Related Experiment Videos
Modulation of granulocyte colony-stimulating factor receptors on murine peritoneal exudate macrophages by tumor
J H Shieh1, R H Peterson, M A Moore
1James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Abstract:
Modulation of granulocyte CSF (G-CSF) receptors on murine peritoneal exudate macrophages (PEM) by various cytokines was investigated. At 4 degrees C, 125I-G-CSF receptor binding on PEM reached a plateau after 6 h and was specifically competed by unlabeled human rG-CSF but not by other cytokines, including human rG-CSF-1, murine recombinant granulocyte-macrophage CSF, murine rIFN-gamma, human rIL-1 beta, and murine rTNF-alpha. 125I-G-CSF bound to PEM has a half-life of 30 min at 37 degrees C. Preincubation of PEM with murine rTNF, murine recombinant granulocyte-macrophage CSF, CSF-1, or G-CSF for 30 min at 37 degrees C resulted in partial reduction of 125I-G-CSF binding capacity, whereas IL-1 or IFN-gamma did not inhibit G-CSF binding. Further studies indicated that reduction of G-CSF binding caused by TNF was a dose- and time-dependent process and did not require FCS. The reduction was transient, and receptor binding was recovered by incubation at 37 degrees C for 8 h. The recovery of G-CSF binding was inhibited in the presence of cycloheximide. In addition, G-CSF binding studies suggested that the TNF-induced decrease in G-CSF binding to PEM was probably due to a reduction in receptor number rather than receptor affinity. Modulation of G-CSFR by TNF was also observed on nonelicited macrophages from various strains of mice. Our results demonstrate a physiologic response of G-CSFR on macrophages that is modulated by TNF. This phenomenon may play an important, as yet unknown, role in the macrophage inflammatory response.
Insights
Tumor necrosis factor (TNF) temporarily reduces granulocyte colony-stimulating factor (G-CSF) receptors on macrophages by decreasing receptor numbers. This G-CSF receptor modulation by TNF may impact macrophage inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Granulocyte colony-stimulating factor (G-CSF) receptors are crucial for myeloid cell function.
- Cytokines modulate immune cell responses, but their effect on G-CSF receptor expression on macrophages is not fully understood.
Purpose of the Study:
- To investigate the modulation of G-CSF receptors on murine peritoneal exudate macrophages (PEM) by various cytokines.
- To elucidate the mechanism and physiological relevance of G-CSF receptor modulation by TNF.
Main Methods:
- Radioligand binding assays using 125I-G-CSF to quantify receptor binding on PEM.
- Competition assays with unlabeled G-CSF and other cytokines (TNF, GM-CSF, IL-1, IFN-gamma).
- Assessment of receptor binding kinetics, half-life, and modulation after cytokine preincubation.
- Investigation of the mechanism (receptor number vs. affinity) and recovery of G-CSF binding.
Main Results:
- 125I-G-CSF specifically bound to PEM and was competed by unlabeled G-CSF, but not other tested cytokines.
- Preincubation with TNF, GM-CSF, CSF-1, or G-CSF partially reduced 125I-G-CSF binding capacity.
- TNF-induced reduction in G-CSF binding was dose- and time-dependent, transient, and likely due to decreased receptor number, not affinity.
- Recovery of G-CSF binding was inhibited by cycloheximide, suggesting de novo protein synthesis is required.
Conclusions:
- Tumor necrosis factor (TNF) physiologically modulates G-CSF receptors on macrophages.
- The TNF-induced decrease in G-CSF binding is mediated by a reduction in receptor number.
- This modulation may play a significant role in the macrophage inflammatory response.