Increased agitation intensity increases CD13 receptor surface content and mRNA levels, and alters the metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry and Northern blotting were used to examine the effects of hydrodynamic forces in stirred tank bioreactors on CD13 receptor surface content and mRNA levels of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 300 or 400 rpm reduced the apparent HL60 growth rate in a dose-dependent manner. This step increase in agitation rate (to 300 or 400 rpm) also increased the CD13 receptor surface content on averge by 30% and 100%, respectively. This increase in CD13 receptor surface content was correlated with a 10% and a 60% increase in CD13 mRNA levels. We also observed a significant and very reproducible drop in CD13 expression over the course of a batch bioreactor run (80 rpm). Although we have no explanation for this, we show that the decrease in CD13 receptor surface content can be (at least partially, if not fully) explained by the corresponding decrease in CD13 mRNA. HL60 cell cultures agitated at 300 and 400 rpm exhibited glucose consumption and lactate production rates that were approximately 40% and 90% greater than values of the cultures agitated at 80 rpm. The physiological and practical implications of these results are discussed.     

Fluid-mechanical forces in agitated bioreactors reduce the CD13 and CD33 surface protein content of HL60 cells

Flow cytometry was used to examine the effect of hydrodynamic forces in surface aerated stirred tank bioreactors on the quantity of CD13 and CD33 surface proteins of Hl60 (human promyelocytic leukemia) cells. A step increase in agitation of the 2-L bioreactors from 80 to 400 rpm reduced the apparent growth rate and the average CD13 and CD33 content per HL60 cell. The effects on the two surface proteins were observed within 30-60 min following the increase in the agitation and preceded observed effects on cell growth by at least 10 h. Upon reduction of the agitation rate back to 80 rpm, the CD13 and CD33 content recovered (in ca. 10 h) for CD13 and ca. 29h for (CD33) to the levels of the control culture whose agitation rate was maintained at 80rpm. The CD13 and CD33 cell content was reduced even at agitation rates (270 rpm) that did not affect cell proliferation. Pluronic F68 (a commonly used shear protectant) had a protective effect on the CD33 content per cell of cultures subjected to hydrodynamic injury but no effect on the CD13 cell content. Possible bioprocessing and physiological implications of these findings are discussed (c) 1993 Wiley & Sons, Inc.     

Serum increases the CD13 receptor expression, reduces the transduction of fluid-mechanical forces, and alters the metabolism of HL60 cells cultured in agitated bioreactors

The effects of serum medium concentration on the CD13 receptor surface content and mRNA levels of HL60 (human promyelocytic leukemia) cells were examined using flow cytometry and Northern blotting. Increasing the serum concentration from 2.5% to 10% and from 5% to 10% increased the CD13 receptor surface content of HL60 cells by 100% and 25%, respectively, in spinner flasks agitated at 60 rpm. In bioreactors at 80 rpm, increasing the serum concentration from 2.5% to 10% and from 5% to 10% increased the CD13 receptor surface content by 60% and 35%, respectively. This increase in CD13 receptor surface content was correlated with a 30% and a 20% increase in CD13 mRNA levels. Increasing serum concentrations also increased the average HL60 cell size under non-damaging conditions (60 rpm in spinner flasks, 80 rpm in bioreactors). Under conditions of agitation at 300 rpm in 2 L bioreactors, increasing serum concentrations (2.5% vs. 10%, 5% vs. 10%) allowed for higher HL60 apparent growth rates, but decreased the CD13 receptor surface content and mRNA levels. In view of our earlier findings on the effects of agitation on the CD13 antigen, these data suggest that serum reduces the transduction of mechanical forces that affect CD13 expression. At 300 rpm, HL60 cells cultured in 10% serum exhibited glucose consumption and lactate production rates that were approximately 50% and 60% lower than the values of cells cultured in 5% and 2.5% serum, respectively.     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

Shear rate and microturbulence effects on the synthesis of proteases by Jacaratia mexicana cells cultured in a bubble column, airlift, and stirred tank bioreactors

