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The Journal of Immunology, 2007, 178: 3973-3982.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fc{alpha}RI (CD89) Alleles Determine the Proinflammatory Potential of Serum IgA1

Jianming Wu*, Chuanyi Ji*, Fenglong Xie*, Carl D. Langefeld{dagger}, Kun Qian*, Andrew W. Gibson*, Jeffrey C. Edberg* and Robert P. Kimberly2,*

* Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and {dagger} Department of Public Health Sciences, Wake Forest University, Winston-Salem, NC 27157


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human IgA FcR (Fc{alpha}RI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A->G) (rs16986050), which changes codon 248 from AGC (Ser248) to GGC (Gly248) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different Fc{alpha}RI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-{alpha}) in murine macrophage P388D1 cells. In the absence of FcR {gamma}-chain association in P388D1 cells, the Ser248-Fc{alpha}RI allele does not mediate cytokine production, but the Gly248-Fc{alpha}RI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with Fc{alpha}RI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly248-Fc{alpha}RI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that Fc{alpha}RI (CD89) {alpha}-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunoglobulin A has a central role in host defense at mucosal surfaces, but its role in systemic immunity is less clear. In the intestinal lamina propria the B lineage cells produce polymeric IgA, which exists almost exclusively as a dimer joined by a J chain polypeptide and linked to the secretory component to form a complex called secretory IgA. Secretory IgA can bind Fc{alpha}RI (CD89) but cannot trigger phagocytosis (1, 2). Nevertheless, through Fc{alpha}RI secretory IgA is able to initiate certain inflammatory responses such as a respiratory burst in polymorphonuclear leukocytes and degranulation of eosinophils (2, 3). In human serum, IgA, the second most abundant Ig after IgG, makes up about one-fifth of the total Ig, is primarily of the IgA1 subclass, and exists mainly in monomeric form (>95%) (2). The two human IgA subclasses, IgA1 and IgA2, differ by an additional 13-aa sequence with O-linked glycosylation sites in the hinge region of IgA1 (4). The lack of 13 aa in the hinge confers on IgA2 its resistance to digestion by the bacterial proteases produced by microorganisms such as Streptococcus mutans, Neisseria meningitidis, and Haemophilus influenzae and may be the rationale for the predominance of IgA2 in mucosal secretions (5).

IgA-mediated immune effector responses such as phagocytosis, Ab-dependent cell-mediated cytotoxicity, respiratory burst, and cytokine release are primarily mediated through Fc{alpha}RI (CD89), an IgA-specific receptor. Fc{alpha}RI is a type I transmembrane (TM)3 receptor expressed on cells of the myeloid lineage including neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells (6). Both monomeric and dimeric IgA can bind to Fc{alpha}RI, and IgA immune complexes can activate phagocytosis and other immune responses through the clustering of Fc{alpha}RI (7).

The gene encoding human Fc{alpha}RI is located in the leukocyte receptor cluster on human chromosome 19q13.4 along with NK cell inhibitory receptors and leukocyte Ig-like receptors (8, 9). The Fc{alpha}RI gene consists of five exons and spans ~12 kilobases. The signal peptide is encoded by first two exons, whereas the extracellular (EC) domains (EC1 and EC2) are encoded by exons 3 and 4, respectively. The last exon (exon 5) encodes the TM region and the short cytoplasmic (CYT) tail (10). Several alternatively spliced variants of cDNAs have been described, and a shorter version lacking 66 bp encoding EC2 is expressed on alveolar macrophages in vivo (11, 12, 13, 14, 15, 16). A significant decrease in the ratio of the EC2 deletion splice variant to the full-length Fc{alpha}RI has been found in neutrophils from pneumonia patients compared with those of healthy individuals, suggesting that the alternatively spliced Fc{alpha}RI isoform might have physiologic relevance in IgA-mediated host defense (16, 17).

