Springer Seminars in Immunopathology

, 28:377

Molecular and functional characteristics of the Fcα/μR, a novel Fc receptor for IgM and IgA

Authors

    • Department of Immunology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences and Center for TARAUniversity of Tsukuba
  • Shin-ichiro Honda
    • Department of Immunology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences and Center for TARAUniversity of Tsukuba
Review

DOI: 10.1007/s00281-006-0050-3

Cite this article as:
Shibuya, A. & Honda, S. Springer Semin Immun (2006) 28: 377. doi:10.1007/s00281-006-0050-3

Abstract

IgM is the first antibody to be produced in a humoral immune response and is a major isotope of natural antibodies and may play an important role in innate immunity. On the other hand, IgA is a secreted antibody at the mucosal membrane such as the gastrointestinal and respiratory tracts and protects from initial invasion of microbes. However, how these antibodies are involved in immunity has been poorly elucidated. We previously identified a novel Fc receptor for IgA and IgM, designated Fcα/μ receptor (Fcα/μR), whose gene is closely located at the polymeric immunoglobulin receptor (poly-IgR), also a receptor for IgA and IgM, in the Fc receptor gene cluster on the chromosome 1. In contrast to the the poly-IgR that is expressed on epithelial, but not hematopoietic, cells, Fcα/μR is constitutively expressed on the majority of B lymphocytes and macrophages in the spleen and at the center of the secondary lymphoid follicles. The Fcα/μR mediates endocytosis Staphylococcus aureus /anti-S. aureus IgM antibody immune complexes by B lymphocytes, for which the dileucine motif in the cytoplasmic tail of the mouse Fcα/μR is responsible. These results reveal a new mechanism in the primary stage of immune defense against microbes.

Introduction

Receptors for the Fc portions of immunoglobulin (Fc receptors) play important roles for a wide array of immune responses. The binding of the Fc portions of antibodies or immune complexes to cell surface Fc receptors on hematopoietic cells can trigger or inhibit immunologic functions, including antibody-dependent cellular cytotoxicity, mast cell degranulation, phagocytosis, cell proliferation, antibody secretion, and enhancement of antigen presentation [7, 28, 29]. Considerable progress has been made in elucidating the structural and functional diversity of the Fc receptors for IgG and IgE [8, 12, 27, 33, 3537, 39]. Results obtained in Fc receptor-deficient mice show an important role for those receptors on host immunity, allergy, or autoimmunity. Analysis of mice lacking the gene for the subunit of the high-affinity IgE receptor, Fcε receptor I (FcεRI), revealed a defect in IgE-triggered passive anaphylaxis due to the absence of the FcεRI on mast cells and basophils [8]. Similarly, mice with a disrupted γ gene from the FcεRI or the IgG Fc receptor I (FcγRI) and III (FcγRIII) exhibit identical phenotypes [35]. Moreover, FcεRI-γ gene knockout mice lack IgG-mediated phagocytosis by macrophages [35] and the ability to mount the Arthus reaction [34]. In contrast to these triggering Fc receptors, the Fcγ receptor IIb (FcγRIIb) inhibits BCR-triggered lymphocyte proliferation, antibody secretion, and lymphokine secretion [29].

IgM is the first antibody to be produced in a humoral immune response and may play an important role in the primary stage of immunity [2]. In addition, IgM is a major isotope of natural antibodies, which exist in naive animals before infections and are involved in the prevention of pathogen dissemination to vital organs [3, 10]. It is also widely accepted that the IgM immune complex is a strong activator for the classical complement cascade by efficient interaction between antigen-binding IgM and C1q subunit [4]. On the other hand, IgA is produced 2∼3 g/day per adult and exists in sera and in the mucosal membrane. IgA is a secreted antibody at the mucosal membrane such as the gastrointestinal and respiratory tracts, and it protects from initial invasion of microbes. IgA binds to microbes and toxins present in the lumen and neutralizes them by blocking their entry into the host. However, how these antibodies are involved in immunity has been poorly elucidated. Functional Fc receptors for IgM (FcμR) have been reported on subpopulations of human and rodent T, B, and NK cells [11, 19, 22, 24, 25, 38]. In contrast to the well-defined Fcγ and Fcε receptors, a gene encoding an Fcμ receptor has not been previously identified, although a polymeric Ig receptor able to bind IgM and IgA, which transports these antibodies across epithelial cells into the lumen, has been described [21]. Similarly, although a human Fcα receptor (FcαR) (CD89) gene has been reported [18], a homologous rodent CD89 has not yet been found. We identified a novel Fc receptor for IgA and IgM, designated the Fcα/μR [31]. This review will focus on the molecular and functional characteristics of the Fcα/μR.