Cysteine proteases from Jacaratia mexicana, an endemic Mexican plant, could compete in industrial applications with papain. Currently the only way to obtain these proteases is by extracting them from the wild plant. An alternative source of these enzymes is by J. mexicana suspension culture. In this work, this culture was carried out in airlift, bubble column and stirred tank bioreactors, and the effects of shear rate and microturbulence on cell growth, protein accumulation and proteolytic activity were determined. The shear rates in the stirred tank, bubble column and airlift bioreactors were 274 1/s, 13 1/s and 36 1/s respectively, and microturbulences (symbolized by λ, in units of μm) were 46, 79, and 77 μm, respectively. Protein levels and proteolytic activity were linearly correlated with both shear rate and microturbulence. A higher shear rate and a more intensive microturbulence occurred in the stirred tank, producing higher protein accumulation and higher proteolytic activity compared with those of the other two bioreactor systems. Higher shear rate and microturbulence had an elicitor effect on protease synthesis, because microturbulence in stirred tank bioreactors was lower than the average length of J. mexicana cells. Furthermore, cells in the stirred tank were smaller and thinner than those grown in shake flask, bubble column and airlift bioreactors. In summary, proteases were produced by J. mexicana cell cultures in a stirred tank under conditions of high shear rate and intensive microturbulence, which are similar to those which occur in industrial stirred tanks. These results encourage continuation of the process development for large scale production of these proteases by this technology.     

Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme

The binding of 125I-labeled human hemopexin to human leukemia HL60 cell at 4 degrees C was saturable with time and with increasing concentrations of 125I-hemopexin. Scatchard analysis of the binding data revealed the presence of approximately 42,000 binding sites/cell with an apparent dissociation constant (Kd) of 1.0 X 10(-9) M. When cells were incubated with radioactive hemopexin at 37 degrees C, 125I-hemopexin was rapidly bound and then was dissociated after the release of heme. Treatment of surface-bound 125I-hemopexin with divalent lysine-directed cross-linking disuccinimidyl suberate revealed a membrane polypeptide of about 80,000 Da, to which hemopexin is cross-linked. To examine the fate of the internalized heme, lysates from the cells previously incubated with [59Fe]heme-hemopexin complex were analyzed by CM-cellulose and Sephacryl S-200 column chromatography. A considerable amount of the radioactivity was present in the fraction which co-eluted with the myeloperoxidase activity. When myeloperoxidase was isolated from the cells incubated with [59Fe]heme-hemopexin complex by immunoprecipitation with anti-myeloperoxidase antibody, radiolabeled iron associated with myeloperoxidase increased with time, and more than 30% of the radioactivity in the cells was present in the myeloperoxidase. These results indicate that the binding of hemopexin to the surface receptors triggers a release of heme and that this heme is incorporated into the intracellular myeloperoxidase.     

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.     

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.     

Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.     

Effects of lipid A content, hydrocortisone and polymyxin B on endotoxin-induced decreases in in vivo drug metabolism

Using hexobarbital sleeping and zoxazolamine paralysis time as indices of in vivo hepatic drug metabolism, the effects of endotoxin on drug action appear to be time- and dose-dependent and the lipid A moiety of endotoxin appears to be responsible for its inhibitory effects. These studies have also demonstrated that polymyxin B can ameliorate the adverse effects of endotoxin on drug metabolism. Since hydrocortisone protects mice from endotoxin lethality, but does not alter the prolongation of hexobarbital sleeping time caused by endotoxin, it is possible to separate the lethal effects of endotoxin from its effects on drug metabolism.     

