Role of the IgM Fc Receptor in Immunity and Tolerance

Immunoglobulin (Ig) M is the first antibody isotype to appear during evolution, ontogeny and immune responses. IgM not only serves as the first line of host defense against infections but also plays an important role in immune regulation and immunological tolerance. For many years, IgM is thought to function by binding to antigen and activating complement system. With the discovery of the IgM Fc receptor (FcμR), it is now clear that IgM can also elicit its function through FcμR. In this review, we will describe the molecular characteristics of FcμR, its role in B cell development, maturation and activation, humoral immune responses, host defense, and immunological tolerance. We will also discuss the functional relationship between IgM-complement and IgM-FcμR pathways in regulating immunity and tolerance. Finally, we will discuss the potential involvement of FcμR in human diseases.


INTRODUCTION
B cells produce different classes of antibodies (Ab), including IgM, IgD, IgG, IgA, and IgE. Ab constitutes a variable F(ab) region that binds to antigen (Ag) and a constant Fc region that mediates effector function. Cellular receptors for the Fc region mediate a variety of functions including phagocytosis of Ab-opsonized pathogens and induction of cellular cytotoxicity. Recent studies have unveiled three Fc receptors for IgM, including Fcα/µ receptor (Fcα/µR), polymeric immunoglobulin receptor (pIgR), and Fcµ receptor (FcµR). Fcα/µR, pIgR, and FcµR are all type I transmembrane proteins belonging to the immunoglobulin (Ig) gene superfamily. Fcα/µR is expressed by both hematopoietic and non-hematopoietic cells (1,2), and has been shown to play an important role in humoral immune responses, especially in pro-inflammatory functions of marginal zone B cells in sepsis (3). pIgR is expressed on the basolateral surface of ciliated epithelial cell in the mucosal epithelium (4,5), but not in hematopoietic cells (6). The main function of pIgR is to transport dimeric IgA and polymeric IgM from the lamina propria across the epithelial barrier to mucosal surfaces (7). FcµR was discovered relatively recently and its function has not been fully elucidated. Here we summarize the results of FcµR published over the past several years, and discuss how it contributes to immunity and tolerance.

