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Updated: May 11, 2026
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Comparative study of the effect of LPS on the function of BALB/c and C57BL/6 peritoneal macrophages
Sara Soudi1, Ahmad Zavaran-Hosseini, Zuhair Muhammad Hassan
11. Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Objective:
Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide (LPS) stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response.
Materials And Methods:
In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1µg/ml LPS. At different time points after LPS treatment, nitric oxide (NO), interferon gamma (IFN-λ), interleukin 4 (IL-4),transforming growth factor β1(TGF-β1), interleukin 17 (IL-17), and interleukin 10(IL-10) production were measured in the supernatants of all macrophage cultures. Indoleamine 2, 3 dioxygenase (IDO) and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-(4,5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent.
Results:
According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-λ, while BALB/c macrophages produced more TGF-β1 and IL-4. There was no significant difference in IDO activity between strains (p≤0.05). BALB/c mice produced more NO inthe first 24 hours after LPS treatment,but C57BL/6 produced more NO at 72 hours post-LPS treatment. Macrophages from both strains hada suppressor effect on splenocyte proliferation, but this effect was stronger in BALB/c mice.
Conclusion:
The results show that macrophages from different genetic backgrounds respond differently to the same stimulus in aspects of type, intensity, and time of response. The consideration of these aspects will enableresearchers to use correct treatment programs for immune-regulation or immunotherapy.
Insights
Macrophages from different mouse strains exhibit distinct responses to lipopolysaccharide (LPS) stimulation, impacting immune cell behavior and cytokine profiles. Understanding these genetic differences is crucial for effective immunotherapy and immune regulation strategies.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages are key immune cells that modulate their environment and surrounding immune cells upon stimulation.
- Different macrophage origins lead to varied immune responses and non-uniform immunological outcomes.
Purpose of the Study:
- To compare the behavior of peritoneal macrophages from BALB/c mice (type 2 immune response indicator) and C57BL/6 mice (type 1 immune response indicator) following lipopolysaccharide (LPS) stimulation.
- To analyze the differential production of key immune mediators and their effects on splenocyte proliferation.
Main Methods:
- Peritoneal macrophages from thioglycolate-stimulated BALB/c and C57BL/6 mice were treated with LPS (1µg/ml).
- Nitric oxide (NO), interferon gamma (IFN-λ), interleukin 4 (IL-4), transforming growth factor β1 (TGF-β1), interleukin 17 (IL-17), and interleukin 10 (IL-10) were measured in culture supernatants.
- Indoleamine 2, 3 dioxygenase (IDO) activity, phagocytic activity, and the effect on splenocyte proliferation (using MTT assay) were assessed.
Main Results:
- Distinct cytokine patterns were observed: C57BL/6 macrophages produced more IL-17, IL-10, and IFN-λ, while BALB/c macrophages produced more TGF-β1 and IL-4.
- Nitric oxide (NO) production varied, with BALB/c showing higher levels initially (24 hours) and C57BL/6 at later time points (72 hours).
- Both macrophage types suppressed splenocyte proliferation, with a more pronounced effect observed in BALB/c mice. No significant difference in IDO activity was found between strains.
Conclusions:
- Macrophages from different genetic backgrounds (strains) exhibit differential responses to LPS in terms of type, intensity, and timing.
- These strain-specific macrophage behaviors highlight the importance of genetic background in immune responses.
- Understanding these differences is essential for developing targeted immunoregulatory and immunotherapy strategies.