Fc{alpha}RI shares sequence similarity with the leukocyte Ig-like receptors, killer inhibitory receptors, and other members of the leukocyte receptor cluster (8, 9). Unlike the classical low affinity Fc{gamma}RIII and high affinity Fc{epsilon}RI, which bind their ligands via the membrane proximal EC Ig-like domain (EC2) and form 1:1 receptor:Fc complexes through interactions with hinge-proximal Fc regions (18, 19), Fc{alpha}RI has its ligand binding site on the membrane distal EC1 domain and forms a 2:1 complex with an Fc{alpha} dimer by binding to each CH2–CH3 domain interface (20, 21, 22, 23, 24, 25). Although Fc{alpha}RI is only distantly related to Fc{gamma}R and Fc{epsilon}RI proteins, it does share some structural similarities and can associate with the FcR {gamma}-chain through its TM domain. Unlike Fc{gamma}RIII and Fc{epsilon}RI, however, Fc{alpha}RI is often expressed in the absence of {gamma}-chain pairing (26, 27) and the function of this nonpaired Fc{alpha}RI has not been defined. Indeed, the net function of Fc{alpha}RI per se is unresolved, with some investigators reporting a capacity to initiate a proinflammatory program (28, 29, 30) while others report a noninflammatory profile (31, 32, 33, 34, 35, 36).

Functionally important single nucleotide polymorphisms (SNPs) in the coding regions of classical Fc{gamma}Rs have provided significant insight into their biological functions (37, 38). As part of an effort to understand the role of naturally occurring polymorphisms in Ig receptors, we sequenced the entire coding region of Fc{alpha}RI from a large number of donors and confirmed a common nonsynonymous SNP (SNP 844A->G) within the Fc{alpha}RI CYT domain (39). Using this natural sequence variation as a biological probe, we have demonstrated that the two different alleles of the CYT domain of CD89 {alpha}-chain alter receptor signaling and its biological functions. In addition, we found that, in contrast to the Ser248-Fc{alpha}RI allele, the Gly248 {alpha}-chain allele of Fc{alpha}RI is able to mediate certain biological functions in the absence of the FcR {gamma}-chain. Thus, this functionally important, nonsynonymous SNP in the CYT domain not only plays an important role in the regulation of Fc{alpha}RI function but may also explain some of the divergent reports on the proinflammatory vs anti-inflammatory potential of Fc{alpha}RI (6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors

Anti-coagulated peripheral blood was obtained from healthy normal volunteers and from SLE patients fulfilling the revised American College of Rheumatology criteria for SLE (40). SLE patients and controls were recruited as part of the University of Alabama at Birmingham DISCOVERY cohort. The human studies were reviewed and approved by the Institutional Review Board of the University of Alabama at Birmingham, and all donors provided written informed consent.

Reagents

All mAbs used in this study were murine in origin. mAbs A59 and A77 are murine IgG1 (mIgG1) specific for Fc{alpha}RI (CD89; gift from Dr. H. Kubagawa, University of Alabama at Birmingham, Birmingham, AL), and the 32.2 (mIgG1) mAb is specific for the Fc{gamma}RI (CD64; American Type Culture Collection). The mAbs specific for CD89 and CD64 were produced and purified from the hybridoma cultures at the University of Alabama at Birmingham Epitope Recognition and Immunoreagent Core Facility. The F(ab')2 of the mAbs A59 and 32.2 were generated at Rockland Immunochemicals, and the purity of these Ab F(ab')2 preparations was monitored by SDS gel staining and Western blotting assays. Anti-human Fc{alpha}RI mAb MIP8a (mIgG1) was purchased from Serotec. Anti-Fc{gamma}RI-FITC (mAb 197 (mIgG2a) and mAb 22.2 (mIgG1)) were from Medarex. F(ab')2 of mIgG and FITC-conjugated and unconjugated goat anti-mouse IgG (H+L) were obtained from Jackson ImmunoResearch. Rabbit anti-Lyn polyclonal Ab was purchased from Santa Cruz Biotechnology. Rabbit anti {gamma}-chain polyclonal Ab was obtained from Upstate Biotech. An AminoLink kit (cyanogen bromide-activated Sepharose 4B coupling gel and reagents) was purchased from Pierce. Protein G-agarose and a protease inhibitor mixture were from Roche Diagnostics. Cytokine detection ELISA kits and substrates were from BD Pharmingen. Human IgA was obtained from Sigma-Aldrich (catalog no. I 1010).

Nucleic acid isolation

Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems). Briefly, 300 µl of blood was lysed in 900 µl of the red blood lysis solution. The PBLs were pelleted and lysed with 300 µl of cell lysis solution. The RNase A solution was added to the cell lysate and incubated at 37°C for 15 min. Proteins were precipitated by adding 100 µl of Protein Precipitation solution (Gentra Systems). DNA in the supernatant was precipitated with 300 µl of 100% isopropanol and washed with 70% ethanol once. Total RNA was isolated from 107 PBL by using TRIzol total RNA isolation reagent (Invitrogen Life Technologies). Five micrograms of total RNA was used to synthesize cDNA with the SuperScript preamplification system (Invitrogen Life Technologies).

