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Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes
Published on: May 31, 2018
IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes
L Borish1, J J Mascali, L J Rosenwasser
1Department of Medicine, University of Colorado Health Sciences Center, National Jewish Center for Immunology and Respiratory Medicine, Denver 80206.
Insights
Monocytes produce Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor-alpha (TNF-alpha) in response to IgE immune complexes. This IgE-dependent cytokine release from monocytes may drive allergic inflammation.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Biology
Background:
- Monocytes play a crucial role in immune responses.
- Allergic reactions involve complex inflammatory pathways.
- The role of IgE in monocyte activation was not fully understood.
Purpose of the Study:
- To investigate the production of pro-inflammatory cytokines by circulating monocytes in response to IgE.
- To determine if IgE immune complexes stimulate monocytes to release IL-1 beta and TNF-alpha.
Main Methods:
- Monocytes were stimulated with IgE/alpha IgE immune complexes.
- Interleukin-1 beta (IL-1 beta) production was measured by biological assay (T cell proliferation) and ELISA.
- Tumor Necrosis Factor-alpha (TNF-alpha) production was quantified using ELISA.
- RNA hybridization analysis was performed to assess de novo synthesis of IL-1 beta.
Main Results:
- IgE immune complexes significantly increased IL-1 beta and TNF-alpha production by monocytes.
- IL-1 beta production was confirmed to be de novo synthesis, with RNA expression peaking at 2 hours.
- Biological assays showed increased T cell proliferation in response to monocyte supernatants, indicating functional IL-1 beta.
- No significant IL-1 alpha secretion was detected.
Conclusions:
- Circulating monocytes are capable of IgE-dependent production of IL-1 beta and TNF-alpha.
- This cytokine release represents de novo synthesis and may contribute to the inflammatory processes observed in allergic responses.
Abstract:
These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.
