Mouse IgA Fc receptor on CD3+ T cells. Molecular forms of IgA that bind to a 38-kDa Fc alpha R protein and development of an anti-Fc alpha R antisera

Previous studies have shown that splenic T cells from mice that bear IgA myelomas, as well as certain T cell lines, express receptors for the Fc of IgA, and are termed Fc alpha R. In this study, we have isolated and characterized two CD3+ T cell lines derived by fusion of murine Peyer's patch (PP) CD4+ T cells with the BW 5147 lymphoma cell line. These cell lines, designated PPT4-6 and PPT4-16, were shown to bind monomeric or dimeric IgA, whereas the fusion partner did not bind either form of IgA. However, polymeric IgA (m.w. 600,000) bound equally well to all three cell lines. Similar results were also obtained with two known Fc alpha R+ T cell lines, ThHA1 nos. 9 and 10. Immunoprecipitation studies with IgA on PPT4-16 and ThHA1 no. 9 have shown that IgA binds to a 38-kDa protein. A rabbit antiserum was prepared to a 38-kDa fraction of Fc alpha R+ T cell membranes, and heterophilic antibody was removed from the antiserum by adsorption with mouse thymocytes, BW 5147 and R1.1 lymphoma. The antiserum bound to both PPT4-16 and ThHA1 no. 9 as well as to other Fc alpha R+ T cells, but did not bind to thymocytes or to the T lymphomas R1.1 or BW 5147. The antiserum appeared specific for the Fc alpha R, because it failed to block binding of anti-CD3 (145 2C11) or other surface molecule-specific antibodies. Further, competitive inhibition studies with IgA and anti-Fc alpha R (38 kDa) showed that preincubation of Fc alpha R+ T cells with the anti-38-kDa protein completely eliminated IgA binding, whereas IgA partially blocked the binding of the anti-Fc alpha R antibodies to the cell membrane. Immunoisolation with the anti-Fc alpha R antibody of radioiodinated cell membrane proteins from Fc alpha R+ T cells, but not from Fc alpha R- cells, gave a distinct band at 38 kDa. To further test the specificity of this antiserum, we have isolated T cells from spleens of IgA-myeloma bearing mice, and tested the phenotype and IgA binding. A subset consisting of 15 to 20% of CD3+, CD8+ T cells was found that bound monomeric or dimeric IgA. Further, the anti-Fc alpha R antiserum also recognized this CD8+ T cell subset, and preincubation of the cells with antibody resulted in their failure to bind IgA. Our results indicate that the Fc alpha R on T cell lines derived from PP is a 38-kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R)

Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding.     

Human immature dendritic cells efficiently bind and take up secretory IgA without the induction of maturation.

Immature dendritic cells (DC) reside in peripheral tissues, where they pick up and process incoming pathogens via scavenger receptors or FcR such as FcgammaR and FcepsilonR. At mucosal surfaces, IgA is the main Ig to protect the body from incoming pathogens. In addition, DC are present in high numbers at these sites. We detected expression of FcalphaR (CD89) on the CD14+ population of CD34+ progenitor-derived DC and on monocyte-derived DC (MoDC). However, CD89 expression was strongly decreased upon differentiation from monocyte to DC. We found only minimal binding of serum IgA to MoDC but strong binding of secretory IgA (SIgA). The SIgA binding to MoDC could not be blocked by anti-CD89 blocking Abs. DC efficiently internalized SIgA, but not serum IgA, and uptake of SIgA could be blocked by specific sugars or partially by Ab reactive with mannose receptor. Importantly, binding and uptake of SIgA was not accompanied by signs of DC maturation, such as increased expression of CD86 and CD83 or induction of cytokine secretion. These data indicate that SIgA can interact with DC not via CD89, but via carbohydrate-recognizing receptors like mannose receptor and suggest that uptake of SIgA-containing immune complexes by immature DC may be a mechanism to modulate mucosal immune responses.     

C/EBP alpha and Ets protein family members regulate the human myeloid IgA Fc receptor (Fc alpha R,CD89) promoter

Fc alpha R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc alpha R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Nonhuman primate IgA: genetic heterogeneity and interactions with CD89