Rapid prenatal and postnatal detection of inborn errors of propionate,methylmalonate, and cobalamin metabolism: A sensitive assay using cultured cells

Summary A sensitive, reliable, and easily performed procedure is described for the prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism using cultured amniotic cells and skin fibroblasts. With this assay, control fibroblast lines incorporated a mean of 6.89 nanoatoms ¹⁴C/mg protein from [1-¹⁴C]propionate into trichloroacetic acid (TCA)-precipitable cell material in 10h. Twenty-five mutant fibroblast lines from patients with propionicacidemia or one of the methylmalonicacidemias fixed 0.04 to 0.93 nanoatoms ¹⁴C/mg. Considerable variation was observed, both among and within discrete mutant classes, with respect to the residual amount of propionate pathway activity, possibly reflecting further molecular heterogeneity in these disorders.We applied this procedure to cultured amniotic cells from controls and 4 midtrimester pregnancies at risk for methylmalonicacidemia and diagnosed one fetus with a methylmalonyl CoA apomutase defect and 3 fetuses which were unaffected.Presented in part at the annual meeting of the Society for Pediatric Research, St. Louis, Missouri, April 1976.     

Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells

Streptococcal surface dehydrogenase (SDH) is a multifunctional, anchorless protein present on the surface of group A Streptococcus (GAS). It plays a regulatory role in GAS-mediated intracellular signaling events in human pharyngeal cells. Using ligand-binding assays, we have identified an approximately 55 kDa protein as an SDH-specific receptor protein on the surface of Detroit human pharyngeal cells. LC-MS/MS analyses identified this SDH-binding pharyngeal cell-surface-exposed membrane-bound protein as uPAR (urokinase plasminogen activator receptor)/CD87. Ligand-binding assays also revealed that only the N-terminal domain (D1) of uPAR bound to SDH. uPAR-D1 more specifically bound to the C-terminal alpha-helix and two immediate flanking regions of the S-loop of the SDH molecule. Site-directed mutagenesis in GAS resulting in SDH with altered C-terminal ends, and the removal of uPAR from pharyngeal cells by phosphatidylinositol-phopsholipase C treatment decreased GAS ability to adhere to pharyngeal cells. When compared to uninfected Detroit pharyngeal cells, GAS-infected pharyngeal cells showed a transient but a significant increase in the expression of uPAR-specific mRNA, and a prolonged recycling process of uPAR on the cell surface. Together, these results indicate that the specific streptococcal surface protein-pharyngeal cell receptor interaction mediated by SDH and uPAR is modulated during GAS infection of human pharyngeal cells. This interaction significantly contributes to bacterial adherence and thus may play a significant role in GAS pathogenesis by regulating intracellular signaling events in pharyngeal cells.     

Activity of the aldehyde and alcohol of gibberellins A12 and A14, two derivatives of gibberellin A15 and four decomposition products of gibberellin A3 in 13 plant bioassays

Summary The biological activities of the aldehyde and alcohol of gibberellins (GAs) A₁₂ and A₁₄, 3-OH-GA₁₅, 3-OH-GA₁₅ wrong lactone (i.e. GA₃₇ wrong lactone) and the four major decomposition products of GA₃ (isogibberellic, allogibberic, epiallogibberic and 9(11)-dehydroallogibberic acids) were tested over a wide range of concentrations on 13 plant bioassays in order to ascertain certain of the structural requirements for biological activity. Generally modification of the basic GA-molecule decreased its activity in all assays except for derivatives of GA₁₂ and GA₁₄ (suggesting conversion of these derivatives to more polar, active GAs). Modification of the 3-OH from the usual 3 to 3 configuration markedly reduced activity. Neither the presence of an inverted lactone ring (i.e. 3-OH-GA₁₅ wrong lactone) nor changes to the lactone ring of GA₃ (410) to form iso-GA₃ (42) appreciably reduce activity. Further decomposition of GA₃ to allogibberic and 9(11)-dehydroallogibberic acid reduced activity only slightly, but epimerization of allogibberic acid at C-9 essentially eliminated biological activity.     

A rapid redistribution of the transferrin receptor to the cell surface of HL-60 cells and K562 cells upon treatment with dimethyl sulfoxide due to slowing of endocytosis

Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 degrees C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluorescein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid-myeloid cell line, showed a 25-65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor.     

A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33.