MOLECULAR CHARACTERISTICS OF FCµR
The existence of a receptor for IgM was noted more than 40 years ago (8)(9)(10)(11)(12)(13)(14)(15)(16). Biochemical analysis revealed that human FcµR had a molecular weight of ∼60-kDa (17). Molecular cloning of FCMR, the gene encoding human FcµR, revealed that it is a single copy gene located on chromosome 1q32.2, adjacent to two other IgM associated Fc receptor genes, polymeric Ig receptor gene (PIGR) and the gene of FcR for IgA and IgM (FCAMR) (18). Human FcµR is a type I transmembrane protein of 390 amino acids (aa), composed of a 234-aa extracellular domain, a 21-aa transmembrane segment, and a 118-aa cytoplasmic tail (19,20). BW5147 T cells ectopically expressing human FcµR exhibited specific binding to IgM but not any other Ab isotypes, demonstrating that FcµR is the bona-fide receptor for IgM (18). Unlike many other FcRs, the cytoplasmic tail of human FcµR does not contain any immunoreceptor tyrosine-based activation (ITAM) or inhibitory (ITIM) motifs. Instead, it contains conserved serine and tyrosine residues, which match the recently described Igtail tyrosine (ITT) motif (21,22). Crosslinking human FcµR with either anti-FcµR monoclonal antibodies or preformed IgM immune complexes triggered the phosphorylation of these serine and tyrosine residues in FcµR-overexpressing BW5147 T cells, suggesting that FcµR could serve as an ITT phosphorylation molecule to interact with and influence the B cell receptor (BCR) signaling (23). Human FcµR is predominantly expressed by B, T, and NK cells, but not by monocytes, granulocytes, erythrocytes, and platelets (18). Human FcµR binds more efficiently to the Fc portion of IgM reactive with surface proteins than to the Fc portion of free IgM (24), suggesting that FcµR might modulate the signal of B, T, and NK cell surface receptors or proteins recognized by natural or immune IgM.
The mouse FcµR gene (Fcmr) is also a single copy gene located on chromosome 1 (56.89 cM), adjacent to Pigr and Fcamr (25). Although mouse and human FcµR have similar molecular structure, they share only 54% aa identity. Mouse FcµR also specifically binds to IgM (25,26). Unlike human FcµR, we found that mouse FcµR is predominantly expressed in B lymphocytes by both microarray of a panel of immune cell types and FACS analyses (25,27,28). However, others have reported that monocytes, macrophages, granulocytes, and dendritic cells also express FcµR (29,30 (28,31). FcµR expression level is indistinguishable between B1 (CD5 + ) and B2 (CD5 − ) cells in the spleen. In the peritoneal cavity, FcµR expression level in each B cell subsets follows the order: (31,32). In addition, FcµR expression is very low in pro-B (B220 + CD43 + ) and pre-B (B220 + CD43 − IgM − ) cells, and slightly upregulated in immature B cells (B220 dull IgM + ) in the bone marrow (BM) (27,31,33). FcµR expression in the germinal center (GC) B cells (CD95 + GL7 + ) is much lower than that in naïve B cells (27), suggesting that FcµR is down-modulated during GC reaction. FcµR is expressed at higher levels in plasmablasts compared to plasma cells. Intriguingly, FcµR is also expressed by IgG-or IgApositive B cells, suggesting that it may play a role in switched B cells (32).
It is intriguing that genes encoding FcµR, Fcα/µR, and pIgR are located in the same chromosomal region (18,25), suggesting that these genes are evolutionarily related and might have derived from a common ancestor gene. However, in contrast to FcµR which only binds to IgM, Fcα/µR binds both IgM and IgA (3,34,35). Moreover, pIgR binds both IgM and IgA via their associated J chains and is essential for the transcytosis of polymeric IgA and IgM to the gut (36). The expression pattern is also quite different among these receptors. FcµR is predominantly expressed by B cells in mice and by B, T, and NK cells in humans (18,25). In contrast, Fcα/µR is expressed by macrophages, B cells, intestinal lamina propria and several other cell types (35), and pIgR is mainly expressed on the intestinal epithelial cells (4,5). Although FcµR was originally designated as Fas apoptotic inhibitory molecule 3 or TOSO (37), it is now clear that both human and mouse FcµR have no inhibitory activity against Fas-mediated apoptosis (38,39).  (33,48). A comparison of the phenotypes of Fcmr −/− mice generated and/or analyzed by different groups is shown in Table 1.

FCµR IN B CELL DEVELOPMENT AND MATURATION
B cell development proceeds from pro-B, pre-B to immature B cells in BM (49). Immature B cells then migrate to the periphery where they further differentiate into various mature B cell subsets that play distinct roles. The survival and maturation of B cells are dependent on the strength of tonic BCR signal (50,51 (31)(32)(33). More recently, we found reduced tonic BCR signaling in FcµR-deficient MZB, which we think led to their decreased numbers in KO mice (28). In contrast, Honjo et al. suggested that the reduction of MZB in KO mice was due to their rapid differentiation into plasma cells (41). Lee et al. found decreased numbers of B cells in the spleen and lymph nodes (47). Choi et al. found that B-1a were increased but B-2 were decreased in the peritoneal cavity and that FOB were decreased in the   However, tyrosine 315, as well as the entire intracellular domain, was shown to be required for inhibiting an IgM anti-FAS Abinduced apoptosis (24). It remains to be investigated how FcµR specifically affects the late phase of BCR signaling and whether these tyrosine and serine residues are involved. It is well-known that B cells express FcγRIIB, which inhibits BCR signaling and B cell activation upon binding IgG-Ag immune complexes, which then results in colligation of FcγRIIB and the BCR. Therefore, B cells express two types of Fc receptors, FcµR and FcγRIIB, which promotes and inhibits BCR signaling and B cell activation, respectively (Figure 1). More recently, Nguyen et al. reported that FcµR limited tonic BCR signaling in immature B cells by regulating the expression of IgM BCR (33). Therefore, FcµR regulates both the cell surface expression and the function of BCR.

