CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells' immune functions

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• S100A8 and S100A9 proteins are key regulators of myeloid immune cells, but their precise immunologic roles are not fully understood.
• Cluster of differentiation 68 (CD68) is a protein found on myeloid cells, and its interaction with extracellular proteins is under investigation.

Purpose of the Study:

• To determine if CD68 binds to extracellular S100A8 and/or S100A9.
• To investigate the role of CD68-S100 protein interactions in regulating myeloid cell immune functions.

Main Methods:

• Enzyme-linked immunosorbent assay (ELISA) and affinity chromatography to assess binding between recombinant S100 proteins and CD68.
• Flow cytometry to confirm binding and evaluate the impact of deglycosylation on S100 protein binding to macrophages.
• In vitro assays using anti-CD68 antibodies, small interfering RNA (siRNA) targeting CD68, and stimulation with S100 proteins or LPS to analyze immune responses.

Main Results:

• Recombinant S100A8 and S100A9 were shown to bind to CD68, but not to CD14, confirmed by ELISA and flow cytometry.
• Deglycosylation of macrophages reduced the binding of S100A8 and S100A9, suggesting glycosylation is important for this interaction.
• Anti-CD68 antibody treatment suppressed S100A8 and S100A9 expression, while CD68 knockdown inhibited macrophage activation by S100 proteins.
• S100A9 stimulation suppressed IL-1β mRNA expression, and S100A8/S100A9 modulated other cytokine mRNAs (IL-6, IL-10, TNF-α).
• In LPS-stimulated macrophages, CD68 colocalized with S100A8 and S100A9, with increased levels of all three molecules.

Conclusions:

• CD68 on macrophages directly binds to extracellular S100A8 and S100A9.
• This interaction plays a significant role in regulating the immune functions of myeloid cells, including cytokine production and activation pathways.