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Identity of the elusive IgM Fc receptor (FcmuR) in humans.

Kubagawa H, Oka S, Kubagawa Y, Torii I, Takayama E, Kang DW, Gartland GL, Bertoli LF, Mori H, Takatsu H, Kitamura T, Ohno H, Wang JY - J. Exp. Med. (2009)

Bottom Line: Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

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Evaluation of the Ig isotype specificity of the FcμR. (A) FcμR cDNA–transduced BW5147 T cells were preincubated with various concentrations of inhibitor paraproteins of human origin (IgM, IgG1-4, IgA1-2, IgD, IgE, Fabμ, and Fc5μ) and incubated with 4 µg/ml of biotin-labeled human IgMκ. Bound biotinylated IgM was detected by addition of PE-labeled SA. Stained cells were analyzed by flow cytometry. Results are expressed as the percent mean fluorescence intensity (MFI) estimated as follows: 100 × ([X of IgM binding with inhibitors − X of background control]/[X of IgM binding without inhibitors − X of background control]), where X indicates the MFI values. Because there were no significant differences among each subclass of IgG and IgA, the results from all four IgG subclasses and two IgA subclasses have been combined as IgG and IgA, and the mean values are presented for simplicity. (B) Representative binding inhibition profiles. FcμR+ BW5147 T cells were incubated first with an eightfold excess of the indicated inhibitor proteins and then with 4 µg/ml of biotin-labeled human IgMκ. The dotted, dashed, and continuous lines indicate the immunofluorescence profiles for background controls, IgM binding without inhibitors, and IgM binding with the test inhibitors, respectively. (C) Control and FcμR+ BW5147 cells were incubated with culture supernatants containing the indicated concentrations of monomeric (m) or pentameric (p) IgM anti–mouse RBC mAb before developing with biotin-labeled anti–mouse κ mAb and APC-SA. These experiments were performed at least twice.
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fig2: Evaluation of the Ig isotype specificity of the FcμR. (A) FcμR cDNA–transduced BW5147 T cells were preincubated with various concentrations of inhibitor paraproteins of human origin (IgM, IgG1-4, IgA1-2, IgD, IgE, Fabμ, and Fc5μ) and incubated with 4 µg/ml of biotin-labeled human IgMκ. Bound biotinylated IgM was detected by addition of PE-labeled SA. Stained cells were analyzed by flow cytometry. Results are expressed as the percent mean fluorescence intensity (MFI) estimated as follows: 100 × ([X of IgM binding with inhibitors − X of background control]/[X of IgM binding without inhibitors − X of background control]), where X indicates the MFI values. Because there were no significant differences among each subclass of IgG and IgA, the results from all four IgG subclasses and two IgA subclasses have been combined as IgG and IgA, and the mean values are presented for simplicity. (B) Representative binding inhibition profiles. FcμR+ BW5147 T cells were incubated first with an eightfold excess of the indicated inhibitor proteins and then with 4 µg/ml of biotin-labeled human IgMκ. The dotted, dashed, and continuous lines indicate the immunofluorescence profiles for background controls, IgM binding without inhibitors, and IgM binding with the test inhibitors, respectively. (C) Control and FcμR+ BW5147 cells were incubated with culture supernatants containing the indicated concentrations of monomeric (m) or pentameric (p) IgM anti–mouse RBC mAb before developing with biotin-labeled anti–mouse κ mAb and APC-SA. These experiments were performed at least twice.

Mentions: A quantitative inhibition immunofluorescence assay with various Ig isotypes and IgM fragments as inhibitors revealed that IgM and its Fc5µ fragments consisting mostly of Cμ3/Cμ4 domains inhibited the binding of a biotin-labeled human IgM to FcμR+ BW5147 T cells in a dose-dependent manner, whereas the Fabµ fragments and other human Ig isotypes (IgG1-4, IgA1,2, IgD, and IgE) did not, thereby confirming the Fcμ specificity of the FcμR (Fig. 2 A). Fig. 2 B shows a representative inhibition profile for IgM binding with an eightfold excess of inhibitors. The inability of FcμR to bind polymeric IgA clearly indicates that FcμR is distinct from pIgR and Fcα/µR, both of which are shown to bind IgM and polymeric IgA. Moreover, the lack of binding to aggregated IgG further confirms the unique IgM isotype specificity of this receptor. Interestingly, mouse IgM bound better to the human FcμR than human IgM, and essentially identical FcμR binding was observed with IgMκ and IgMλ ligands irrespective of the presence of Ca2+/Mg2+. The affinity of IgM/FcμR binding was estimated by Scatchard plot analysis using 125I-labeled human IgM and FcμR+ BW5147 T cells. Assuming a 1:1 stoichiometry of pentameric IgM ligand to FcμR, this analysis revealed a strikingly high binding affinity of 10.8 ± 9.2 nM (mean ± SD from four experiments with two different human IgM myeloma proteins). Pretreatment of FcμR+ cells with neuraminidase slightly enhanced IgM binding, suggesting a role of sialic acid in this interaction, as reported previously by others (Pricop et al., 1993). Higher concentrations (>100-fold) were required for binding of IgM monomers to the FcμR+ cells than IgM pentamers, indicating the importance of IgM ligand configuration (Fig. 2 C). Collectively, these results indicate that the previously identified FAIM3/TOSO is an authentic FcμR with exclusive and high affinity binding specificity for the Fc portion of IgM.


