Related Experiment Videos
Interferon-gamma and tumor necrosis factor-alpha enhance p60src expression in human macrophages and myelomonocytic
Abstract:
We investigated modulation of p60src expression in human mononuclear phagocytes. By analysis of [35S]methionine-labelled cells we found that synthesis of p60src is higher in human monocytes compared to macrophages derived from in vitro cultivation of monocytes. Western blot analysis showed that expression of p60src in monocyte-derived macrophages can be enhanced if monocytes are differentiated into macrophages in the presence of interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha). Enhanced p60src expression caused by IFN-gamma or TNF-alpha correlated with an enhanced autophosphorylating kinase activity assayed in anti-p60src immune precipitates. In vivo phosphorylation of p60src and analysis of phosphopeptides by tryptic digestion showed that treatment with cytokines did not affect the pattern of phosphorylation of distinct phosphopeptides. The human monocytic cell lines, U937 and HL-60, induced to differentiate along the monocytic pathway by IFN-gamma, or a combination of IFN-gamma and TNF-alpha, expressed higher amounts of the p60src, but not of the p59fyn or p62yes, kinase activity. These findings show that p60src is modulated in the course of differentiation of human monocytes to macrophages, and that macrophage-activating cytokines increase p60src expression in human monocyte-derived macrophages.
Insights
p60src expression is higher in monocytes than macrophages. Macrophage-activating cytokines, like interferon-gamma and tumor necrosis factor-alpha, increase p60src expression and kinase activity in human monocyte-derived macrophages.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- p60src is a proto-oncogene tyrosine kinase.
- Mononuclear phagocytes, including monocytes and macrophages, play critical roles in immunity and inflammation.
- The regulation of p60src during monocyte differentiation into macrophages is not fully understood.
Purpose of the Study:
- To investigate the modulation of p60src expression during human monocyte differentiation into macrophages.
- To determine the effect of macrophage-activating cytokines on p60src expression and activity.
- To compare p60src expression in monocytes versus macrophages.
Main Methods:
- Analysis of [35S]methionine-labeled cells to assess protein synthesis.
- Western blot analysis to quantify p60src expression.
- Assay of autophosphorylating kinase activity in anti-p60src immune precipitates.
- In vivo phosphorylation studies and tryptic phosphopeptide mapping.
- Use of human monocytic cell lines (U937 and HL-60) induced to differentiate.
Main Results:
- p60src synthesis was higher in human monocytes compared to in vitro-derived macrophages.
- Interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) enhanced p60src expression in monocyte-derived macrophages.
- Enhanced p60src expression correlated with increased autophosphorylating kinase activity.
- Cytokine treatment did not alter the pattern of p60src phosphorylation.
- Differentiated U937 and HL-60 cell lines showed increased p60src expression but not p59fyn or p62yes activity.
Conclusions:
- p60src expression is modulated during human monocyte to macrophage differentiation.
- Macrophage-activating cytokines (IFN-gamma, TNF-alpha) upregulate p60src expression and kinase activity in human monocyte-derived macrophages.
- These findings highlight a cytokine-dependent regulation of p60src in the mononuclear phagocyte system.