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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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Role of FcμR in Fas-mediated apoptosis of Jurkat T cells. (A) Jurkat cells transduced without (none) or with the bicistronic retroviral construct containing GFP cDNA only (GFP) or both FcμR and GFP cDNAs (FcμR/GFP) were incubated with biotin-labeled isotype-matched control mAb (left), HM14 anti-FcμR mAb (middle), or human IgM (right), and then with APC-SA before analysis by FACSCalibur. Note the comparable levels of GFP in both GFP and FcμR/GFP transductants, and the expression of FcμR on the FcμR/GFP transductant as determined by anti-FcμR reactivity and IgM ligand binding. (B) These three cell lines were incubated at 37°C for 24 h with agonistic anti–human Fas mAbs of mouse IgMκ (CH11 clone; 10 ng/ml) or IgG3κ isotype (2R2 clone; 0.3 µg/ml). Cells were stained with 7-AAD and APC-labeled annexin V before identification of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by FACSCalibur. Note the resistance of FcμR/GFP transductant to IgM but not IgG3 anti-Fas mAb–induced apoptosis. Numbers indicate percentages of cells. These experiments were performed more than three times.
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fig6: Role of FcμR in Fas-mediated apoptosis of Jurkat T cells. (A) Jurkat cells transduced without (none) or with the bicistronic retroviral construct containing GFP cDNA only (GFP) or both FcμR and GFP cDNAs (FcμR/GFP) were incubated with biotin-labeled isotype-matched control mAb (left), HM14 anti-FcμR mAb (middle), or human IgM (right), and then with APC-SA before analysis by FACSCalibur. Note the comparable levels of GFP in both GFP and FcμR/GFP transductants, and the expression of FcμR on the FcμR/GFP transductant as determined by anti-FcμR reactivity and IgM ligand binding. (B) These three cell lines were incubated at 37°C for 24 h with agonistic anti–human Fas mAbs of mouse IgMκ (CH11 clone; 10 ng/ml) or IgG3κ isotype (2R2 clone; 0.3 µg/ml). Cells were stained with 7-AAD and APC-labeled annexin V before identification of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by FACSCalibur. Note the resistance of FcμR/GFP transductant to IgM but not IgG3 anti-Fas mAb–induced apoptosis. Numbers indicate percentages of cells. These experiments were performed more than three times.

Mentions: To determine whether FcμR inhibits Fas-mediated apoptosis as originally described for FAIM3/TOSO (Hitoshi et al., 1998), retroviral constructs containing both FcμR and GFP cDNAs or only the GFP cDNA were transduced into the apoptosis-prone Jurkat human T cell line. Cells expressing comparable levels of GFP were enriched from each transductant by FACS, and the FcμR/GFP transductant was found to express relatively high levels of cell-surface FcμR as determined by both receptor-specific mAbs (see next section) and IgM ligand binding (Fig. 6 A). The resultant FcμR+GFP+ or GFP+ Jurkat cells and nontransduced Jurkat cells as an additional control were then subjected to apoptosis assays using agonistic anti-Fas mAbs of the IgM or IgG3 isotype. Cross-linkage of Fas with the IgM antibody induced robust early (annexin V+/7-aminoactinomycin D [7-AAD]−) and late (annexin V+/7-AAD+) apoptotic cells as well as dead cells (annexin V−/7-AAD+) in the nontransduced Jurkat cells and the GFP+ cells, but not in the FcμR+/GFP+ cells (Fig. 6 B). This result is consistent with the previously reported antiapoptotic activity of FAIM3/TOSO (Hitoshi et al., 1998). It should be noted, however, that addition of control IgM of either human or mouse origin at a 100-fold molar excess into these cultures did not make the FcμR+/GFP+ cells susceptible to IgM anti-Fas mAb-induced apoptosis, suggesting that the simultaneous dual binding to Fas and FcμR (i.e., cis interaction) is dominant over the single binding to FcμR (i.e., trans interaction) in this apoptosis model (Fig. S4). Unlike the effect seen with IgM anti-Fas mAb, ligation of Fas receptor with the IgG3 antibody induced apoptosis in all three cell types, including the FcμR+GFP+ cells. Notably, ligation of FcμR and Fas with the corresponding mAbs either in the absence (i.e., separate ligation of each receptor) or presence of a common secondary reagent (i.e., coligation of both receptors) had no demonstrable effects on the IgG3 anti-Fas mAb–induced apoptosis of FcμR+GFP+ cells. Essentially identical results using IgM versus IgG3 anti-Fas mAb were also obtained with EBV-transformed B cell lines expressing both endogenous FcμR and Fas on their cell surface (unpublished data). Collectively, these findings indicate that FcμR has no intrinsic activity to inhibit Fas-mediated apoptosis, but they raise the interesting possibility that IgM anti-Fas autoantibody, if present in individuals with autoimmune disorders, could interrupt Fas-mediated signaling via FcμR in vivo.