RT-PCR and cDNA sequencing

To facilitate heterozygote detection, a dye primer strategy was adopted for fluorescence-based automated cycle sequencing of PCR products on an ABI 377 sequencer (ABI PRISM dye primer cycle sequencing 21M13 FS and M13REV FS ready reaction kits; Applied Biosystems). Two sets of overlapping primers with either M13 universal or reverse primer sequence tags (underlined letters in the sequences below) at the appropriate 5' ends were used to amplify the whole coding region starting from nt 40 and ending at nt 903 of the Fc{alpha}RI cDNA (GenBank accession no. X54150). The first set of primers amplifies from nt 34 to nt 563, yielding a PCR product 585 bp in length including the underlined M13 sequences: 5'-TGTAAAACGACGGCAGTAGCACGATGGACCCCAAACAG-3' (annealing between nt 34 and nt 54) and 5'-CAGGAAACAGCTATGACCGGTGTTCCCCACTTTGGTGC-3' (annealing between nt 542 and nt 563). The second set of primers amplifies from nt 458 to nt 934, yielding a the PCR product 513 bp in length including the underlined M13 sequences: 5'-TGTAAAACGACGGCCAGTAGAATATTTCCCTCACGTGC-3' (annealing between nt 458 and nt 497) and 5'-CAGGAAACAGCTATGACCCTGGCTCCTCTCTGCCTTCAC-3' (annealing between nt 914 and nt 934). The PCR was performed in a 9600 PCR system with 2 µl of cDNA synthesized with the SuperScript preamplification system, 200 nM each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 2.5 U of TaqDNA polymerase in a 50-µl reaction volume starting at 95°C for 5 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min with a final extension at 72°C for 7 min. The PCR product was purified with the QIAquick gel extraction kit (Qiagen). All PCR products were sequenced in both directions.

Genotyping Fc{alpha}RI CYT domain SNPs by direct sequencing

To accelerate the genotyping of Fc{alpha}RI SNPs, a PCR of genomic DNA was designed for the fifth exon encoding for the TM segment and the CYT tail of Fc{alpha}RI (Fig. 1A). The forward primer 5'-CCATCCACCAAGATTACACGA-3' anneals between nt 692 and nt 712 and the reverse primer 5'-CTGGCTCCTCTCTGCCTTCAC-3' anneals between nt 914 and nt 934 (GenBank accession no. X54150). The PCR was performed with 200 ng of DNA, 200 nM each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 2.5 U of TaqDNA polymerase in a 50 µl reaction volume starting at 95°C for 5 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 1 min with a final extension at 72°C for 7 min. All the PCR products were purified from a 2.5% agarose gel with the QIAquick gel extraction kit. The purified genomic PCR products from different donors were sequenced from both directions using the BigDye terminator sequencing kits on an ABI 377 (ABI PRISM BigDye terminator cycle sequencing ready reaction kits; Applied Biosystems).


Figure 1
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  		FIGURE 1. Fc{alpha}RI coding region polymorphic nucleotide positions and their respective amino acid codon changes. A, The Fc{alpha}RI coding region was amplified and sequenced from nucleotide position 34 to position 934 with two sets of overlapping primers indicated with arrowheads. The respective SNP sites, nucleotide substitutions, and amino acid codon changes are shown. UTR, untranslated region. B, Chromatograms of SNP 844 in the cytoplasmic (CY) domain of Fc{alpha}RI showing donors homozygous for SNP 844G (lower row), homozygous for 844A (upper row), and heterozygous for 844G->A (middle row).