Identification of Fcα/μR

To identify an Fc receptor for IgM, we screened COS-7 cells transfected with a cDNA library prepared from the mouse T cell leukemia BW5147 by using a mouse IgM monoclonal antibody (mAb) as a probe. We isolated a cDNA clone of 2,361 bp that contains an open reading frame encoding a type I transmembrane protein [31]. A pair of cysteine residues in the extracellular domain was flanked by the consensus sequence for Ig-like domains, indicating that this molecule, designated mFcα/μR, is a member of the Ig supergene family (Fig. 1). A human cDNA with homology to the mFcα/μR (hFcα/μR) was identified in a database [31] and was found to encode a protein with 49% amino acid identity to the mFcα/μR. These genes were mapped by fluorescence in situ hybridization (FISH) to syntenic regions of mouse chromosome 1 (1F) and human chromosome 1 (1q32.3) near several other Fc receptors, including Fcγ receptors I, II, and III, Fcε receptor, and the polymeric Ig receptor (Fig. 2).
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Fig. 1

Molecular characteristics of the Fcα/μ receptor. The cDNA of Fcα/μR contains an open reading frame encoding a type I transmembrane protein with a 32-amino acid (aa) leader sequence, 423 aa extracellular domain, 20 aa transmembrane domain, and 60 aa cytoplasmic region. The extracellular domain has four potential sites for N-linked glycosylation, suggesting that the receptor is a glycoprotein. A pair of cysteine residues in the extracellular domain was flanked by the consensus sequence for Ig-like domains, indicating that Fcα/μR is a member of the Ig supergene family. The cytoplasmic tail of mouse, but not human, Fcα/μR contains dileucine motif

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Fig. 2

The Fcα/μR gene is located in the Fc receptor gene cluster in chromosome 1. The human Fcα/μR gene was mapped by FISH to chromosome 1 (1q32.3), near several other Fc receptors, including Fcγ receptors I, II, III, Fcε receptor, and the polymeric Ig receptor. The mouse Fcα/μR gene was mapped to the syntenic region of mouse chromosome 1 (1F). The human Fcα/μR gene is located just 12 kbp apart from the locus of the Poly-IgR gene

The Fcα/μR is unique, demonstrating no significant homology to other proteins or nucleotide sequences in databases. However, we observed a motif in the Ig-like domain conserved in the first Ig-like domain of the human, bovine, and rodent polymeric Ig receptor, which binds to the Fc of IgA and IgM (Fig. 3). Overall, the Fcα/μR has less than 10% amino acid homology with the mouse and human polymeric Ig receptor.
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Fig. 3

The amino acid sequences of the Ig-like domain. The conserved amino acid sequences in the Ig-like domain of the Fcα/μR and in the first Ig-like domain of poly-IgR are shown in each species indicated. The peptide in the box is involved in IgA and IgM binding

Because FcγR has rich diversity of structures encoded by corresponding genes on chromosome 1, a question has been raised whether the Fcα/μR is a single or multiple gene-family member [16]. To address this issue, we searched the Fcα/μR gene in a database. The Fcα/μR gene containing the entire coding regions was found in the locus from the 194644 to 194653 kbp on chromosome 1 (GenBank and DDBJ accession number: NT004787). However, we could not detect any gene fragments with significant homology with Fcα/μR in other chromosomal regions, suggesting that the Fcα/μR is a single gene-family member [32]. Comparison of human Fcα/μR cDNA with the genome sequence on chromosome 1 in the database revealed that the human Fcα/μR gene is composed of 6 exons. It is of interest that the human Fcα/μR gene is located just 12 kbp apart from the locus of the Poly-IgR gene [32]. Although Fcα/μR and poly-IgR have less than 10% amino acid homology each other, both Fc receptors have a conserved motif in the Ig-like domain, which binds to the Fc of IgA and IgM [31], suggesting the phylogenetic and functional relationship between Fcα/μR and Poly-IgR.

