Functions of IgM fc receptor (FcµR) related to autoimmunity.

Unlike Fc receptors for switched immunoglobulin (Ig) isotypes, Fc receptor for IgM (FcµR) is selectively expressed by lymphocytes. The ablation of the FcµR gene in mice impairs B cell tolerance as evidenced by concomitant production of autoantibodies of IgM and IgG isotypes. In this essay, we reiterate the autoimmune phenotypes observed in mutant mice, ie IgM homeostasis, dysregulated humoral immune responses including autoantibodies, and Mott cell formation. We also propose the potential phenotypes in individuals with FCMR deficiency and the model for FcµR-mediated regulation of self-reactive B cells.

Enhanced Mott cell formation linked with IgM Fc receptor (FcµR) deficiency.

In previous studies, Mott cells, an unusual form of plasma cells containing Ig-inclusion bodies, were frequently observed in peripheral lymphoid tissues in our IgM Fc receptor (FcµR)-deficient (KO) mouse strain. Because of discrepancies in the reported phenotypes of different Fcmr KO mouse strains, we here examined two additional available mutant strains and confirmed that such enhanced Mott-cell formation was a general phenomenon associated with FcµR deficiency. Splenic B cells from Fcmr KO mice clearly generated more Mott cells than those from WT mice when stimulated in vitro with LPS alone or a B-1, but not B-2, activation cocktail. Nucleotide sequence analysis of the Ig variable regions of a single IgMλ+ Mott-hybridoma clone developed from splenic B-1 B cells of Fcmr KO mice revealed the near (VH) or complete (Vλ) identity with the corresponding germline gene segments and the addition of six or five nucleotides at the VH/DH and DH/JH junctions, respectively. Transduction of an FcµR cDNA into the Mott hybridoma significantly reduced cells containing IgM-inclusion bodies with a concomitant increase in IgM secretion, leading to secreted IgM binding to FcµR expressed on Mott transductants. These findings suggest a regulatory role of FcµR in the formation of Mott cells and IgM-inclusion bodies.

Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms.

IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.

Soluble Fc Receptor for IgM in Sera From Subsets of Patients With Chronic Lymphocytic Leukemia as Determined by a New Mouse Monoclonal Antibody.

The FcR for IgM (FcµR) is the newest member of the FcR family, selectively expressed by lymphocytes, and distinct from FcRs for switched Ig isotypes that are expressed by various immune cell types and non-hematopoietic cells. From studies of Fcmr-ablated mice, FcµR was shown to have a regulatory function in B-cell tolerance, as evidenced by high serum titers of autoantibodies of the IgM and IgG isotypes in mutant mice. In our previous studies, both cell-surface and serum FcµR levels were elevated in patients with chronic lymphocytic leukemia (CLL), where antigen-independent self-ligation of BCR is a hallmark of the neoplastic B cells. This was assessed by sandwich ELISA using two different ectodomain-specific mAbs. To determine whether the serum FcµR is derived from cleavage of its cell-surface receptor (shedding) or its alternative splicing to skip the transmembrane exon resulting in a 70-aa unique hydrophilic C-terminus (soluble), we developed a new mouse IgG1κ mAb specific for human soluble FcµR (solFcµR) by taking advantages of the unique nature of transductant stably producing His-tagged solFcµR and of an in vivo differential immunization. His-tagged solFcµR attached to exosomes and plasma membranes, allowing immunization and initial hybridoma screening without purification of solFcµR. Differential immunization with tolerogen (membrane FcµR) and immunogen (solFcµR) also facilitated to generate solFcµR-specific hybridomas. The resultant solFcµR-specific mAb reacted with serum FcµR in subsets of CLL patients. This mAb, along with another ectodomain-specific mAb, will be used for verifying the hypothesis that the production of solFcµR is the consequence of chronic stimulation of BCR.

[B lymphocytes and plasma cells as drivers of rheumatic diseases]. / B-Lymphozyten und Plasmazellen als Treiber rheumatischer Erkrankungen.

Various research groups at the German Rheumatism Research Center in Berlin, in close cooperation with the Department of Rheumatology and Clinical Immunology of the Medical Clinic at the Charité, have made important contributions to the significance of B cells and plasma cells in rheumatic diseases, which are relevant not only for rheumatology but for all clinical specialties in which antibody-mediated diseases play a role. In particular, the research addresses impaired B cell homeostasis, the importance of the IgM Fc receptor in the regulation of autoimmunity, the role of long-lived memory plasma cells in maintaining autoimmunity and ensuring its survival in specific niches organized by stromal cells in bone marrow and inflamed tissues. The research results have contributed to a better understanding of the immunological and molecular mechanisms in rheumatic diseases and their treatment. The identification of the long-lived memory plasma cell has led to promising treatment approaches with curative potential in autoimmune diseases.

