Biochemical characterisation of porcine polymorphonuclear leucocytes: comparison with human polymorphonuclear leucocytes

Polymorphonuclear leucocytes were isolated from pig blood relatively free from other cells and were characterised biochemically and morphologically and compared with human PMNLs. The activities of 16 enzymes of porcine and human PMNLs were measured and compared. Alkaline phosphatase, acid phosphatase, phosphodiesterase, gamma-glutamyl transpeptidase, NADH-cytochrome c oxidoreductase, malate dehydrogenase and acetylcholinesterase had higher specific activities in procine than in human cells. Alkaline phosphatase has an 87-fold higher specific activity in porcine than in human cells. beta-glucuronidase, lysozyme, beta-galactosidase, N-acetyl-glucosaminidase, beta-glucosidase, myeloperoxidase and catalase had higher specific activities in human than in porcine cells. beta-glucuronidase and myeloperoxidase showed over a 1000- and a 13-fold higher specific activity, respectively, in human than in porcine cells. Porcine PMNLs are readily available in large numbers and are recommended for studies of phagocytosis, chemotaxis and membrane biochemistry.     

Generation and functional analysis of distinct macrophage sub-populations from goldfish (Carassius auratus L.) kidney leukocyte cultures

Three distinct sub-populations of macrophages derived from goldfish kidney leukocyte cultures were generated and characterised. The sub-populations designated as R1, R2 and R3-type macrophages had distinct morphological, cytochemical and flow cytometric profiles, and also differed in their anti-microbial functions after activation with macrophage activation factors (MAF) and bacterial lipopolysaccharide (LPS). The R1-type macrophages were small cells that contained acid phosphatase, but lacked myeloperoxidase and non-specific esterase. The R2-type macrophages were morphologically similar to mature tissue macrophages of mammals, and were positive for acid phosphatase, myeloperoxidase and non-specific esterase. The R3-type macrophages were round cells with eccentrically placed nuclei and resembled mammalian monocytes. This sub-population stained for acid phosphatase, myeloperoxidase and non-specific esterase. The R2 and R3-type macrophages exhibited distinct functional responses after activation with MAF and/or LPS. R2-type macrophages were potent producers of nitric oxide, while R3-type macrophages produced little or no nitric oxide after activation with MAF and LPS. The R2 and R3-type macrophages also exhibited unique respiratory burst responses (ROI) after treatment with MAF and/or LPS. After treatment with MAF and LPS, activated R2 macrophages were primed for ROI after only 6 h of stimulation with the activating agents, and continued to exhibit a strong ROI response for an extended cultivation period (48 h). In contrast, activated R3-type macrophages showed an early ROI response (6 h after treatment with MAF and LPS), which decreased significantly by 48 h after treatment with the activating agents. Our results suggest that the analysis of the mechanisms of induction of fish anti-microbial responses may be dependent upon the concerted actions of functionally distinct macrophage sub-populations.     

Biochemical and functional analyses of a secreted H-2Ld molecule.

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.     

Control of kidney differentiation by soluble factors secreted by the embryonic liver and the yolk sac

Since transferrin is necessary for the differentiation of the embryonic kidney in organ culture, we have suggested that the component is a growth factor for in vivo development as well. In the present study we demonstrate that transferrin is present in the serum of 11-day-old mouse embryos, at the time when kidney differentiation starts. We have also tested whether various embryonic tissues can replace transferrin as stimulators of the differentiation and proliferation of the metanephric mesenchyme. We used a transfilter model system where nephrogenic mesenchymes are cultured with spinal cord, a known inductor of kidney tubules. The embryonic liver could not replace the spinal cord as an inducer of tubular differentiation. However, when the kidney mesenchymes were cultured together with both the spinal cord and the liver, the mesenchymes proliferated and differentiated also in the absence of exogenous transferrin. In such cocultures the spinal cord had to be in close contact with the mesenchyme while the embryonic liver could be located several cell layers apart. The liver-mediated stimulation of proliferation of the induced mesenchyme could be inhibited by anti-transferrin antibodies. Immunoprecipitation and immunoblotting with these antibodies of the liver-conditioned medium demonstrated that the 11-day mouse liver produces transferrin. Other potential mitogens produced by liver cells, alpha-fetoprotein, or multiplication stimulating activity, did not in any way stimulate the proliferation of induced mesenchymes. These studies suggest that the mitogen in the liver medium is transferrin. This is supported by data which show that another embryonic transferring producer, the visceral yolk sac, can replace the effect of the liver, whereas a tissue not producing transferrin, the salivary mesenchyme, cannot. In conclusion, an essential function of the inducer is to make the mesenchyme responsive to transferrin. The liver and the yolk sac stimulate early kidney differentiation by producing the soluble factor, transferrin, but they are ineffective as inductors of the transferrin responsiveness.     

