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Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-α production in a receptor for advanced glycation end
Benfang Helen Ruan1, Xin Li, Aaron R Winkler
1Inflammation and Immunology, Pfizer Biotherapeutics Research and Development, Cambridge, MA 02140, USA.
Abstract:
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
Insights
The receptor for advanced glycation end products (RAGE) binds C3a and CpG DNA, enhancing innate immune responses. This RAGE-mediated synergy in IFN-α production suggests a role in autoimmune diseases.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The receptor for advanced glycation end products (RAGE) is a multiligand receptor involved in various diseases, including autoimmune conditions.
- Understanding RAGE's pathogenic mechanisms requires identifying its ligands and their interactions.
Purpose of the Study:
- To identify novel ligands for RAGE.
- To investigate the synergistic effects of C3a and CpG DNA on RAGE-mediated immune responses.
- To elucidate the role of RAGE in the context of autoimmune disease pathogenesis.
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA) to determine binding affinity (EC50).
- Surface plasmon resonance and fluorescence anisotropy to analyze complex formation.
- Interferon-alpha (IFN-α) production assays in human peripheral blood mononuclear cells (PBMCs) and RAGE-deficient mouse bone marrow cells.
Main Results:
- C3a binds to RAGE with high affinity, which is significantly enhanced by human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs).
- A ternary complex of RAGE, C3a, and hCpGAs was demonstrated.
- C3a and hCpGAs synergistically increased IFN-α production in a RAGE-dependent manner, stimulating innate immune responses.
Conclusions:
- RAGE acts as a direct receptor for both C3a and CpGA.
- The interaction between C3a and CpGA via RAGE amplifies IFN-α production and innate immunity.
- These findings highlight a potential role for RAGE in autoimmune diseases characterized by the accumulation of immunostimulatory DNA and C3a.
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