ROLE OF FCµR IN HUMORAL IMMUNE RESPONSES
The basal Ig levels reflect the immune homeostasis at the steady state. We found that basal serum IgM levels were elevated in the absence of FcµR in a gene dosage-dependent manner, suggesting that a portion of the serum IgM actually binds to the FcµR in WT mice (27). Nguyen et al. found the same results and attributed the high IgM level to the elevated numbers and hyper-activation of B1 cells in the spleen (33). In addition, Honjo et al. found that IgM levels were elevated and that the IgG3 levels were slightly elevated in KO mice (32). In contrast, Choi et al. reported that only IgG1 levels were reduced in 3-month old mice and IgG3 and IgA levels were slightly elevated in 6-month old mice (31). Therefore, FcµR-deficient mice generated by different groups all exhibited increased levels of serum IgM and/or IgG3 ( Table 1). These results implicate a role for FcµR in B cell homeostasis. We found that KO mice had significantly decreased production of NP-specific IgG1 during both primary and secondary responses against a T-dependent (T-D) Ag, NP-CGG (27,28), likely due to impaired GC formation and reduced memory and plasma cell differentiation. Similarly, Honjo et al. found impaired primary IgG1 and secondary IgM anti-CGG responses, but normal Ab affinity maturation (32). During humoral immune responses to T-D Ag, Ag-specific IgM is first produced, which is followed by the production of Agspecific IgG. Based on our results that FcµR is required for efficient Ab production and the earlier findings that FcγRIIB inhibits B cell activation and Ab production, we propose an autoregulatory mechanism for T-D humoral immune responses [(27) and Figure 2]. During the early phase of the response, when the amount of Ag-specific IgM is greater than that of Ag-specific IgG, B cell activation is enhanced by FcµR-mediated positive signals. However, during the later phase of the response, when the amount of Ag-specific IgG is greater than that of Ag-specific IgM, further B cell activation is suppressed by FcγRIIB-mediated inhibitory signal (Figure 2). B cell activation and Ab production can thus be positively and negatively regulated by Ag-specific IgM and IgG present in the local environment, respectively.
Consistent with the reduced survival in FcµR-deficient B cells after BCR crosslinking, FcµR KO mice had decreased Ab production against a type 2 T-independent (T-I) Ag, NP-FICOLL (27), since response to this type of Ag is largely dependent on BCR signal. Additionally, we found that FcµR KO mice had impaired Ab production against a type 1 T-I Ag, NP-LPS (28), which activates B cells through both BCR and toll-like receptor 4. Moreover, we found that MZB in KO were not activated upon LPS injection (28). Since MZB cells are thought to participate in the response to LPS, the reduced Ab production to NP-LPS immunization could be due to both a reduction in the number of MZB cells and their impaired response to LPS. Our results are consistent with the earlier finding by Lang et al. that FcµR-deficient mice had reduced LPS response in vivo (29). Choi et al. found elevated numbers of GC B cells and accelerated plasma cell formation during type 1 and 2 T-I immune responses and secondary T-D immune responses (31). In addition, the plasma cell formation in primary T-D immune response was also increased (summarized in Table 1). The reason for the discrepancies among results from different groups is unclear but could in part be attributable to the differences in the targeting strategy, the immunization protocol, and the genetic background as well as rearing environment of these mutant mice. Collectively, these results suggest that FcµR regulates humoral immune responses.