Nonhuman primates are extremely valuable animal models for a variety of human diseases. However, it is now becoming evident that these models, although widely used, are still uncharacterized. The major role that nonhuman primate species play in AIDS research as well as in the testing of Ab-based therapeutics requires the full characterization of structure and function of their Ab molecules. IgA is the Ab class mostly involved in protection at mucosal surfaces. By binding to its specific Fc receptor CD89, IgA plays additional and poorly understood roles in immunity. Therefore, Ig heavy alpha (IGHA) constant (C) genes were cloned and sequenced in four different species (rhesus macaques, pig-tailed macaques, baboons, and sooty mangabeys). Sequence analysis confirmed the high degree of intraspecies polymorphism present in nonhuman primates. Individual animals were either homozygous or heterozygous for IGHA genes. Highly variable hinge regions were shared by animals of different geographic origins and were present in different combinations in heterozygous animals. Therefore, it appears that although highly heterogeneous, hinge sequences are present only in limited numbers in various nonhuman primate populations. A macaque recombinant IgA molecule was generated and used to assess its interaction with a recombinant macaque CD89. Macaque CD89 was able to bind its native ligand as well as human IgA1 and IgA2. Presence of Ag enhanced macaque IgA binding and blocking of macaque CD89 N-glycosylation reduced CD89 expression. Together, our results suggest that, despite the presence of IgA polymorphism, nonhuman primates appear suitable for studies that involve the IgA/CD89 system.     

Antisera to the secretory component recognize the murine Fc receptor for IgA

Studies were undertaken to determine a possible structural relationship between the secretory component (SC) and the receptor for IgA (Fc alpha R). An IgA-mediated rosetting technique was used to assess the presence of Fc alpha R+ cells in various lymphoid tissues from normal BALB/c mice and mice bearing an IgA plasmacytoma (MOPC 315). Tissues from the MOPC 315-bearing BALB/c mice were found to have a significantly higher percentage of Fc alpha R+ cells; thus, nonadherent spleen cells from MOPC 315-bearing mice were used as a source of Fc alpha R+ cells in these studies. The cells were preincubated with anti-SC and then assayed for the ability of IgA to bind to the Fc alpha R. Antisera to SC from various species inhibited the formation of IgA-mediated rosettes, although preincubation of the Fc alpha R+ cells with antisera directed against other cell surface molecules (e.g., Thy1.2, Lyt1, Lyt2, Fc gamma R, MHC class I and II) or preimmune sera had no significant effect on IgA-mediated rosette formation. Preabsorption of the anti-SC with secretory IgA or with free SC removed the inhibitory effect; preabsorption with myeloma IgA had no effect. These data suggest that SC and Fc alpha R are related serologically and may be structurally related, possible in the IgA-binding region.     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

Fc alpha RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.

The FcR for IgA CD89/FcalphaRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.     

The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Crosslinking of the human Fc receptor for IgA (FcalphaRI/CD89) triggers FcR gamma-chain-dependent shedding of soluble CD89

CD89/FcalphaRI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR gamma-chain subunit. Shedding of sCD89 was dependent on signaling via the gamma-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.     

Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor

Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants containing IBF alpha. These studies show that Th HA cells express Fc alpha R with less Fc mu R, and the solubilized form of Fc alpha R exhibits IBF alpha-like activity. The significance of FcR expression by Th cell clones and cell lines and the relationship of soluble Fc alpha R and IBF alpha for IgA response regulation are discussed.     

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.     

Molecular heterogeneity of Fc alpha receptors detected by receptor-specific monoclonal antibodies.

Fc alpha receptors (Fc alpha R) were isolated from a human monocytic cell line and used to raise four mAb with receptor specificity. The antibodies were used to identify the types of white blood cells that express Fc alpha R and the molecular heterogeneity of the receptor molecules. Nonpolymorphic epitopes, outside of the Fc alpha-binding site, were recognized only on blood cells of granulocyte and monocyte/macrophage lineages. The molecules identified, both by the antibodies and by the IgA ligand, were glycoproteins ranging in relative molecular mass from 55 to 75 kDa. However, one antibody detected a subpopulation of Fc alpha R molecules characterized by relatively restricted size heterogeneity. A complex glycosylation pattern was revealed by the resolution of discrete 32- and 36-kDa molecular species after removal of N-linked oligosaccharides and by evidence for O-linked carbohydrate moieties on at least a portion of the Fc alpha R molecules. In biosynthetic studies, all four anti-Fc alpha R antibodies and the IgA ligand bound a single 32-kDa core protein present in tunicamycin-treated cells, and the exceptional antibody again recognized molecules with relatively restricted glycosylation in the nontreated cells. These antibodies and native IgA ligands thus provide complementary reagents for definition of the complex structure and function of Fc alpha R in systemic IgA antibody responses.     

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.     

Disulfide bonding of secretory component to a single monomer subunit in human secretory IgA.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.     

Pentraxins and IgA share a binding hot‐spot on FcαRI

The pentraxins, C‐reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C‐and F‐strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA‐binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA‐binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA‐binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C′‐strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC‐ and FG‐loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.     

HIV-1 replication in Langerhans and interstitial dendritic cells is inhibited by neutralizing and Fc-mediated inhibitory antibodies

Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34(+) cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1.