 
Generation of the Fc{alpha}RI expression constructs

The human Fc{alpha}RI (CD89) expression constructs were generated by cloning KpnI/EcoRI-flanked RT-PCR products with 864 bp of the Fc{alpha}RI coding region into the eukaryotic expression vector pcDNA3.1 (Invitrogen Life Technologies). The KpnI/EcoRI-flanked RT-PCR products from a heterozygous SNP 844G->A donor were generated from human mixed mononuclear cell cDNA synthesized using the SuperScript preamplification system (Invitrogen Life Technologies), with the upper primer 5'-TAATAGGGTACCATGGACCCCAAACAGACCACCCTC-3' (underlined and bold nucleotides are the KpnI cutting site) annealing between nucleotide positions 40 and 62 and lower primer 5'-TTATTAGAATTCTTACTTGCAGACACTTGGTGTTCGT-3' (underlined and bold nucleotides are EcoRI cutting site) annealing between nucleotide positions 879 and 904. RT-PCR was performed with KOD HiFi DNA polymerase (EMD Biosciences; distributed by Novagen). A point mutation at nucleotide position 728, which changes amino acid Arg209 to Leu209 in a TM segment, was made with the QuikChange site-directed mutagenesis kit (Stratagene) following the vendor’s instruction. For Arg209 to Leu209 constructs (nt 728 changes), the sense primer 5'-AGAACTTGATCCTCATGGCCGTGGC-3' and antisense primer 5'-GCCACGGCCATGAGGATCAAGTTCT-3' were used on the template plasmids from Fc{alpha}RI expression constructs with either the 844A (Ser248-Fc{alpha}RI) allele or the 844G (Gly248-Fc{alpha}RI) allele (underlined, bold, and italicized letters are intentional mutations). The sequences of all the cloned constructs were confirmed by direct sequencing from both directions on an ABI 377 sequencer with the ABI BigDye terminator cycle sequencing kit (Applied Biosystems).

Generation of the CD64EC-TM/CD89CYT chimeric receptor expression constructs

The expression constructs for a chimeric receptor comprised of human Fc{gamma}RI (CD64) EC domains and a TM segment (CD64EC-TM) fused with Fc{alpha}RI (CD89) CYT domain (CD89CYT) were generated by overlapping PCR to create KpnI/BamHI-flanked RT-PCR products of chimeric cDNA. The resultant CD64EC-TM/CD89CYT coding region was inserted into pcDNA3.1 (Invitrogen Life Technologies). The CD64EC-TM cDNA fragment was amplified from human cDNA with the upper primer 5'-TAA CGGGGTACCGGAGACAACATGTGGTTCTTGACAACT-3' (underlined and bold nucleotides are the KpnI cutting site) annealing between nucleotide positions 48 and 54 and the lower primer 5'-ATGGCTGTGCCAATTTTCTATTGTCACCCAGAGAAC-3' annealing between nucleotide positions 958 and 975 (CD64; GenBank accession no. X14356). The CD89CYT cDNA fragment was amplified with the upper primer 5'-GTTCTCTGGGTGACAATAGAAAATTGGCACAGCCAT-3' annealing between nucleotide positions 778 and 795 and the lower primer 5'-TTACTAGGATCCTTACTTGCAGACACTTGGTGTTCGT-3' (underlined and bold nucleotides are the BamHI cutting site) annealing between nucleotide positions 879 and 904 (Fc{alpha}RI GenBank accession no. X54150). The PCR fragments of CD64EC-TM and CD89CYT were purified with QIAquick gel extraction kit and mixed together with the upper primer 5'-TAACGGGGTACCGGAGACAACATGTGGTTCTTGACAACT-3' and the lower primer 5'-TTACTAGGATCCTTACTTGCAGACACTTGGTGTTCGT-3' (underlined and bold nucleotides are the KpnI and BamHI cutting sites) to generate RT-PCR products for the chimeric receptor. RT-PCR was performed with KOD HiFi DNA polymerase (EMD Biosciences; distributed by Novagen). The sequences of all cloned constructs were confirmed by direct sequencing from both directions on an ABI 377 sequencer with ABI BigDye terminator cycle sequencing kit (Applied Biosystems).

Generation of expression constructs for the GST-CD89CYT fusion protein

The GST/Fc{alpha}RI (CD89) CYT domain fusion protein (GST-CD89CYT) expression constructs were generated through cloning BamHI/EcoRI-flanked RT-PCR products with 126 bp of the CD89 CYT domain coding region (41 aa residues) into pGEX2T vector (Amersham Biosciences). The BamHI/EcoRI-flanked RT-PCR products were amplified with PBL cDNA from a heterozygous SNP 844 donor using the upper primer 5'-CCGCGTGGATCCATGGACCCCAAACAGACCACCCTC-3' (underlined and bold nucleotides are the BamHI cutting site) annealing between nucleotide positions 778 and 797 and the lower primer 5'-TTATTAGAATTCTTACTTGCAGACACTTGGTGTTCGT-3' (underlined and bold nucleotides are the EcoRI cutting site) annealing between nucleotide positions 879 and 9044. RT-PCR was performed with TaqDNA polymerase (Invitrogen Life Technologies). The GST fusion constructs were transformed into DH5{alpha} Escherichia coli and the fusion proteins were produced and purified with glutathione-Sepharose beads (Amersham Biosciences) according to the vendor’s instructions. The sequences of all cloned constructs were confirmed by sequencing from both directions on an ABI 377 sequencer with an ABI BigDye terminator cycle sequencing kit (Applied Biosystems).