Analyses of IgA and IgM binding to the Fcα/μR by monoclonal antibodies

To examine the cell surface expression of the Fcα/μR protein, we generated a monoclonal antibody (mAb) against the mFcα/μR by immunizing a rat with the Ba/F3 cells transfected with mFcα/μR. Out of more than 2,000 hybridomas screened, we obtained only a single clone that produced a mAb against mFcα/μR, which we designated TX6. Although TX6 mAb effectively stained cell surface mFcα/μR, as determined by flow cytometry, it was inappropriate to use for immunohistochemistry and immunoblotting. Therefore, we had been trying to further generate mAbs against mFcα/μR by immunizing rats with the Ba/F3 transfectant expressing mFcα/μR several times. However, they were unsuccessful. Furthermore, we could never generate anti-human Fcα/μR (hFcα/μR) mAb by immunizing mice with the hFcα/μR antigens. Because amino acid sequences of extracellular domain of Fcα/μR are highly conserved among human, mouse, and rat (80.2% identity between mouse and rat and 51.3% between mouse and human) [31], it was considered that the immunological tolerance against Fcα/μR antigens may be a cause of difficulty for establishing anti-human and anti-mouse Fcα/μR mAbs. To avoid immunological tolerance against mFcα/μR antigens, we immunized a mouse deficient in Fcα/μR gene, instead of rat or other species of animals, for generation of anti-mFcα/μR with the mFcα/μR-expressing Ba/F3 transfectant and successfully generated 12 anti-mFcα/μR mAbs, which were designated TX57 to TX68 [5].

These mAbs could be divided into five groups (group I, II, III, IV, and V) that differed from each other in their recognition sites (Table 1). It was of interest that the group III (TX58, TX59, TX60, TX61, and TX66) mAbs, but not the others, cross-reacted with hFcα/μR, suggesting that these mAbs recognize an epitope conserved also in hFcα/μR. Moreover, group I (TX57) and the groups III (TX58, TX59, TX60, TX61, and TX66) and IV (TX67 and TX68) totally and partially inhibited the binding of both IgM and IgA to the mFcα/μR, respectively. These results suggested that the epitopes in the mFcα/μR recognized by the mAbs in the groups I, III, and IV are both the IgA and IgM binding site of the mFcα/μR or physically related to the ligand binding site. These blocking mAbs bound to a peptide derived from the Ig-like domain of mFcα/μR [5], which is conserved not only in human, mouse, and rat Fcα/μR but also in polymeric Ig receptor (poly-IgR; Fig. 3), which recognizes the J chain of IgA and IgM. These results suggest that IgA and IgM bind to an epitope in the conserved amino acids in the Ig-like domain of mFcα/μR and poly-IgR. On the other hand, although the binding site of IgA and IgM by the Fcα/μR has not yet been determined, these results suggested that the Fcα/μR might recognize the same (i.e., J chain) or closely related epitope of IgA and IgM as the poly-IgR.
Table 1

Summary of the characteristics of anti-mFcα/μR mAbs

Group

I

II

III

IV

V

Isotype

TX57

TX64

TX58

TX59

TX60

TX61

TX66

TX67

TX68

TX62

TX63

TX65

IgG1

IgG1

IgG1

IgG1

IgG1

IgG1

IgG1

IgG1

IgG1

IgG2a

IgG2a

IgG2a

Blocking capacity of IgM binding

++

+

+

+

+

+

+

+

Blocking capacity of IgA binding

++

+

+

+

+

+

+

+

Cross−reactivity of human Fcα/μR

+

+

+

+

+

Binding to the motif peptide

+

+

+

+

+

+

+

+

Expression of Fcα/μR transcript

RT-PCR analyses demonstrated that the Fcα/μR transcripts is expressed in a variety of hematopoietic and non-hematopoietic tissues including thymus, spleen, liver, kidney, small and large intestines, and placenta [31, 32]. Northern blot analyses further demonstrated that the Fcα/μR transcripts is abundantly expressed in kidney, small intestine, and lymph node [30]. Although several splicing variants in the exons 1 and 2 of the Fcα/μR were identified in human and mouse kidney (our unpublished observation), the molecular and functional characteristics of the splicing variants and the original Fcα/μR in the kidney have remained undetermined. The abundant expression of the Fcα/μR transcripts in the kidney suggests a possibility that the Fcα/μR is involved in IgA nephropathy, which is the most common form of glomerulonephritis and characterized by the deposition of IgA immune complexes in the glomerular mesangium. McDonald et al. [20] reported that human mesangial cell lines expressed the Fcα/μR transcripts. However, since it has remained unclear whether the Fcα/μR protein and its transcript are really expressed on primary mesangial cells, careful examination should be required to determine the involvement of the Fcα/μR in the pathogenesis of IgA nephropathy.

