Soluble CD16 binds peripheral blood mononuclear cells and inhibits pokeweed-mitogen-induced responses
C Teillaud1, J Galon, M T Zilber
1Laboratoire d'Immunologie Cellulaire et Clinique, Unité INSERM 255, Institut Curie, Paris, France.
Insights
Soluble CD16 (sCD16), derived from neutrophils, binds to human immune cells and inhibits their proliferation and antibody production. This suggests neutrophils regulate immune responses through sCD16.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophils express Fc gamma RII (CD32) and Fc gamma RIIIB (CD16) low-affinity IgG receptors.
- Soluble CD16 (sCD16) is generated from neutrophil Fc gamma RIIIB via proteolysis near the cell surface.
Purpose of the Study:
- To produce recombinant soluble CD16 (sCD16) in eukaryotic cells.
- To investigate the functional roles and binding properties of sCD16.
- To assess the impact of sCD16 on human peripheral blood mononuclear cell (PBMC) responses.
Main Methods:
- Production of recombinant sCD16 in eukaryotic cells.
- Binding assays with human IgG subclasses and F(ab')2 fragments.
- In vitro proliferation and antibody production assays using pokeweed mitogen (PWM) stimulated PBMCs.
- Flow cytometry (FACS) analysis to determine sCD16 binding to PBMCs.
Main Results:
- Purified recombinant sCD16 (48 kD) specifically bound human IgG1 and IgG3.
- sCD16 inhibited PWM-induced PBMC proliferation and IgM/IgG production in a time- and dose-dependent manner.
- FACS analysis confirmed sCD16 specifically binds to monocytes, B cells, and T cell subsets within PBMCs.
Conclusions:
- Soluble CD16 (sCD16) exhibits cell-binding and antiproliferative capacities.
- Neutrophils may regulate immune responses through the release of sCD16.
- sCD16 exerts its biological functions through direct interactions with mononuclear blood cells.
Abstract:
Human neutrophils express two types of low affinity receptors for IgG, Fc gamma RII or CD32 and Fc gamma RIIIB or CD16. Human serum contains soluble CD16 (sCD16), which is produced by proteolysis of neutrophil Fc gamma RIIIB, the cleavage site being located close to the cell surface. In order to assess the functional roles of sCD16, we have produced, in eukaryotic cells, a recombinant sCD16 containing the extracellular region of Fc gamma RIIIB. Purified sCD16, of molecular mass of 48 kD, bound human IgG1 and IgG3 but not IgG2, IgG4, or F(ab')2. It inhibited, in a time and dose-dependent fashion, proliferation and IgM and IgG production of human peripheral blood mononuclear cells (PBMC) stimulated by pokeweed mitogen (PWM) in vitro. FACS analysis showed that biotinylated sCD16 bound specifically to a fraction (35%) of PBMC, which corresponds to monocytes and to subsets of B and T lymphocytes. Moreover, sCD16 did not modify the staining of PBMC by FITC-coupled PWM. Thus, the biologic function(s) of sCD16 on PWM-induced responses are exerted through direct and specific interaction(s) with mononuclear blood cells and not with PWM. In conclusion, neutrophils may play a regulatory role on immune responses via the production of soluble forms of CD16 with cell-binding and antiproliferative capacities.
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