Murine Macrophage-like Cell Line RAW Research Articles

Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages

The biosynthesis and processing of cachetin/tumor necrosis-factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipopolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa presequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.

Lipopolysaccharide Induces Hyporesponsiveness to Its Own Action in RAW 264.7 Cells

Bacterial endotoxin (lipopolysaccharide, LPS) has the property of inducing hyporesponsiveness or tolerance to its own effects. This phenomenon has been demonstrated in man and experimental animals. The cellular changes that contribute to LPS tolerance are not understood. One mechanism of tolerance could involve a diminished response to LPS by key effector cells such as macrophages. Here we describe experiments designed to determine the mechanism whereby LPS produces a hyporesponsive state to its own effects. Because of the importance of the monokine known as tumor necrosis factor-alpha (TNF-alpha) in mediating many of the diverse effects of LPS, we have studied induction of TNF-alpha at the mRNA and activity level in the murine macrophage-like cell line RAW 264.7. Hyporesponsiveness can be induced by exposure of RAW 264.7 cells to low doses of LPS for more than 6 h prior to challenge with a second, normally stimulatory dose of LPS. This hyporesponsiveness is characterized by a diminished ability of LPS to increase steady state levels of TNF-alpha mRNA, is not due to an increased rate of TNF-alpha mRNA degradation, and is specific for LPS since LPS-pretreated and control cells produce similar amounts of TNF-alpha in response to challenge with heat-killed Staphylococcal aureus. The presence of indomethacin during the primary and/or challenge LPS treatment has no effect on the induction of acquired hyporesponsiveness. Thus, cyclooxygenase products are probably not involved in the development of LPS-induced hyporesponsiveness. These studies provide the basis for a better understanding of the cellular mechanisms that contribute to LPS tolerance.

Interferon action: Effects of mouse α and β interferons on rosette formation, phagocytosis, and surface-antigen expression of cells of the macrophage-type line RAW 309Cr.1

Treatment with an essentially pure mouse α or β interferon boosts the binding and phagocytosis of opsonized sheep red blood cells by cells of the murine macrophage-like cell line RAW 309Cr.l. The kinetics and the dose dependence of the effects of the two interferons are very similar. The effects depend on continued RNA and protein synthesis, and they diminish after the removal of interferon from the medium. Studies with agents specifically binding FcRI receptors (i.e., IgG2a) and FcRII receptors (i.e., the Fab fragment of the antireceptor monoclonal antibody 2.4G2) revealed a three- to fivefold increase in the level of FcRI receptors per cell and an about twofold increase in that of FcRII receptors per cell after treatment with interferon. The enhanced binding and phagocytosis of opsonized sheep red blood cells by interferon treatment are apparently a consequence of the increased number of Fc receptors. As revealed by studies involving the binding to the cells of labeled monoclonal antibodies to several cell surface antigens, the level of the H-2D d surface antigen is also selectively increased three- to fourfold in the cells after exposure to interferon.