J774 Mouse Macrophage Cell Research Articles

Influence of high density lipoprotein on esterified cholesterol stores in macrophages and hepatoma cells.

The ability of high density lipoproteins (HDL) to induce the clearance of cholesteryl esters from cultured cells has been explored. Studies using the J774 mouse macrophage cell line showed that these cells are not stimulated to clear esterified cholesterol upon exposure to HDL. This was observed over a wide range of HDL concentrations (10 to 1000 micrograms/ml HDL protein), and the lack of stimulation was not influenced by a number of factors relating to the preparation of the HDL, such as HDL subfraction, varying extents of lecithin:cholesterol acyltransferase modification, or heparin-Sepharose chromatography to remove particles containing apo E. Neither the method of loading the cells with esterified cholesterol nor the physical state of the lipid droplets affected the inability of HDL to elicit esterified cholesterol clearance. In the presence of the acyl CoA:cholesterol acyltransferase inhibitor, Sandoz 58-035, where a high level of intracellular free cholesterol was generated, efflux of only a small fraction of the excess free cholesterol to HDL was observed. J774 cells were able to clear esterified cholesterol efficiently in the presence of cholesterol-free apolipoprotein HDL/phospholipid particles, indicating that the cells have the capacity to clear esterified cholesterol. Fu5AH hepatoma cells and P388.D1 mouse macrophage cells also failed to clear esterified cholesterol in response to HDL. In contrast, mouse peritoneal macrophages cleared esterified cholesterol efficiently to HDL, indicating that there are fundamental differences between mouse peritoneal macrophages and the other cells types studied in regard to cholesterol metabolism as influenced by HDL.

Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line.

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages.

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.

Fc-receptor variants of a mouse macrophage cell line

Variants of the J774 mouse macrophage cell line that lack immunologically important membrane receptors were isolated. After mutagenesis, variants were selected in a metrizamide gradient that separated cells heavily rosetted with sheep erythrocytes (E) coated with rabbit anti-E IgG (EIgG) from poorly rosetted cells. Stable variants that exhibited altered binding were found with a frequency of <10(-7), and five clones were studied in detail. The variants failed to bind E opsonized with a monoclonal mouse IgG2b anti-E antibody but bound monomeric IgG2a normally when compared to the parental J774 line (K(a) 4 degrees C= approximately 1x10(8) M(-1); approximately 2x10(5) sites per cell). This demonstrates the independence of the receptor for mouse IgG2b complexes (FcRII) from the trypsin-sensitive receptor for mouse IgG2a monomer (FcRI). The variants bound an average of 10-15 EIgG per cell, compared to >20 per cell for J774. After trypsinization, three variants bound only three to five EIgG per cell; the J774 line was not affected by this treatment. Monomeric IgG2a could inhibit the binding of soluble rabbit IgG-antigen complexes to the variants but not to the parent line. Finally, E coated with IgM and complement (EIgMC) were bound poorly by all the variants, relative to the J774 parent. These results show that rabbit IgG complexes are bound by both FcRI and FcRII on mouse macrophages. The impairment of EIgMC rosetting in the variants suggests that the C3b receptor and FcRII are related.

Influence of external ATP on permeability and metabolism of dog red blood cells.

Influence of external ATP on permeability and metabolism of dog red blood cells.