Superinduction by cycloheximide of mitogen-induced secreted proteins produced by Balb/c 3T3 cells

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2-4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called "superinducible proteins" (SIPs) is increased 2-5-fold. Superinduction of the SIPs is seen also in response to polyribol-polyriboC, the classical inducer of interferons. None of the SIPs, however, are immuno-precipitated by anti-beta-interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectable in cellular fractions. Their induction is, however, correlated with the ability of the polypeptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibit 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth or differentiation signal between cells.     

Secretion of stromelysin by cultured dermal papilla cells: differential regulation by growth factors and functional role in mitogen-induced cell proliferation.

To understand better the molecular nature of the epithelial-mesenchymal interactions that govern folliculogenesis and hair growth, we have studied the behavior of cultured rat dermal papilla cells (rDP), the mesenchymal component of the hair follicle. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) both potentiated the growth of rDP in culture, and transforming growth factor-beta (TGF-beta) inhibited rDP proliferation. Biosynthetic labeling studies demonstrated that both PDGF and bFGF induced synthesis of a major secreted protein(s) with Mr = 55-60 kD. It was noted that PDGF and bFGF differentially regulated synthesis of this major secreted protein; PDGF-mediated induction was found to be transient, while bFGF allowed prolonged synthesis of the protein. Sodium dodecyl sulfate (SDS)-substrate gel analysis of rDP-conditioned media revealed that this protein is a metalloproteinase with casienolytic activity and Mr approximately 51 kD (unreduced). We have identified the growth factor-regulated rDP protein as the matrix metalloproteinase stromelysin by immunoprecipitation. Northern analysis established that increased secretion of stromelysin was accompanied by an increased expression of stromelysin-specific mRNA. Remarkably, stromelysin antisera interfere with stimulation of dermal papilla cell growth, demonstrating that stromelysin production serves a functional role in mitogen-induced proliferation in these cells. These findings provide insight into the mechanism by which the connective tissue remodeling required for formation of hair embryonically and the postembryonic hair cycle may be regulated.     

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis

ObjectiveHighly elevated plasma levels of interleukin-10 (IL-10) are causally associated with “Disappearing HDL Syndrome” and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages.Methods and resultsMacrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting.Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01).ConclusionsInterleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to “Disappearing HDL Syndrome”.     

Biochemical enrichment of lymphokines secreted by a cloned helper T lymphocyte

The Mls-reactive murine helper T cell clone L2 produces at least 10 lymphokine activities affecting at least five distinct target cells. Culture conditions can be optimized to enable production of high levels of colony-stimulating factors (CSFs) by lectin stimulation of L2 cells under serum-free conditions. Selective enrichment of three lymphokine activities--interleukin 2, CSF, and interferon--can be achieved by using a combination of phenyl-Sepharose and hydroxyapatite chromatography. At least two types of CSF can be separated by concanavalin A-Sepharose chromatography. The CSF that does not bind to concanavalin A-Sepharose can be enriched to a specific activity of at least 5 X 10(8) U/mg of protein by using ion exchange high-pressure liquid chromatography.     

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.     

Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1) regulates mononuclear cell proliferation, differentiation, and survival. The functions of CSF-1 are well documented in mammals; however, little is known about CSF-1 biology in lower vertebrates. This is the first report on the identification and functional characterization of a fish CSF-1 molecule expressed highly in the spleen and in phorbol 12-myristate 13-acetate-stimulated monocytes. Goldfish CSF-1 is a 199-amino acid protein that possesses the required cysteine residues to form important intra-chain and inter-chain disulfide bonds that allow CSF-1 to form a functional homodimer and to interact with its high affinity receptor, CSF-1R. Recombinant goldfish CSF-1 formed a homodimer and bound to the soluble goldfish CSF-1R. The addition of the recombinant CSF-1 to sorted goldfish progenitor cells, monocytes, and macrophages induced the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells. The proliferation of these cells was abrogated by addition of an anti-CSF-1R antibody as well as the soluble CSF-1R. The ability of the soluble CSF-1R to inhibit CSF-1-induced proliferation represents a novel mechanism for the regulation of CSF-1 function.     

