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Fc-receptor variants of a mouse macrophage cell line
Abstract:
Variants of the J774 mouse macrophage cell line that lack immunologically important membrane receptors were isolated. After mutagenesis, variants were selected in a metrizamide gradient that separated cells heavily rosetted with sheep erythrocytes (E) coated with rabbit anti-E IgG (EIgG) from poorly rosetted cells. Stable variants that exhibited altered binding were found with a frequency of <10(-7), and five clones were studied in detail. The variants failed to bind E opsonized with a monoclonal mouse IgG2b anti-E antibody but bound monomeric IgG2a normally when compared to the parental J774 line (K(a) 4 degrees C= approximately 1x10(8) M(-1); approximately 2x10(5) sites per cell). This demonstrates the independence of the receptor for mouse IgG2b complexes (FcRII) from the trypsin-sensitive receptor for mouse IgG2a monomer (FcRI). The variants bound an average of 10-15 EIgG per cell, compared to >20 per cell for J774. After trypsinization, three variants bound only three to five EIgG per cell; the J774 line was not affected by this treatment. Monomeric IgG2a could inhibit the binding of soluble rabbit IgG-antigen complexes to the variants but not to the parent line. Finally, E coated with IgM and complement (EIgMC) were bound poorly by all the variants, relative to the J774 parent. These results show that rabbit IgG complexes are bound by both FcRI and FcRII on mouse macrophages. The impairment of EIgMC rosetting in the variants suggests that the C3b receptor and FcRII are related.
Insights
Researchers isolated macrophage variants lacking key receptors, revealing distinct pathways for IgG2b (FcRII) and IgG2a (FcRI) binding. This study clarifies macrophage immune receptor function and potential links between FcRII and complement receptors.
Area of Science:
- Immunology
- Cell Biology
- Macrophage Biology
Background:
- Macrophage Fc receptors mediate crucial immune functions by binding antibody-opsonized targets.
- Understanding the distinct roles and potential cross-reactivity of different Fc receptor subtypes is essential for deciphering immune responses.
Purpose of the Study:
- To isolate and characterize J774 macrophage variants with defects in specific Fc receptor-mediated binding.
- To delineate the functional independence of the mouse IgG2b Fc receptor (FcRII) from the IgG2a Fc receptor (FcRI).
- To investigate potential relationships between FcRII and complement receptors.
Main Methods:
- Isolation of J774 macrophage variants using metrizamide density gradients after mutagenesis.
- Selection based on altered binding of sheep erythrocytes (E) opsonized with rabbit anti-E IgG (EIgG).
- Characterization of Fc receptor binding affinities and capacities for monomeric and complexed IgG, and EIgMC, in parental and variant cell lines.
Main Results:
- Isolated variants showed significantly reduced binding of IgG2b-opsonized targets, indicating impaired FcRII function.
- These variants retained normal binding of monomeric IgG2a, demonstrating FcRI independence.
- Impaired binding of complement-opsonized erythrocytes (EIgMC) in variants suggests a link between FcRII and complement receptors.
Conclusions:
- Mouse macrophage FcRII and FcRI are functionally distinct receptors.
- FcRII plays a role in binding IgG2b complexes, while FcRI binds monomeric IgG2a.
- The data suggest a potential relationship or shared component between FcRII and the C3b receptor.