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Functional analysis of complement receptor 1 using a new monoclonal antibody, KuN241
J M Mathew1, B Naziruddin, B Duffy
1Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110.
Insights
A novel monoclonal antibody, KuN241, targets complement receptor 1 (CR1) on immune cells. KuN241 binding to CR1 and Fc gamma RII triggers a unique transmembrane signaling pathway.
Area of Science:
- Immunology
- Cell Biology
Background:
- Monoclonal antibodies (MAbs) are crucial tools in immunology.
- Complement receptor 1 (CR1) plays a role in immune cell function.
- Transmembrane signaling pathways are critical for cellular communication.
Purpose of the Study:
- To characterize the antigen recognized by the MAb KuN241.
- To investigate the signaling mechanism induced by MAb KuN241 binding.
Main Methods:
- Immunoprecipitation and cell surface expression analysis.
- Functional assays measuring intracellular calcium ([Ca2+]i) levels.
- In vivo and in vitro cell activation studies.
Main Results:
- KuN241 recognizes complement receptor 1 (CR1) on monocytes, PMNs, B cells, and T cells.
- CR1 expression is downregulated on B cells after PWM activation and on PMNs/monocytes after PMA treatment.
- KuN241 binding induces a transient increase in intracellular calcium ([Ca2+]i) in PMNs and monocytes, mediated by dual binding to CR1 and Fc gamma RII.
Conclusions:
- KuN241 is a specific MAb for CR1.
- A novel transmembrane signaling mechanism involves dual binding of KuN241 to CR1 and Fc gamma RII.
- This signaling pathway offers new insights into immune cell activation.
Abstract:
A monoclonal antibody (MAb), KuN241, recognized an antigen present on all monocytes, polymorphonuclear leukocytes (PMNs), B cells, and a subpopulation of T cells. Cell surface expression pattern and immunoprecipitation studies indicated the molecule recognized by KuN241 to be complement receptor 1 (CR1). This was confirmed by sequential immunoprecipitation using E11, a CR1-specific monoclonal, and by immunoprecipitation analysis of a truncated transfection product of CR1. In vivo activation of PBLs for 7 days with pokeweed mitogen (PWM) abrogated surface expression of CR1 on B cells. Overnight culture with phorbol 12-myristate 13-acetate (PMA) downregulated the expression of CR1 detected by KuN241 both on PMNs and monocytes. Addition of MAb KuN241 to purified PMN and monocytes resulted in a transient increase in intracellular calcium ([Ca2+]i). This was blocked by the Fab fragment of MAb KuFc79 directed against the Fc gamma receptor. Further, Fab fragment of KuN241 cross-linked with F(ab')2 goat anti-mouse Ig failed to increase [Ca2+]i levels. Taken together, these results suggested a novel mechanism for transmembrane signaling events by dual binding of KuN241, the Fab region to CR1 and the Fc region of Fc gamma RII.