The siglecs (sialic acid-binding Ig-like lectins) are a distinct subset of the Ig superfamily with adhesion-molecule-like structure. We describe here a novel member of the siglec protein family that shares a similar structure including five Ig-like domains, a transmembrane domain, and a cytoplasmic tail containing two ITIM-signaling motifs. Siglec-10 was identified through database mining of an asthmatic eosinophil EST library. Using the Stanford G3 radiation hybrid panel we were able to localize the genomic sequence of siglec-10 within the cluster of genes on chromosome 19q13.3-4 that encode other siglec family members. We have demonstrated that siglec-10 is an immune system-restricted membrane-bound protein that is highly expressed in peripheral blood leukocytes as demonstrated by Northern, RT-PCR and flow cytometry. Binding assays determined that the extracellular domain of siglec-10 was capable of binding to peripheral blood leukocytes. The cytoplasmic tail of siglec-10 contains four tyrosines, two of which are embedded in ITIM-signaling motifs (Y597 and Y667) and are likely involved in intracellular signaling. The ability of tyrosine kinases to phosphorylate the cytoplasmic tyrosines was evaluated by kinase assay using wild-type siglec-10 cytoplasmic domain and Y-->F mutants. The majority of the phosphorylation could be attributed to Y597 andY667. Further experiments with cell extracts suggest that SHP-1 interacts with Y667 and SHP-2 interacts with Y667 in addition to another tyrosine. This is very similar to CD33, which also binds the phosphatases SHP-1 and SHP-2, therefore siglec-10, as CD33, may be characterized as an inhibitory receptor.     

Effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae cells. Studies by 13C-, 1H-NMR and biochemical methods

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1-13C]glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using 13C-, 1H-NMR and biochemical methods. The 13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 1H-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 microM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The 13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (approximately 1.0) is practically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 microM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.     

Werner's syndrome: A review of recent research with an analysis of connective tissue metabolism,growth control of cultured cells,and chromosomal aberrations

Summary Werner's syndrome is a rare, autosomal recessive condition with multiple progeroid features, but it is an imitation of aging rather than accelerated or premature senescence. Somatic chromosome aberrations occur in multipe tissues in vivo and in vitro, and there is an increased incidence of neoplasia. Thus, Werner's syndrome can be classified in the group of chromosome instability syndromes. Recent findings provide additional support for the concept that there is aberration of connective tissue metabolism in Werner's syndrome, but it is unclear whether this is a primary or secondary manifestation of the underlying genetic defect. Abnormal growth characteristics are observed in cultured skin fibroblast-like cells and this provides another avenue for current research. Identification of the basic genetic defect in Werner's syndrome might clarify our understanding of the normal aging process in general, or might elucidate specific aspects such as the development of neoplasia, atherosclerosis, diabetes, or osteoporosis.     

Effect of chronic viral infection on epitope selection, cytokine production, and surface phenotype of CD8 T cells and the role of IFN-gamma receptor in immune regulation

Regulation of CD8 T cell responses in chronic viral infections is not well understood. In this study, we have compared the CD8 T cell responses to immunodominant and subdominant epitopes during an acute and a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The epitope hierarchy of the primary CD8 T cell response was similar in acute and chronic LCMV infections. However, strikingly, the epitope hierarchy of the primary CD8 T cell response was conserved in the T cell memory only in an acute but not in a chronic LCMV infection. Interestingly, in an acute infection, increasing the viral dose caused significant changes in the epitope hierarchy of the LCMV-specific memory CD8 T cell pool, with no effect on the primary CD8 T cell response. Functional and phenotypic analyses revealed that exposure of CD8 T cells to extended periods of antigenic stimulation could lead to long-term defects in cytokine production and alteration in expression of cell surface L-selectin (CD62L). Whereas expression of CD44 was minimally altered, a greater proportion of LCMV-specific memory CD8 T cells were CD62L(low) in mice that have recovered from a chronic LCMV infection, compared with acutely infected mice. Mechanistic studies showed that IFN-gammaR deficiency altered the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells without significantly affecting the immunodominance of the primary CD8 T cell response in an acute infection. Taken together, these findings should further our understanding about the regulation of T cell responses in human chronic viral infections.