Transforming growth factor-alpha expression in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: responses to lipopolysaccharide and 12-O-tetradecanoylphorbol-13-acetate

Abstract

Recent findings in our laboratory indicated that peritoneal macrophages (MPs) elicited from phorbol ester-sensitive SENCAR mice generated significant amounts of superoxide when stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; negligible responses were observed for MPs derived from relatively resistant B6C3F1 mice. We hypothesized a similar strain-dependent secretion of transforming growth factor-alpha (TGF-alpha) by TPA-stimulated MPs. TGF-alpha secreted by MPs was quantitated by competitive enzyme-linked immunosorbent assay. After 72 h (maximal secretion), for MPs derived from B6C3F1 mice, in vitro exposure to 10 microgram/mL lipopolysaccharide (LPS; non-lipid-A-detoxified) resulted in maximal induction (708 pg/mL), in vivo exposure to intraperitoneally (i.p.) administered TPA (2 micrograms/mouse) alone resulted in a minimal response (32 pg/mL), and prior in vivo exposure to TPA significantly inhibited (more than 90% suppression) the LPS-stimulated MP response in culture (i.e., to 62 pg/mL). Although significant amounts of TGF-alpha could be detected in both SENCAR- and B6C3F1-derived MPs (i.e., approximately 2-3 ng/5 x 10(6) cells), SENCAR MPs did not secrete TGF-alpha in response to either TPA or LPS. In addition, the use of the semiquantitative reverse transcription-polymerase chain reaction to detect TGF-alpha-specific mRNA did not support the strain dependency observed for LPS-stimulated TGF-alpha secretion, i.e., detectable transcripts were observed in MP RNA derived from both strains. In conclusion, although TPA itself demonstrated negligible effects on TGF-alpha expression in murine MPs, prior in vivo exposure inhibited LPS-stimulated transcriptional activation and intracellular TGF-alpha production. The negligible TGF-alpha secretion determined for LPS-stimulated SENCAR-derived MPs suggested the possibility of a strain-specific defect in the posttranslational processing of the proTGF-alpha molecule.