FcµR IN INFECTIOUS IMMUNITY
As summarized in Table 1, FcµR-deficient mice generated a higher titer of anti-phosphorylcholine Ab and a lower titer of anti-protein Ab than did WT mice when infected with a low dose of live non-encapsulated strain of Streptococcus pneumoniae (R36A) (32). However, a high dose of pathogen infection induced no significant difference in Ab production between WT and KO mice. We found that FcµR protected mice against sepsis induced by Citrobacter rodentium, a gram-negative bacterium that has LPS on the outer membrane (28). Similarly, Lang et al. found that the absence of FcµR resulted in limited cytokine production after FIGURE 3 | A model for FcµR-mediated immunity and tolerance. FcµR promotes the survival and activation of mature B cells by interacting with the BCR and potentiating foreign Ag-triggered BCR signaling (left). By analogy, FcµR might also promote self Ag-triggered BCR signaling in immature B cells and contribute to the deletion/anergy of autoreactive immature B cells in the BM (right). Ag-specific IgM/IgG are illustrated in the scheme shown in Figures 1, 2 and 4 to suggest that those reactions occur during an immune response. In contrast, IgM shown in this scheme is not Ag-specific to implicate that these reactions can occur in the absence of Ag-specific IgM.
Listeria monocytogenes (a gram-positive bacterium) infection and increased death of the infected KO mice (29). They also found that FcµR was required for the control of persistenceprone virus infection in a lymphocytic choriomeningitis virus model system (44). In addition, Yu et al. reported that FcµR deficiency resulted in increased numbers of IL-10-producing B cells, which mediated regulation of T cell immunity during influenza infection (45). On the contrary, Nguyen et al. found that FcµR expression on B cells, but not Fcα/uR expression or complement activation, was important for the antiviral IgG responses (48). B cell-specific KO mice lacked robust clonal expansion of influenza hemagglutinin-specific B cells early after infection and developed fewer IgG plasma cells and memory B cells in the spleen and BM, compared with WT mice (48). These results suggest that FcµR has important roles in B cell responses to protein and non-protein determinants of live pathogens and in cooperating with other immune cells to protect the mice against infection.

FCµR IN B CELL TOLERANCE
B cell central tolerance ensures autoreactive immature B cells to undergo clonal deletion, anergy or receptor editing while peripheral tolerance functions to delete autoreactive B cells generated during GC reaction. We and others found that KO generated autoreactive antibodies including anti-dsDNA, rheumatoid factor, and anti-nuclear antibodies (27,32,33,41,45). Honjo et al. crossed FcµR-deficient mice with the Fasdeficient autoimmune-prone B6.MRL Fas lpr/lpr mice (B6/lpr), and found that the double mutant mice had accelerated development of autoreactive Ab including anti-dsDNA and anti-Sm Ab (41). They also found enhanced formation of Mott cells, aberrant plasma cells which accumulate large amount of Ig in the rough endoplasmic reticulum, in KO mice. Nevertheless, KO mice with autoimmune-prone background have normal kidney function and equal mortality compared to control group (41). Brenner et al. reported that KO mice were protected from the development of severe experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Their results suggested that FcµR regulated the function of dendritic and regulatory T cells (30). Collectively, a common feature of KO and BKO generated by different groups is the production of various autoantibodies ( Table 1). It remains to be investigated how FcµR regulates B cell tolerance. We have shown that FcµR promotes B cell survival and activation by interacting with BCR and potentiating Ag-triggered BCR signaling (Figure 3, left panel). By analogy, we think that FcµR might also promote self Ag-triggered BCR signaling in immature B cells and contribute to the deletion/anergy of autoreactive immature B cells in the BM (Figure 3, right panel). Further studies are required to clarify whether and how FcµR contributes to B cell central or peripheral tolerance.