Differences between Human and Mouse IgM Fc Receptor (FcµR).

Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since the bona fide IgM Fc receptor (FcµR) was identified in humans by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. In this short essay, we describe the differences between human and mouse FcµRs in terms of their identification processes, cellular distributions and ligand binding activities with emphasis on our recent findings from the mutational analysis of human FcµR. We have identified at least three sites of human FcµR, i.e., Asn66 in the CDR2, Lys79 to Arg83 in the DE loop and Asn109 in the CDR3, responsible for its constitutive IgM-ligand binding. Results of computational structural modeling analysis are consistent with these mutational data and a model of the ligand binding, Ig-like domain of human FcµR is proposed. Serendipitously, substitution of Glu41 and Met42 in the CDR1 of human FcµR with mouse equivalents Gln and Leu, either single or more prominently in combination, enhances both the receptor expression and IgM binding. These findings would help in the future development of preventive and therapeutic interventions targeting FcµR.

A rheostat sets B-cell receptor repertoire selection to distinguish self from non-self.

In bone marrow VDJ-recombination continuously generates original repertoires of immature B cells expressing IgM-B cell receptor (BcR), in which each cell recognizes the wide variety of self and non-self antigens with an individually different spectrum of avidities. High avidity self-reactive B cells try to edit their BcRs by secondary or multiple VL-rearrangements to JL-rearrangements. If they do not manage to change their self reactivity, they are deleted by apoptosis. Low avidity self-reactive B cells are anergized, while B cells with no avidity to self are ignored. A rheostat crosslinking antigen-binding BcRs, self antigen complexed with pentameric IgM and Fcµ-receptor monitors high, low or no binding. PI3K and PTEN are the effectors of this self antigen-sensing device. In mature B cells this rheostat continues to function in the activation of resting B cells by foreign antigens which crosslink BcR, antigen and pentameric IgM with Fcµ-receptors.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

Identification of Amino Acid Residues in Human IgM Fc Receptor (FcµR) Critical for IgM Binding.

Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.

Functional Roles of the IgM Fc Receptor in the Immune System.

It is now evident from studies of mice unable to secrete IgM that both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since identification of its Fc receptor (FcµR) by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. Unlike Fc receptors for switched Ig isotypes (e.g., FcγRs, FcεRs, FcαR, Fcα/µR, pIgR, FcRn), FcµR is selectively expressed by lymphocytes: B, T, and NK cells in humans and only B cells in mice. FcµR may have dual signaling ability: one through a potential as yet unidentified adaptor protein non-covalently associating with the FcµR ligand-binding chain via a His in transmembrane segment and the other through its own Tyr and Ser residues in the cytoplasmic tail. FcµR binds pentameric and hexameric IgM with a high avidity of ~10 nM in solution, but more efficiently binds IgM when it is attached to a membrane component via its Fab region on the same cell surface (cis engagement). Four different laboratories have generated Fcmr-ablated mice and eight different groups of investigators have examined the resultant phenotypes. There have been some clear discrepancies reported that appear to be due to factors including differences in the exons of Fcmr that were targeted to generate the knockouts. One common feature among these different mutant mice, however, is their propensity to produce autoantibodies of both IgM and IgG isotypes. In this review, we briefly describe recent findings concerning the functions of FcµR in both mice and humans and propose a model for how FcµR plays a regulatory role in B cell tolerance.

Fcµ Receptor Promotes the Survival and Activation of Marginal Zone B Cells and Protects Mice against Bacterial Sepsis.

The marginal zone B cells (MZB) are located at the interface between the circulation and lymphoid tissue and as a gatekeeper play important roles in both innate and adaptive immune responses. We have previously found that MZB are significantly reduced in mice deficient in the IgM Fc receptor (FcµR) but how FcµR regulates the development and function of MZB remains unknown. In this study, we found that both marginal zone precursor (MZP) and MZB were decreased in FcµR-/- mice. The reduction of MZP and MZB was not due to impaired proliferation of these cells but rather due to their increased death. Further analysis revealed that FcµR-/- MZB had reduced tonic BCR signal, as evidenced by their decreased levels of phosphorylated SYK and AKT relative to WT MZB. MZB in FcµR-/- mice responded poorly to LPS in vivo when compared with MZB in WT mice. Consistent with the reduced proportion of MZB and their impaired response to LPS, antibody production against the type 1 T-independent Ag, NP-LPS, was significantly reduced in FcµR-/- mice. Moreover, FcµR-/- mice were highly susceptible to Citrobacter rodentium-induced sepsis. These results reveal a critical role for FcµR in the survival and activation of MZB and in protection against acute bacterial infection.

Authentic IgM Fc Receptor (FcµR).