Identity of the elusive IgM Fc receptor (FcmuR) in humans.

Kubagawa H, Oka S, Kubagawa Y, Torii I, Takayama E, Kang DW, Gartland GL, Bertoli LF, Mori H, Takatsu H, Kitamura T, Ohno H, Wang JY - J. Exp. Med. (2009)

Evaluation of the Ig isotype specificity of the FcμR. (A) FcμR cDNA–transduced BW5147 T cells were preincubated with various concentrations of inhibitor paraproteins of human origin (IgM, IgG1-4, IgA1-2, IgD, IgE, Fabμ, and Fc5μ) and incubated with 4 µg/ml of biotin-labeled human IgMκ. Bound biotinylated IgM was detected by addition of PE-labeled SA. Stained cells were analyzed by flow cytometry. Results are expressed as the percent mean fluorescence intensity (MFI) estimated as follows: 100 × ([X of IgM binding with inhibitors − X of background control]/[X of IgM binding without inhibitors − X of background control]), where X indicates the MFI values. Because there were no significant differences among each subclass of IgG and IgA, the results from all four IgG subclasses and two IgA subclasses have been combined as IgG and IgA, and the mean values are presented for simplicity. (B) Representative binding inhibition profiles. FcμR+ BW5147 T cells were incubated first with an eightfold excess of the indicated inhibitor proteins and then with 4 µg/ml of biotin-labeled human IgMκ. The dotted, dashed, and continuous lines indicate the immunofluorescence profiles for background controls, IgM binding without inhibitors, and IgM binding with the test inhibitors, respectively. (C) Control and FcμR+ BW5147 cells were incubated with culture supernatants containing the indicated concentrations of monomeric (m) or pentameric (p) IgM anti–mouse RBC mAb before developing with biotin-labeled anti–mouse κ mAb and APC-SA. These experiments were performed at least twice.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806608&req=5

fig2: Evaluation of the Ig isotype specificity of the FcμR. (A) FcμR cDNA–transduced BW5147 T cells were preincubated with various concentrations of inhibitor paraproteins of human origin (IgM, IgG1-4, IgA1-2, IgD, IgE, Fabμ, and Fc5μ) and incubated with 4 µg/ml of biotin-labeled human IgMκ. Bound biotinylated IgM was detected by addition of PE-labeled SA. Stained cells were analyzed by flow cytometry. Results are expressed as the percent mean fluorescence intensity (MFI) estimated as follows: 100 × ([X of IgM binding with inhibitors − X of background control]/[X of IgM binding without inhibitors − X of background control]), where X indicates the MFI values. Because there were no significant differences among each subclass of IgG and IgA, the results from all four IgG subclasses and two IgA subclasses have been combined as IgG and IgA, and the mean values are presented for simplicity. (B) Representative binding inhibition profiles. FcμR+ BW5147 T cells were incubated first with an eightfold excess of the indicated inhibitor proteins and then with 4 µg/ml of biotin-labeled human IgMκ. The dotted, dashed, and continuous lines indicate the immunofluorescence profiles for background controls, IgM binding without inhibitors, and IgM binding with the test inhibitors, respectively. (C) Control and FcμR+ BW5147 cells were incubated with culture supernatants containing the indicated concentrations of monomeric (m) or pentameric (p) IgM anti–mouse RBC mAb before developing with biotin-labeled anti–mouse κ mAb and APC-SA. These experiments were performed at least twice.
Mentions: A quantitative inhibition immunofluorescence assay with various Ig isotypes and IgM fragments as inhibitors revealed that IgM and its Fc5µ fragments consisting mostly of Cμ3/Cμ4 domains inhibited the binding of a biotin-labeled human IgM to FcμR+ BW5147 T cells in a dose-dependent manner, whereas the Fabµ fragments and other human Ig isotypes (IgG1-4, IgA1,2, IgD, and IgE) did not, thereby confirming the Fcμ specificity of the FcμR (Fig. 2 A). Fig. 2 B shows a representative inhibition profile for IgM binding with an eightfold excess of inhibitors. The inability of FcμR to bind polymeric IgA clearly indicates that FcμR is distinct from pIgR and Fcα/µR, both of which are shown to bind IgM and polymeric IgA. Moreover, the lack of binding to aggregated IgG further confirms the unique IgM isotype specificity of this receptor. Interestingly, mouse IgM bound better to the human FcμR than human IgM, and essentially identical FcμR binding was observed with IgMκ and IgMλ ligands irrespective of the presence of Ca2+/Mg2+. The affinity of IgM/FcμR binding was estimated by Scatchard plot analysis using 125I-labeled human IgM and FcμR+ BW5147 T cells. Assuming a 1:1 stoichiometry of pentameric IgM ligand to FcμR, this analysis revealed a strikingly high binding affinity of 10.8 ± 9.2 nM (mean ± SD from four experiments with two different human IgM myeloma proteins). Pretreatment of FcμR+ cells with neuraminidase slightly enhanced IgM binding, suggesting a role of sialic acid in this interaction, as reported previously by others (Pricop et al., 1993). Higher concentrations (>100-fold) were required for binding of IgM monomers to the FcμR+ cells than IgM pentamers, indicating the importance of IgM ligand configuration (Fig. 2 C). Collectively, these results indicate that the previously identified FAIM3/TOSO is an authentic FcμR with exclusive and high affinity binding specificity for the Fc portion of IgM.

Bottom Line: Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Show MeSH