FUNCTIONAL RELATIONSHIP BETWEEN IgM-COMPLEMENT AND IgM-FCµR PATHWAYS
IgM is the first Ab to appear during evolution and the only isotype produced by all species of jawed vertebrates (54)(55)(56). It is also the first isotype produced during a T-D immune response and is the first line of host defense (57). IgM is not only an effector molecule, but also regulates humoral immune response. Earlier studies suggested that IgM promotes the production of antigen-specific IgG via activating complement. However, a recent study by Heyman's group demonstrated that mice expressing a mutant IgM unable to activate complement (Cµ13) had completely normal humoral immune responses (58), thus raising the possibly that in addition to complement activation, there are alternative pathways by which IgM elicits its function. As discussed above, IgM can elicit its function through FcµR. Therefore, both IgM-FcµR and IgM-complement pathways function to regulate B cell survival and activation (Figure 4). It remains to be investigated whether these two pathways function cooperatively, independently, or competitively.

FCµR IN HUMAN DISEASES
Human FcµR was shown to be overexpressed and associated with the anti-apoptotic characteristic in chronic lymphocytic leukemia (CLL) (59,60). CLL is a malignancy of mature IgM + B cells that exhibit features of polyreactive, partially anergized B cells related to memory B cells (60). Several studies showed that FCMR expression in CLL was significantly higher than that in healthy controls and other B cell lymphoproliferative diseases (59,(61)(62)(63). In addition, CLL patients also had higher serum titers of FcµR compared with healthy donors. The serum FcµR, a 40-kDa soluble form of the receptor generated by alternative splicing, was produced by both CLL B and non-CLL B cells (64). Cox regression analysis indicated that high expression of FCMR was an independent indicator for shorter treatmentfree survival in CLL (64). Thus, FcµR is associated with the disease progression and patient survival and may serve as a prognostic factor. Interestingly, FcµR can even be used as a target for a more selective treatment of CLL by T cells expressing a chimeric antigen receptor (CAR-T), and initial studies have implicated a superior therapeutic index with anti-FcµR CAR-T cells for the treatment of CLL compared with the currently used therapies (65).
The reason that causes FcµR upregulation in CLL remains unclear. A negative correlation was observed between age and FcµR expression (59). In addition, overexpression of FCMR seemed to promote the chromosomal abnormalities (61). These shreds of evidence suggest that FcµR expression is related to the degree of genomic activity. Intriguingly, surface FcµR levels were also significantly elevated in the non-CLL B cells and T cells, suggesting that abnormal expression of FcµR is associated with systemic gene regulation (64). FcµR expression is significantly upregulated by BCR stimulation but decreased by CD40 ligation, which suggested that autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL (63). Besides, FcµR expression on CLL cells is downmodulated at both the mRNA and protein levels by TLR7 and TLR9 agonists (60). This study also revealed that FcµR not only localized to the cell membrane but also accumulated in the trans-Golgi network (60). FcµR may internalize IgM-Ag complexes and thus serve as a receptor for the delivery of therapeutic Ab-drug conjugates into CLL cells (60). In addition, based on the findings in mice, human FcµR may have some roles in TNFα-mediated liver damage (47), malaria vaccine promotion (46), and the function of pancreatic islets (66).

CONCLUSION
IgM is an old immunoglobulin isotype, which can bind to Ag with high avidity and activate the complement cascade. Its authentic and specific Fc receptor (FcµR) is the last one to be explored after Fcα/µR and pIgR. Although there are some discrepancies regarding the function of FcµR published by different groups, the following common abnormal phenotypes have been observed: (1) alterations in B cell maturation and differentiation; (2) impaired humoral immune responses; (3) autoantibody production. In addition, FcµR appears to contribute to the initiation/progression of human CLL and has recently been tested as a therapeutic target for treating CLL. Yet still many questions remain to be answered, including the function of FcµR in the generation, maintenance and activation of memory B cells, and in host defense mediated by natural IgM produced by B-1 and Ag-specific IgM produced by B-2 cells. Further studies are required to fully uncover the function of FcµR in immunity and tolerance.

AUTHOR CONTRIBUTIONS
JL provided a draft of the manuscript. YW completed the references. EX and RH provided all the figures. QL revised the manuscript. HO corrected the manuscript. J-YW designed the outline and made the final corrections of the manuscript.