Related Experiment Videos
Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxynucleotides
L Jin1, D P Raymond, T D Crabtree
1Surgical Infectious Disease Laboratory, Department of Surgery, and Department of Internal Medicine, University of Virginia, Charlottesville, VA 22906, USA.
Abstract:
The immunomodulatory role of unmethylated cytosine-guanine sequences (CpG) in bacterial DNA has been well documented. We have previously demonstrated that murine macrophage-like RAW 264.7 cells respond to CpG DNA with an increase in the proinflammatory cytokine, TNF-alpha, in both a dose-dependent and time-dependent manner. In addition, CpG DNA stimulates a significant, though delayed, secretion of the anti-inflammatory cytokine IL-10. Because TNF-alpha and TNFR (TNFRI and II) expression are tightly regulated responses, we hypothesized that CpG containing oligodeoxynucleotide (CpG ODN) would also affect TNFRI and II shedding. Using both murine peritoneal macrophages and RAW 264.7 cells, we demonstrated a significant, time-dependent increase in soluble TNFRI and TNFRII production with CpG ODN stimulation. RAW 264.7 cells treated with CpG ODN had a transient increase in membrane TNFRII expression, but not TNFRI. Both types of TNFR mRNA were also up-regulated by CpG ODN, and addition of the transcriptional inhibitor actinomycin D abrogated the effect of CpG ODN on TNFR mRNA and protein expression. Addition of anti-IL-10 and anti-TNF-alpha Abs did not change these results. The addition of plate-bound anti-TNF receptor Abs to this system increased the amount of bioactive TNF, implying that these receptors are acting as inhibitors of TNF activity. These results suggest that the de novo, non-IL-10- and non-TNF-alpha-dependent transcription, translation, and shedding of TNFRs are additional potential counterinflammatory effects of CpG DNA.
Insights
CpG DNA stimulates the production and shedding of TNF receptors (TNFRs) in macrophages. This process, independent of IL-10 and TNF-alpha, suggests a novel counterinflammatory mechanism for CpG DNA.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Unmethylated cytosine-guanine sequences (CpG) in bacterial DNA are known immunomodulators.
- CpG DNA previously shown to increase pro-inflammatory TNF-alpha and anti-inflammatory IL-10 in murine macrophages.
- TNF-alpha and its receptors (TNFRs) have tightly regulated expression patterns.
Purpose of the Study:
- To investigate the effect of CpG oligodeoxynucleotides (CpG ODN) on TNF receptor (TNFR) shedding.
- To determine if CpG ODN influences the expression of TNFRI and TNFRII at the mRNA and protein levels.
- To elucidate the role of IL-10 and TNF-alpha in CpG ODN-induced TNFR modulation.
Main Methods:
- Stimulation of murine peritoneal macrophages and RAW 264.7 cells with CpG ODN.
- Measurement of soluble TNFRI and TNFRII production.
- Analysis of membrane TNFRII and TNFRI expression.
- Assessment of TNFR mRNA levels using transcriptional inhibitors (actinomycin D).
- Investigation using anti-IL-10 and anti-TNF-alpha antibodies.
Main Results:
- CpG ODN significantly increased soluble TNFRI and TNFRII production in a time-dependent manner.
- Transient increase in membrane TNFRII expression, but not TNFRI, was observed.
- CpG ODN up-regulated both TNFR mRNA and protein expression, dependent on de novo transcription.
- The observed effects were independent of IL-10 and TNF-alpha.
- Plate-bound anti-TNFR antibodies increased bioactive TNF, suggesting TNFRs act as TNF inhibitors.
Conclusions:
- CpG ODN induces de novo transcription, translation, and shedding of TNF receptors (TNFRs).
- This TNFR modulation is independent of IL-10 and TNF-alpha.
- The shedding and potential inhibitory function of TNFRs represent a novel counterinflammatory effect of CpG DNA.