Expression of Fcα/μR protein

Analysis of spleen cells by flow cytometry by using anti-mFcα/μR mAbs showed that the Fcα/μR is expressed on a subset of B cells and macrophages, but not on granulocytes, T cells, or NK cells in the mouse spleen [31]. Although Fcα/μR is closely related to poly-IgR as described above, the expression pattern of Fcα/μR is quite different from that of poly-IgR, which is expressed on epithelial, but not hematopoietic, cells. During developmental stages of B cells in the bone marrow, IgD+, IgM+ mature B lymphocytes expressed significant amounts of the mFcα/μR. In contrast, it was not expressed on the majority of IgD, IgM+ immature B lymphocytes [30], suggesting that the Fcα/μR might be involved in B cell development. However, recent evidences demonstrated that each lymphocyte population, including B cell subsets, was normal in mice deficient in Fcα/μR gene (Honda et al., submitted for publication), suggesting that Fcα/μR defect did not affect lymphocyte development. Unlike mFcα/μR, hFcα/μR is hardly detected on the cell surface of B cells and monocytes in peripheral blood. Immunohistochemical studies demonstrated that the Fcα/μR is expressed in germinal centers in lymphoid tissues, including spleen, lymph nodes, and Peyer’s patch (our unpublished observation; Fig. 4), strongly suggesting that Fcα/μR may play an important role in humoral immune responses.
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Fig. 4

Expression of Fcα/μ receptor in lymphoid tissues. Mesenteric lymph nodes obtained from mice that had been immunized i.p. with TNP-KLH were stained with phycoerythrin-conjugated anti-Fcα/μR (red) and FITC-conjugated anti-TCR or B220 mAbs (green) and analyzed by fluorescent microscopy. The Fcα/μR was strongly expressed at the center of the secondary lymphoid follicles

Endocytosis of IgM-immune complex by the mFcα/μR

The cytoplasmic domain of the mFcα/μR contains a dileucine motif at residues 519 and 520, which has been implicated in endosome and lysosome targeting of diverse proteins and is involved in agonist-induced internalization [1, 6, 13, 14]. A dileucine motif is involved in FcγRIIB-mediated endocytosis [15]. In fact, Fcα/μR mediated endocytosis of IgM-coated fluorescent beads. However, the fluorescent beads were not detected in any of the Ba/F3 transfectants expressing Fcα/μR mutated (L-A) at residue 519, 520, or both [31]. These results indicate that both leucines at residues 519 and 520 are required for endocytosis by the mFcα/μR.

To examine the biological significance of the mFcα/μR expressed on primary B lymphocytes, mouse spleen cells were incubated with immune complexes composed of FITC-labeled S. aureus bacteria coated with IgM or IgG anti-S. aureus mAbs. B220+ spleen cells cultured with the immune complex of IgM anti-S. aureus and FITC-labeled S. aureus bacteria contained one or more FITC-labeled S. aureus bacteria in the cytoplasm, as determined by immunofluorescent microscopy. By contrast, the IgG immune complex of S. aureus was not detected in B220+ spleen cells [31]. These results indicate that the mFcα/μR mediates endocytosis of IgM-coated microbial pathogens, suggesting that Fcα/μR may be involved in protection against microbial infection by stimulating phagocytosis, as well as in antigen processing and presentation to helper T cells, resulting in linkage from innate to adaptive immune responses.

Concluding remarks

Previous reports demonstrated that mice deficient in the secretory form of IgM (sIgM) exhibit delayed development of specific IgG antibodies to T cell-dependent foreign antigens [9] and dissemination of micropathogens in peripheral organs, but not in secondary lymphoid organs [23]. These reports suggested an idea that the Fcα/μR may play an essential role in the priming of helper T lymphocytes in the secondary lymphoid organs and in immune defense against microbes in peripheral organs. In addition, mice deficient in sIgM also showed the high titer of anti-dsDNA when these mice were challenged with repeated injection with bacterial lipopolysaccharide [10]. Furthermore, sIgM-deficient mice that had been mated with lupus-prone lpr mice developed an earlier onset of autoimmune diseases [3]. On the other hand, IgA deficiency in humans is relatively common and associated with autoimmunity [17, 26]. To explore the in vivo role of Fcα/μR in immune responses, we have recently established Fcα/μR-deficient mice. With these Fcα/μR-deficient mice, we are exploring the possibility that Fcα/μR might have an important role in humoral immune responses against self- and foreign antigens (Honda et al., submitted). We are just starting to explore the functional significance of the Fcα/μR in the immune system, and future studies using the Fcα/μR-deficient mice should contribute to unveil the mystery of how the polymeric immunoglobulins such as IgA and IgM mediate immunity against infections and/or self-antigens.

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© Springer-Verlag 2006