabbits and four calves (Bos taurus) were examined during first and third or fourth infestations and also during the moulting period. The gut caecae were removed and examined by histochemistry and light and electron microscopy. Attachment sites of the nymphs were biopsied from all hosts and cell counts made by light microscopy. Resistance was expressed by reduction in numbers of ticks completing engorgement and reduced engorgement weights. The gut was comprised of a proliferative stem cell; a digestive cell that differentiated into a sessile type ingesting by pinocytosis and a motile type ingesting by phagocytosis; and a cell secreting a glycoprotein with acid phosphatase activity into the lumen. The gut grew during the early stages of feeding to accommodate the expansion during engorgement. On rabbits and cattle resistant to ticks the stem cells were damaged, with moribund nuclei and poorly differentiated cytoplasm. Thus there were fewer digestive and secretory cells and the gut did not expand to accommodate a full blood meal. The attachment sites were dominated by mononuclear cells and neutrophils in both host species at the first infestations but at the third or fourth infestations there was a considerable increase in proportions of eosinophils and basophils. Host granulocytes were traced to the lumen of the tick gut and to motile digestive cells which destroyed them by phagocytosis.