In situ hybridization analysis of macrophage-derived tumor necrosis factor and interleukin-1 mRNA

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) play an intimate role in the initiation and maintenance of inflammatory reactions due to their pluripotent activities. In this paper, we describe the use of an in situ hybridization analysis as an effective means to probe for TNF and IL-1 mRNA levels in primary macrophage cultures and macrophage cell lines. A significant increase in lipopolysaccharide (LPS)-induced TNF mRNA accumulation was demonstrated by in situ hybridization using either a 35S-labeled synthetic oligonucleotide (30-mer) complementary to TNF mRNA or a 35S-randomly primed labeled TNF DNA probe. An augmentation in TNF mRNA accumulation, as assessed by increasing grains/cell, was demonstrated over a wide concentration range of LPS. This accumulation was shown using both immunologically elicited primary macrophage cultures and the macrophage cell line RAW 264.7. Interestingly, the RAW 264.7 constitutively produced TNF in the absence of specific stimulus and this tonic production was observed at the molecular level via in situ hybridization analysis. Specificity of the in situ hybridization technique was shown by a complete loss in binding of 35S-probe after either RNase digestion or competition with "cold-labeled" probe. beta-actin served as a 35S-labeled control probe where the number of actin-specific grains/cell was not altered by stimulating macrophages with LPS. IL-1 alpha mRNA was also increased by LPS stimulation of macrophages as assessed by in situ hybridization. The LPS-dependent increase in macrophage mRNA for TNF and IL-1 alpha, as assessed by in situ hybridization, was confirmed by classical Northern blot analysis as well as the production of biologically-active protein.