Tumoricidal alveolar macrophage and tumor infiltrating macrophage cell lines

Abstract

Continuous alveolar macrophage (AM) and tumor-infiltrated (TIM) cell lines have been generated from C57B16J mice by in vitro infection with the J2 retrovirus carrying the v-raf and v-myc oncogens. Four cloned AM cell lines (AMJ2-C8, AMJ2-C10, AMJ2-C11, AMJ2-C20) and 3 cloned TIM cell lines (TIMJ2-C4, TIMJ2-C7 and TIMJ2-C15) were expanded for further characterization. Flow cytometry detected the product of the raf gene in the cytoplasm of all these cell lines. Studies on the tumoricidal properties of these AM and TIM cell lines demonstrated differences in their response to a panel of known macrophage activators. Four of these cell lines (AMJ2-C8, AMJ2-C10, TIMJ2-C7 and TIMJ2-C15) were activated following exposure to recombinant murine interferon gamma (rMuIFN-gamma) but not lipopolysaccharide (LPS) or muramyl dipeptide (MDP). AMJ2-C20 was only activated by incubation with rMuIFN-gamma plus LPS. AMJ2-C11 and TIMJ2-C4 are the cell lines that most closely resembled the response pattern of the parental AM and TIM, since they could be activated by either the combination of rMuIFN-gamma plus LPS or rMuIFN-gamma plus MDP. Constitutive expression of MHC-class-II antigens was low on AMJ2-C11 or TIMJ2-C4 but was increased following exposure to rMuIFN-gamma. Neither cell line secreted substantial amounts of IL-1 or TNF but both secreted large amounts of IL-6. Thus these cell lines could be powerful tools to study AM and TIM activation and cytotoxicity.

Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines

“…When we sought to reproduce the dual LPS and IFN-7 requirement, we employed a range of LPS concentrations, without IFN-y, and then combined the maximum LPS concentration with 100 U/ml IFN-7. In contrast to the earlier report (19), we found that all three of the AM cell lines could produce TNF and IL-I[3 mRNA as early as 3 h after challenge with 10 gg/ml LPS alone, suggesting that these lines eould produce proinflammatory eytnkines without eostimulation by IFN-y (23). We extended our study to characterize these AM lines and their production of TNK IL-1]3, and IL-6 following stimulation with LPS and/or IFN-y, their TNF production in response to varying concentrations of LPS with and without serum, and their eonstitutive expression of the membrane LPS receptor, CD14 (mCD14) (33).…”

“…AMJ2CS, AM52C11, and AMJ2C20 ceils were generously provided by Dr. Alicia Palleroni, Hoffman LaRoche (Nutley, NJ). These cells had been derived from C57B1/6J mouse AM transformed with recombinant J2 retrovirus (19). Cells were grown in Dulbecco's minimal essential medium (DMEM, BioWhittaker, Walkersville, MD) supplemented with 10% defined fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT) and 5 mM HE-PES buffer (GIBCO, Grand Island, NY) at 37 ° C, in 5% CO2 and 95% humidity.…”

“…These cell lines had been produced by immortalizing primary AM from C57BL/6J mice with the J2 retrovims, a recombinant virus carrying the v-rafand v-myc oncogenes (3). In the report describing the creation of these lines, some aspects of proinflammatory cytokine production were characterized for AMJ2Cll (19). Unlike primary AM, AMJ2Cll were reported to require both interferon-T (IFN-3') and LPS stimulation for induction of TNF and IL-1.…”

“…Our interest in studying posttranseriptional regulatory mechanisms of TNF production in AM led us to obtain three mouse AM lines recently described by Palleroni et al: AMJ2C8, AMJ2C 11, and AMJ2C20 (19). These cell lines had been produced by immortalizing primary AM from C57BL/6J mice with the J2 retrovims, a recombinant virus carrying the v-rafand v-myc oncogenes (3).…”

“…At present, no human AM lines 1To whom eon'espondence should be addressed at Pulmonary Research Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129. have been reported. Recently, howevm; AM cell lines from mice have been described (8,19).…”