Establishment and characterization of murine bone marrow-derived spontaneously immortalized cell lines and clones expressing properties of normal macrophages

Murine bone marrow (BM) cells were cultivated on bacteriological grade culture dishes (BCD) in liquid medium containing L-cell-conditioned medium (LCM). The first month of rapid exponential multiplication was always followed by an interim phase of slow growth, and then by continuous proliferation. These established lines were called Jerusalem bone marrow macrophages (JBM∅). One of these, which had been derived from a C3H/Crg1 female mouse and was designated JBM∅1.1, was studied in more detail. Its cloning efficiency when grown in LCM-containing soft agar was 65%. Of several clones isolated, one, Cl.26, was selected for further culitvation and propagated for about 600 days. Cells from all cultures were surface adherent with limited proliferative capacity on tissue culture plastic. The properties displayed by all cells in a culture of clone include a typical macrophage (M∅)-like morphology, effective ingestion of killed bacteria and zymosan, staining for nonspecific esterase, and expression of Fc receptors and of F4/80 surface antigen. Addition of lymphokine (LK) induced Ia antigen expression on a high percentage of the cells. The JBM∅1.1 cells also secreted high levels of lysozyme, produced a zymosan-induced respiratory burst, and, upon addition of lipopolysaccharide (LPS), released interleukin-1 and tumor necrosis factor. Efficient tumoricidal activity could be induced by LK and LPS. No evidence for the production of colony-stimulating factors, even in the presence of LPS, could be found. The JBM∅1.1 or Cl.26 cells did not develop into tumors following subcutaneous injection in x-irradiated syngeneic or in nu/nu mice and were also incapable of growing in soft agar without LCM. All the properties studied were expressed at similar levels by the 'young' BM-derived M∅ during their first exponential growth phase, as well as by other JBM∅ lines and clones. It is concluded that the established JBM∅ lines consist of homogeneous cell populations which, according to all markers and functions studied, could be classified as non-activated, functional, and mature M∅, resembling in all aspects BM-derived M∅ during their first few weeks of cultivation. This shows that cell lines expressing properties of normal M∅ may develop spontaneously by continuous cultivation of BM cells in growth-containing liquid medium on BCD. Moreover, the complete expression of many properties by all cells in a line and by all clones demostrates that the plurality of M∅ functions may be explained by the versatility of the single M∅ rather than by hypothetical subpopulations expressing different properties. Finally M∅ maturation does not necessarily prevent their proliferative capability.