Since the bona fide Fc receptor for IgM antibody (FcµR) was identified eight years ago, much progress has been made in defining its biochemical nature, cellular distribution, and effector function. However, there are clearly conflicting results, especially about the cellular distribution and function of murine FcµR. In this short article, we will discuss recent findings from us and other investigators along with our interpretations and comments that may help to resolve the existing puzzles and should open new avenues of investigation.

FcµR in human B cell subsets in primary selective IgM deficiency, and regulation of FcµR and production of natural IgM antibodies by IGIV.

IgMFcR (FcµR) are expressed on B cell and B cell subsets. Mice deficient in secreted IgM and FcµR share properties of impaired specific antibody response and autoimmunity with patient with selective IgM deficiency (SIGMD). Intravenous immunoglobulin (IGIV) regulates immune response, including modulation of IgGFc receptors. However, there are no data on the expression of FcµR in patients with SIGMD, and the effects of IGIV on FcµR. In this study, we investigated FcµR expression in naïve marginal zone (MZ), IgM memory, and class-switched memory B cells in patients with selective IgM deficiency and healthy controls. Furthermore, we examined the direct effect of IGIV on FcµR expression and on the upregulation of FcµR by TLR2 agonist (Pam3). Finally, we examined the effect of IVIG on spontaneously produced IgM and natural IgM anti-phosphorylcholine (PC) antibodies by B cells and B1 cells. FcµR expression is significantly decreased in MZ B cells in patients with SIGMD as compared to control. IGIV, at immunomodulatory concentrations, inhibited FcµR upregulation by Pam3 in MZ B cells, and IgM-depleted IGIV inhibited spontaneous secretion of natural IgM anti-PC antibodies and not total IgM by B1 cells. These data suggest that decreased FcµR expression on MZ B cells may play a role in the pathogenesis of SIGMD, and an inhibition of TLR-2-induced upregulation of FcµR by IGIV may be one of the mechanisms of its anti-inflammatory action. IGIV-induced inhibition of natural IgM antibodies may be one of the mechanisms of IGIV-induced immunoregulation.

Nomenclature of Toso, Fas apoptosis inhibitory molecule 3, and IgM FcR.

Hiromi Kubagawa and John E. Coligan coordinated an online meeting to define an appropriate nomenclature for the cell surface glycoprotein presently designated by different names: Toso, Fas apoptosis inhibitory molecule 3 (FAIM3), and IgM FcR (FcµR). FAIM3 and Faim3 are the currently approved symbols for the human and mouse genes, respectively, in the National Center for Biotechnology Information, Ensembl, and other databases. However, recent functional results reported by several groups of investigators strongly support a recommendation for renaming FAIM3/Faim3 as FCMR/Fcmr, a name better reflecting its physiological function as the FcR for IgM. Participants included 12 investigators involved in studying Toso/FAIM3(Faim3)/FµR, representatives from the Human Genome Nomenclature Committee (Ruth Seal) and the Mouse Genome Nomenclature Committee (Monica McAndrews), and an observer from the IgM research field (Michael Carroll). In this article, we provide a brief background of the key research on the Toso/FAIM3(Faim3)/FcµR proteins, focusing on the ligand specificity and functional activity, followed by a brief summary of discussion about adopting a single name for this molecule and its gene and a resulting recommendation for genome nomenclature committees.

Unique ligand-binding property of the human IgM Fc receptor.

The IgM Fc receptor (FcµR) is the newest FcR, and coligation of FcµR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcµR was further examined. FcµR-mediated protection from apoptosis was partially blocked by addition of 10(4) molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcµR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcµR resulted in modulation of Ca(2+) mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcµR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcµR upon IgM binding at 4°C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcµR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcµR and its critical role in receptor function. Hence, FcµR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface.

Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcµR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcµR-bearing, but not FcµR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcµR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcµR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcµR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcµR(+) cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcµR(+) cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcµR binding. Unlike control Jurkat cells, FcµR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcµR, thereby promoting apoptosis of FcµR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcµR.

The old but new IgM Fc receptor (FcµR).

IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcµR) in such effector functions has not been explored until recently. We have identified an authentic FcµR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcµR deficiency in mice. Since the FcµR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcµR/Toso/FAIM3 and we will also comment on this topic.

Enhanced auto-antibody production and Mott cell formation in FcµR-deficient autoimmune mice.

The IgM-Fc receptor (FcµR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcµR(-) B6/lpr than FcµR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcµR(+) B6/lpr mice, were reduced to normal B6 levels in FcµR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcµR(-) B6/lpr mice compared with either FcµR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcµR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcµR(-) mice compared with FcµR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcµR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcµR(-) and FcµR(+) B6/lpr mice. Collectively, these findings suggest that FcµR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice.

The long elusive IgM Fc receptor, FcµR.

IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune "natural" and antigen-induced "immune" IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcµR). We have recently identified a bona fide FcµR in both humans and mice. In this article we briefly review what we have learned so far about FcµR.