RAW264 MACROPHAGES STABLY TRANSFECTED WITH AN HIV-1 LTR REPORTER GENEPROVIDE A SENSITIVE BIOASSAY FOR ANALYSIS OF SIGNALING PATHWAYS IN MACROPHAGES STIMULATED WITH LIPOPOLYSACCHARIDE, TNF-ALPHA OR TAXOL

Bacterial lipopolysaccharide (LPS) modulates expression of a variety o f genes in macrophages, and additionally activates viral promoters inc luding the HIV-1 LTR. The HIV-1 LTR driving the luciferase reporter ge ne was stably transfected into the murine macrophage cell line, RAW264 . In stably transfected cells, luciferase activity was LPS-dependent. As little as 0.01 ng/ml LPS was sufficient to increase luciferase acti vity over basal levels with maximal stimulation resulting in a 10- to 20-fold response. The cells also responded to human and murine tumour necrosis factor (TNF alpha). Endogenous TNF alpha was not involved in LPS responses, since pretreatment with alpha-TNF alpha antibody did no t affect activation. Induction of HIV-1 LTR activity by LPS occurred i ndependently of phorbol myristate acetate (PMA) sensitive protein kina se C (PKC), since depletion of PKC by prolonged exposure to PMA blocke d TNF alpha and PMA responses but was not able to abolish LPS action o n these cells. Taxol (5-20 mu g/ml), a chemotherapeutic agent which mi mics LPS action on macrophages, was also able to increase expression o f the reporter gene driven by the HIV-1 LTR. However, lower doses of t axol that were not sufficient to trans-activate the LTR or to induce T NF alpha expression were cytotoxic to RAW264 cells suggesting that the cytotoxic and LPS-like activities of taxol were not linked. This cell line provides a convenient method for detecting LPS-like activity and is a useful tool for examining LPS and TNF alpha signal ling pathways . (C) 1995 Wiley-Liss, Inc.