Therapeutic factors secreted by mesenchymal stromal cells and tissue repair

Systemic administration of MSCs resulted in remarkable functional improvements in injured tissues without either long-term engraftment or differentiation in many clinical and experimental situations. Emerging evidence suggest that most of the beneficial effects of MSCs could be explained by secretion of soluble factors that have multiple effects including modulation of inflammatory and immune reactions, protection from cell death, and stimulation of endogenous progenitor cells. In this review, we focus on the therapeutic factors that account for the beneficial effects of MSCs in animal models of human diseases.     

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.     

Nature of factors stimulating cobalamin production by Achromobacter cobalamini

The object of this work was to study the nature of factors contained in molasses and maize extract and stimulating cobalaminogenesis in Achromobacter cobalamini. The activity of substrate fractions was analyzed to show that the stimulating substance was precipitated on the cation exchanger and eluted from it with HCl. The factor was found to be an organic nitrogen base readily soluble in water and ethanol but insoluble in ether, chloroform and methanol. It was stable upon heating in concentrated HCl. Betaine in the composition of molasses and choline in the composition of maize extract had similar properties. Their addition to the growth medium produced the same effect as that of molasses and maize extract. It is concluded therefore that cobalaminogenesis is stimulated in A. cobalamini by betaine in molasses and by choline in maize extract.     

Site-specific O-glycosylation of human granulocyte/macrophage colony-stimulating factor secreted by yeast and animal cells.

To compare the site specificity of O-glycosylation in lower and higher eukaryotes, we expressed human granulocyte/macrophage colony-stimulating factor (hGM-CSF) in the yeast Saccharomyces cerevisiae and in COS-1 cells. Analyses of specific hGM-CSF mutants secreted by yeast led to the conclusion that efficient O-glycosylation in yeast requires residues S9 and T10. However, only S9 is used as an attachment point for an extended O-glycosyl chain in a 15.5-kDa hGM-CSF form. A 14.5-kDa hGM-CSF form, secreted by yeast, appears substituted by single mannosyl residues at both positions S9 and T10, indicating that O-glycosylation at T10 inhibits extension of the O-glycosyl chain attached to S9. As in yeast cells, the addition of O-glycosyl chains to hGM-CSF secreted by COS-1 cells requires the presence of S9 and T10 residues. These results demonstrate that, inspite of different biosynthetic routes, the selection of O-glycosylation sites is similar between lower and higher eukaryotes.     

Glycosylation and functional activity of anti-D secreted by two human lymphoblastoid cell lines

Cell lines BTSN4 and BTSN5 were produced by the Epstein-Barr Virus (EBV) transformation of B-lymphocytes from the same human donor. Both secrete an anti-D monoclonal of the IgG1 subclass but these antibodies display vastly different effector activities. Specifically, anti-D from BTSN4 has a far greater activity in both monocyte-and lymphocyte-mediated ADCC reactions and causes a higher percentage of rosettes to be formed with monocyte-like U937 cells. This variation in functional activity is shown to coincide with changes in the structure of the sugar chains attached to the asparagine-297 site on the immunoglobulin heavy chain.Abbreviations ADCC antibody dependent cellular cytotoxicity - EBV Epstein-Barr virus - GlcNAc glucosamine - Gal Galactose - Man Mannose - Fuc Fucose - SA Sialic acid     

Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney

This study was undertaken to clarify the physiological role of catalase in the maintenance of pro/antioxidant balance in goldfish tissues by inhibiting the enzyme in vivo with 3-amino 1,2,4-triazole. Intraperitoneal injection of aminotriazole (0.5 mg/g wet mass) caused a decrease in liver catalase activity by 83% after 24 h that was sustained after 168 h post-injection. In kidney catalase activity was reduced by approximately 50% and 70% at the two time points, respectively. Levels of protein carbonyls were unchanged in liver but rose by 2-fold in kidney after 168 h. Levels of thiobarbituric acid-reactive substances were elevated in both tissues after 24 h but were reversed by 168 h. Glutathione peroxidase and glutathione-S-transferase activities increased in kidney after aminotriazole treatment whereas activities of glutathione peroxidase and glutathione reductase in liver decreased after 24 h but rebounded by 168 h. Liver glucose-6-phosphate dehydrogenase activity was reduced at both time points. Activities of these three enzymes in liver correlated inversely with the levels of lipid damage products (R2=0.65-0.81) suggesting that they may have been oxidatively inactivated. Glutathione-S-transferase activity also correlated inversely with catalase (R2=0.86). Hence, the response to catalase depletion involves compensatory changes in the activities of enzymes of glutathione metabolism.