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Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B
1Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York.
Abstract:
The effects of bacterial lipopolysaccharide (LPS) on macrophage gene expression are mediated in part by its ability to induce activation of transcription factor NF-kappa B. We compared the ability of LPS-treated macrophages from Lpsn (LPS-responsive) C3H/HeN and Lpsd (LPS-hyporesponsive) C3H/HeJ mice to mobilize NF-kappa B by electrophoretic mobility shift assays with oligonucleotide probes containing a unique NF-kappa B sequence from the promoter of inducible nitric oxide synthase (iNOS). In response to ng/ml concentrations of LPS, this probe bound proteins that appeared rapidly in the nuclei of thioglycollate-elicited macrophages and bone marrow-derived macrophage cell lines from both Lpsn and Lpsd mice. Only in macrophages from Lpsn mice, however, was LPS able to induce iNOS or tumor necrosis factor alpha. NF-kappa B-containing DNA-protein complexes from Lpsd macrophages were formed in lesser amounts than from Lpsn macrophages but shared the same composition, insofar as they displayed the same electrophoretic mobilities and content of heterodimers of p50/RelA (p65) and p50/c-rel. Two conclusions emerge from these findings: (1) NF-kappa B activity alone is not sufficient for induction of certain LPS-responsive genes and (2) An LPS-response pathway involving activation of NF-kappa B is preserved in Lpsd mice. The inability of cells from Lpsd mice to induce gene expression in response to LPS thus cannot be attributed to inability to activate NF-kappa B.
Insights
Bacterial lipopolysaccharide (LPS) activates NF-kappa B in both LPS-responsive and hyporesponsive mice. However, only LPS-responsive macrophages induce specific genes, indicating NF-kappa B activation alone is insufficient for gene expression.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Bacterial lipopolysaccharide (LPS) triggers macrophage responses, partly via NF-kappa B activation.
- Genetic variations in mice (C3H/HeN vs. C3H/HeJ) lead to differential LPS responsiveness.
Purpose of the Study:
- To compare NF-kappa B mobilization in LPS-treated macrophages from LPS-responsive (Lpsn) and hyporesponsive (Lpsd) mice.
- To investigate the role of NF-kappa B activation in LPS-induced gene expression.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) using NF-kappa B probes.
- Analysis of macrophages from C3H/HeN (Lpsn) and C3H/HeJ (Lpsd) mice.
- Quantification of NF-kappa B DNA-protein complex formation and composition.
Main Results:
- LPS induced rapid NF-kappa B binding to DNA in both Lpsn and Lpsd macrophages.
- LPS induced iNOS and TNF-alpha only in Lpsn macrophages.
- NF-kappa B complexes in Lpsd macrophages were less abundant but had similar composition (p50/RelA, p50/c-rel heterodimers).
Conclusions:
- NF-kappa B activation is necessary but not sufficient for LPS-induced expression of certain genes.
- The LPS-response pathway activating NF-kappa B is functional in Lpsd mice.
- Lpsd mice's inability to induce LPS-responsive genes is not due to a failure in NF-kappa B activation.