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Updated: Oct 3, 2026
Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Differential expression of tumor necrosis factor-alpha isoforms from lipopolysaccharide- and cytokine-stimulated
1Canadian Red Cross Society, Blood Transfusion Centre, Edmonton, Alberta, Canada.
Abstract:
Tumor necrosis factor-alpha (TNF alpha) is a biologically active cytokine with a wide range of functions, which is primarily expressed by macrophages. It is produced as a biologically active propeptide that becomes processed to the mature form of secreted protein. Previous studies used a mouse macrophage cell line and showed that after stimulation with lipopolysaccharide, TNF alpha propeptide is expressed as multiple isoforms with approximate molecular masses of 26, 29 and 32 kDa. However, little is known of the production of TNF alpha isoforms from normal macrophages or of the effects of cytokines on TNF alpha production by macrophages in the absence of co-stimulation by lipopolysaccharide. We have compared the TNF alpha isoforms produced by cytokine-and lipopolysaccharide-stimulated bone marrow-derived macrophages from mice that normally respond to lipopolysaccharide (C3H/HeN) and mice that are hyporesponsive (C3H/HeJ). We found that the pattern of immunoprecipitated TNF alpha propeptide isoforms expressed depended on the stimulus: lipopolysaccharide, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor. Lipopolysaccharide induced three isoforms of 25, 29 and 35 kDa, supporting previous studies. However, macrophage and granulocyte-macrophage colony-stimulating factors also stimulated cells to express the 24 and 27 kDa isoforms, but not the 35 kDa isoform. In addition, cells stimulated with granulocyte-macrophage colony-stimulating factor expressed a novel 20 kDa propeptide. The results show that granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and lipopolysaccharide differently regulate TNF alpha protein expression and suggest that different isoforms may have different functions.
Insights
Different cytokines, including granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor, stimulate distinct tumor necrosis factor-alpha (TNF alpha) propeptide isoforms in macrophages. These findings suggest varied functions for different TNF alpha isoforms.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF alpha) is a key cytokine produced by macrophages, existing as a propeptide processed into mature forms.
- Previous research identified specific TNF alpha propeptide isoforms (26, 29, 32 kDa) in stimulated mouse macrophage cell lines.
- Limited knowledge exists regarding TNF alpha isoform production in normal macrophages and cytokine effects without lipopolysaccharide co-stimulation.
Purpose of the Study:
- To compare TNF alpha propeptide isoforms produced by normal mouse macrophages stimulated with cytokines or lipopolysaccharide.
- To investigate the differential regulation of TNF alpha production by various stimuli in lipopolysaccharide-responsive and hyporesponsive mice.
- To explore potential functional differences among TNF alpha isoforms.
Main Methods:
- Bone marrow-derived macrophages from C3H/HeN and C3H/HeJ mice were stimulated with lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor.
- TNF alpha propeptide isoforms were identified and quantified using immunoprecipitation.
- Molecular masses of expressed isoforms were determined.
Main Results:
- Lipopolysaccharide stimulation yielded 25, 29, and 35 kDa TNF alpha propeptide isoforms.
- Macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor induced 24 and 27 kDa isoforms, but not the 35 kDa isoform.
- Granulocyte-macrophage colony-stimulating factor also stimulated a novel 20 kDa propeptide isoform.
Conclusions:
- Lipopolysaccharide, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor differentially regulate TNF alpha protein expression in macrophages.
- The distinct patterns of TNF alpha isoforms produced suggest that these isoforms may possess unique biological functions.
- Further research is warranted to elucidate the specific roles of different TNF alpha isoforms.
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