Generation of stable cell lines expressing Fc{alpha}RI

The murine macrophage cell line P388D1 and the rat mast cell line rat basophilic leukemia (RBL)-2H3 were obtained from American Type Culture Collection and maintained in DMEM supplemented with 10% FCS (FBS) and L-glutamine (2 mM) in 5% CO2. Individual transfections were conducted on a 60-mm cell culture dish with the cell density at 80–100% confluence. Fc{alpha}RI expression construct plasmid DNA (8 µg), purified with the Wizard PureFection plasmid DNA purification system (Promega) and the Lipofectamine 2000 reagent (12 µl), were used for transfection according to the vendor’s instructions (Invitrogen Life Technologies). Transfected cells were cultured in DMEM supplemented with 10% FCS and G418 (1 mg/ml) for the selection of stable transfectants. The G418-resistant cells were sorted on a FACSVantage system (BD Biosciences) for the equal expression of all constructs in both the P388D1 and RBL-2H3 cells. At least two independent cell lines derived from independent transfections were prepared to assess receptor function for each of the individual constructs.

Measurement of change in intracellular Ca2+ concentration ([Ca2+]i)

Changes in [Ca2+]i, induced by the cross-linking of Fc{alpha}RI with anti-Fc{alpha}RI mAb (clone A59) F(ab')2 were determined with Indo-1-acetoxymethylester (AM)-loaded (Molecular Probes), stably transfected cells expressing Fc{alpha}RI and an SLM 8000 spectrofluorometer. Briefly, cells were washed once on the plate, lifted gently from plate, and washed again with 10 ml of Ca2+-free and Mg2+-free Hank’s buffer. Washed cells were re-suspended in Iscove’s medium plus 5% FBS at a concentration of 107 cells/ml and incubated with 5 µM Indo-1-AM at 37°C for 40 min. Cell preparations to be opsonized with mAb A59 F(ab')2 were washed, resuspended in Ca2+- and Mg2+-supplemented Hank’s buffer at concentration of 107 cells/ml, and incubated with saturating concentrations of A59 F(ab')2 (5 µg/ml) at 4°C for 30 min. All cells were washed and re-suspended at 5 x 106 cells/ml in 1 mM Ca2+, 1 mM Mg2+, 1 mg/ml BSA, 10 mM HEPES Hank’s buffer, and then 0.4 ml of cells (2 x 106 cells) were immediately transferred to a continuously stirring cell cuvette maintained at 37°C in the SLM 8000. With excitation at 355 nm, the simultaneous fluorescence emission at 405 and 490 nm was measured, integrated, and recorded each second. After establishing a baseline for 60 s, goat F(ab')2 anti-mouse IgG was added at final concentrations of 20 µg/ml, and data acquisition was continued for an additional 3.5 min. Each sample was individually calibrated by lysing cells in 1% Triton X-100 to determine the maximal emission ratio and by adding EDTA (20 mM final concentration) to determine the minimal ratio. The Indo-1 fluorescence emission ratio was converted to [Ca2+]i as described elsewhere (37).

Degranulation assays

RBL-2H3 cells (105 cells/well) expressing either CD89 (Fc{alpha}RI) or CD64EC-TM/CD89CYT chimeric receptors were cultured overnight in 24-well culture plates (Corning). The culture medium were removed from plates and the cells were incubated with DMEM containing anti-human Fc{alpha}RI mAb F(ab')2 (clone A59; final concentration of 5 µg/ml) or anti-human CD64 mAb F(ab-)2 (clone 32.2; final concentration of 2.5 µg/ml) for 45 min at 4°C. The cells were washed with Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES, and 0.1% BSA (pH 7.4)) and then stimulated with goat anti-mouse F(ab')2 (Jackson ImmunoResearch Laboratories) at a final concentration of 20 µg/ml in the Tyrode’s buffer. The supernatants were collected at 0, 15, 30, 45, and 60 min for the measurement of beta-hexosaminidase activity. Cells from control wells were lysed with the same volume of 0.1% Triton X-100 in Tyrode’s buffer for evaluation of the total beta-hexosaminidase activity for each RBL-2H3 stable cell line. Supernatants and cell lysates were incubated with substrate (1.3 mg/ml p-nitrophenyl-N-acetyl beta-D-glucosamine) (Sigma-Aldrich) in 0.1 M sodium citrate (pH 4.5) for 1 h at 37°C. The reaction was stopped by 0.2 M sodium carbonate buffer (pH 10.0) and the enzyme reactivity was evaluated by measuring OD at 405 nm. The percentage of specific beta-hexosaminidase activity released was calculated as follows: percentage release = 100 x average supernatant activity from 4 wells/average cell lysate activity from four control wells.

