Characterization of clonally derived, spontaneously transformed bone marrow macrophage cell lines from lipopolysaccharide hyporesponsive LPS(d) and normal LPS(n) mice

Six macrophage cell lines, each derived from a bone marrow macrophage colony grown in soft agar, were established by expansion of the macrophage clones in liquid culture until spontaneous transformation occurred. Four lines originated from the LPS(d) nonresponder mouse strain C3H/HeJ and two from the LPS(n) responder strain CBA/J. The cell lines adhered to plastic and glass surfaces and displayed typical macrophage functions such as phagocytosis and nonspecific esterase activity. Flow cytometry analyses showed that the lines expressed the macrophage surface markers CD11b, CD13, CD32/16, F4/80, and BM8 constitutively. A moderate expression of the adhesion receptor CD11a, but only a very low expression of its ligand CD54, was observed. A minor fraction of the cells in each line constitutively expressed MHC class II antigen, and its expression could be up‐regulated in each cell line by treatment with interferon‐γ (IFN‐γ). Secretion of the inflammatory mediators nitric oxide (NO), interleukin‐6 (IL‐6), and tumor necrosis factor α (TNF‐α) after induction by three bacterial derivatives, heat‐killed Salmonella typhimurium (HKS), lipopolysaccharide (LPS), and the Mycoplasma fermentans‐derived amphiphilic lipid MDHM, were examined in detail. Not only did the lines differ in the amounts of mediators secreted in response to any one stimulus, but the doses of MDHM or LPS required for 50% maximal induction of NO varied up to 10‐fold among the four LPS(d) cell lines, suggesting considerable functional heterogeneity between the clones. Secretion of large amounts of TNF‐α was induced in all the cell lines by HKS. Although it could be shown that exogenously added TNF‐α acted synergistically with IFN‐γ to induce NO release from the cell lines, an autocrine role for TNF‐α during HKS‐IFN‐γ induction of NO synthesis could not be substantiated. Neutralization of TNF‐α with a specific antibody completely blocked NO induction by exogenous TNF‐α but did not abrogate NO release either by HKS‐IFN‐γ‐induced cells or by macrophages treated with supernatant from an HKS‐IFN‐γ‐activated cell line. These results indicate that the clones are arrested in distinct stages of differentiation and retain some properties of normal untransformed macrophages. They should be helpful tools for investigations into macrophage function. J. Leukoc. Biol. 61: 469–480; 1997.