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Differential Regulation of IL-1β and IL-6 Release in Murine Macrophages
Seishiro Hirano1, Quan Zhou2,3, Akiko Furuyama2
1Center for Health and Environmental Risk Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki, 305-8506, Japan. seishiro@nies.go.jp.
Abstract:
Asbestos and silica (exogenous danger) and adenosine triphosphate (ATP, endogenous danger-signaling molecule) synergistically increase IL-1β release from endotoxin-primed macrophage, which is mediated by NOD-like receptor protein 3 (NLRP3) inflammasome. However, the conversion of pro-IL-1β to its active form seems to depend on the macrophage cell types. In the present study, bone marrow-derived macrophages (BMM) and three murine macrophage cell lines, J774.1, J774A.1, and RAW264.7 were exposed to ATP or fibrous titanium dioxide (FTiO2) in the presence or absence of lipopolysaccharide (LPS), and the concentrations of IL-1β and IL-6 in both cell lysates and in the culture media were measured by immunoblotting to differentiate active form of IL-1β from pro-IL-1β. IL-1β release was synergistically increased when the cells were exposed to both LPS and ATP or FTiO2, while IL-6 was readily released by LPS alone. IL-1β released into the culture medium was pro-IL-1β in J774.1 and RAW264.7, and most of the pro-IL-1β remained inside the cells. In contrast, the active form of IL-1β was released together with pro-IL-1β from J774A.1 and BMM after the co-stimulation. J774A.1 and BMM express apoptosis-associated speck-like protein contains a carboxyl-terminal CARD (ASC) while J774.1 and RAW264.7 do not or only faintly express ASC, and accordingly, caspase-1, which converts pro-IL-1β to its active form, is activated only in J774A.1 and BMM. Collectively, the canonical inflammasome pathway is not activated in J774.1 and RAW264.7, and the apparent synergistical increase of IL-1β in the culture medium mostly reflects the leakage of pro-IL-1β from these cells.
Insights
Adenosine triphosphate (ATP) and danger signals synergistically boost IL-1β release from macrophages via the NLRP3 inflammasome. However, active IL-1β release depends on macrophage cell type and ASC inflammasome component expression.
Area of Science:
- Immunology
- Cell Biology
Background:
- The NOD-like receptor protein 3 (NLRP3) inflammasome mediates the release of IL-1β, a key inflammatory cytokine.
- Endogenous danger signals like adenosine triphosphate (ATP) and exogenous danger signals such as asbestos and silica synergistically enhance IL-1β release from lipopolysaccharide (LPS)-primed macrophages.
- The processing of pro-IL-1β into its active form and subsequent release appear to be cell-type dependent.
Purpose of the Study:
- To investigate the role of macrophage cell type in the synergistic release of IL-1β induced by danger signals and LPS.
- To determine the involvement of the canonical inflammasome pathway, specifically ASC and caspase-1 activation, in IL-1β release from different macrophage populations.
Main Methods:
- Bone marrow-derived macrophages (BMM) and murine macrophage cell lines (J774.1, J774A.1, RAW264.7) were stimulated with LPS, ATP, or fibrous titanium dioxide (FTiO₂).
- Immunoblotting was used to measure pro-IL-1β and IL-1β concentrations in cell lysates and culture media.
- Expression levels of ASC and activation of caspase-1 were assessed in different macrophage types.
Main Results:
- Co-stimulation with LPS and ATP or FTiO₂ synergistically increased IL-1β release in BMM and J774A.1 cells.
- In contrast, J774.1 and RAW264.7 cells primarily released pro-IL-1β, with limited active IL-1β detected.
- ASC and caspase-1 were activated in BMM and J774A.1, but not in J774.1 and RAW264.7, indicating canonical inflammasome pathway activation is cell-type specific.
- IL-6 release was observed with LPS stimulation alone across all cell types.
Conclusions:
- The canonical NLRP3 inflammasome pathway, involving ASC and caspase-1, is essential for the release of active IL-1β in response to synergistic danger signals.
- Macrophage cell type dictates the activation status of the inflammasome and the subsequent release of active IL-1β versus pro-IL-1β.
- Apparent IL-1β release in ASC-deficient cell lines (J774.1, RAW264.7) is primarily due to pro-IL-1β leakage, not inflammasome-mediated processing and release.