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)

Role of FcμR in Fas-mediated apoptosis of Jurkat T cells. (A) Jurkat cells transduced without (none) or with the bicistronic retroviral construct containing GFP cDNA only (GFP) or both FcμR and GFP cDNAs (FcμR/GFP) were incubated with biotin-labeled isotype-matched control mAb (left), HM14 anti-FcμR mAb (middle), or human IgM (right), and then with APC-SA before analysis by FACSCalibur. Note the comparable levels of GFP in both GFP and FcμR/GFP transductants, and the expression of FcμR on the FcμR/GFP transductant as determined by anti-FcμR reactivity and IgM ligand binding. (B) These three cell lines were incubated at 37°C for 24 h with agonistic anti–human Fas mAbs of mouse IgMκ (CH11 clone; 10 ng/ml) or IgG3κ isotype (2R2 clone; 0.3 µg/ml). Cells were stained with 7-AAD and APC-labeled annexin V before identification of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by FACSCalibur. Note the resistance of FcμR/GFP transductant to IgM but not IgG3 anti-Fas mAb–induced apoptosis. Numbers indicate percentages of cells. These experiments were performed more than three times.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig6: Role of FcμR in Fas-mediated apoptosis of Jurkat T cells. (A) Jurkat cells transduced without (none) or with the bicistronic retroviral construct containing GFP cDNA only (GFP) or both FcμR and GFP cDNAs (FcμR/GFP) were incubated with biotin-labeled isotype-matched control mAb (left), HM14 anti-FcμR mAb (middle), or human IgM (right), and then with APC-SA before analysis by FACSCalibur. Note the comparable levels of GFP in both GFP and FcμR/GFP transductants, and the expression of FcμR on the FcμR/GFP transductant as determined by anti-FcμR reactivity and IgM ligand binding. (B) These three cell lines were incubated at 37°C for 24 h with agonistic anti–human Fas mAbs of mouse IgMκ (CH11 clone; 10 ng/ml) or IgG3κ isotype (2R2 clone; 0.3 µg/ml). Cells were stained with 7-AAD and APC-labeled annexin V before identification of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by FACSCalibur. Note the resistance of FcμR/GFP transductant to IgM but not IgG3 anti-Fas mAb–induced apoptosis. Numbers indicate percentages of cells. These experiments were performed more than three times.
Mentions: To determine whether FcμR inhibits Fas-mediated apoptosis as originally described for FAIM3/TOSO (Hitoshi et al., 1998), retroviral constructs containing both FcμR and GFP cDNAs or only the GFP cDNA were transduced into the apoptosis-prone Jurkat human T cell line. Cells expressing comparable levels of GFP were enriched from each transductant by FACS, and the FcμR/GFP transductant was found to express relatively high levels of cell-surface FcμR as determined by both receptor-specific mAbs (see next section) and IgM ligand binding (Fig. 6 A). The resultant FcμR+GFP+ or GFP+ Jurkat cells and nontransduced Jurkat cells as an additional control were then subjected to apoptosis assays using agonistic anti-Fas mAbs of the IgM or IgG3 isotype. Cross-linkage of Fas with the IgM antibody induced robust early (annexin V+/7-aminoactinomycin D [7-AAD]−) and late (annexin V+/7-AAD+) apoptotic cells as well as dead cells (annexin V−/7-AAD+) in the nontransduced Jurkat cells and the GFP+ cells, but not in the FcμR+/GFP+ cells (Fig. 6 B). This result is consistent with the previously reported antiapoptotic activity of FAIM3/TOSO (Hitoshi et al., 1998). It should be noted, however, that addition of control IgM of either human or mouse origin at a 100-fold molar excess into these cultures did not make the FcμR+/GFP+ cells susceptible to IgM anti-Fas mAb-induced apoptosis, suggesting that the simultaneous dual binding to Fas and FcμR (i.e., cis interaction) is dominant over the single binding to FcμR (i.e., trans interaction) in this apoptosis model (Fig. S4). Unlike the effect seen with IgM anti-Fas mAb, ligation of Fas receptor with the IgG3 antibody induced apoptosis in all three cell types, including the FcμR+GFP+ cells. Notably, ligation of FcμR and Fas with the corresponding mAbs either in the absence (i.e., separate ligation of each receptor) or presence of a common secondary reagent (i.e., coligation of both receptors) had no demonstrable effects on the IgG3 anti-Fas mAb–induced apoptosis of FcμR+GFP+ cells. Essentially identical results using IgM versus IgG3 anti-Fas mAb were also obtained with EBV-transformed B cell lines expressing both endogenous FcμR and Fas on their cell surface (unpublished data). Collectively, these findings indicate that FcμR has no intrinsic activity to inhibit Fas-mediated apoptosis, but they raise the interesting possibility that IgM anti-Fas autoantibody, if present in individuals with autoimmune disorders, could interrupt Fas-mediated signaling via FcμR in vivo.

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