Cytokine analysis

Cells were stimulated in 24-well tissue culture plates (Corning) with surface-bound mAb A59 F(ab')2. Wells were coated with either anti-Fc{alpha}RI mAb A59 F(ab')2 (20 µg/ml) or control mIgG F(ab')2 (20 µg/ml) overnight at room temperature. The Fc{alpha}RI-expressing cells were added to coated wells and the culture media were collected after 24 h. The levels of murine IL-6 or TNF-{alpha} in culture medium were quantified by ELISA (BD Pharmingen).

Immunoprecipitation of Fc{alpha}RI and detection of {gamma}-chain

Fc{alpha}RI was immunoprecipitated from the transfected cell lines using either mAb MIP8a (Serotec) or mAb A77 prebound to protein G-agarose. The {gamma}-Chain was detected with polyclonal rabbit anti-{gamma}-chain Abs (Upstate Biotech). Cells (2 x 107 cells/ml) were lysed in PBS containing 1% Nonidet P-40 with a protease inhibitor mixture and used for immunoprecipitation. Immunoprecipitates were analyzed with SDS-PAGE and Western blotting.

Column immunoprecipitation of Fc{alpha}RI and detection of Lyn

Affinity columns were generated through the coupling of the A77 mAb to the cyanogen bromide-activated Sepharose 4B beads. P388D1 cells (108) expressing Fc{alpha}RI were lysed in 5 ml PBS (pH 7.2) containing 1% digitonin plus a protease inhibitor mixture (lysis buffer). The cell lysates were centrifuged at 12000 x g and the supernatants were cleared by passing through an irrelevant mIgG1 isotype control column. The cleared cell lysates were then loaded onto the anti-Fc{alpha}RI (A77) affinity column containing 1 ml of beads and washed with 20 volumes of lysis buffer. Fc{alpha}RI and associated molecules captured on column were eluted with 0.1 M glycine buffer (pH 2.8) containing 0.1% digitonin. The eluted fractions were neutralized with 1 M Tris · HCl buffer (pH 8.0) immediately and analyzed with SDS-PAGE and Western blotting with a rabbit anti-Lyn polyclonal Ab (Santa Cruz Biotechnology) and a mouse anti-CD89 mAb.

Immunoprecipitation of Lyn and detection of Fc{alpha}RI

A rabbit anti-Lyn Ab was used to immunoprecipitate Lyn from P388D1 cells expressing different forms of Fc{alpha}RI. Coimmunoprecipitated Fc{alpha}RI was detected by Western blotting with mAb MIP8a (Serotec). Cells (2 x 107 cells/ml) were lysed in PBS containing 1% Nonidet P-40 with protease inhibitor mixture and used for immunoprecipitation and Western blotting.

Human neutrophil cytokine production assays

Fresh anti-coagulated blood was diluted 1/1 in HBSS and centrifuged through a two-step Ficoll-Hypaque gradient in 50-ml conical tubes (37). Neutrophils were harvested from the lower Ficoll-Hypaque interface and washed three times with HBSS containing 1% RPMI 1640 medium. The residual RBCs were lysed with Puregene red blood lysis solution (Gentra Systems). The resulting neutrophils were washed twice with HBSS. The cells were then counted and resuspended in RPMI 1640 medium supplemented with 10% FBS and 10 mM HEPES (pH 7.2). The neutrophils were stimulated in 12-well tissue culture plates (Corning) previously coated overnight with anti-Fc{alpha}RI mAb A59 F(ab')2 (20 µg/ml), mIgG1 F(ab')2 (20 µg/ml), or human IgA (20 µg/ml). The wells were washed twice with HBSS before use and 2 x 106 neutrophils in 1 ml of culture medium were used for each well. The cells were incubated at 37°C in 5% CO2 for 24 h and the culture supernatants were collected for cytokine determination. The levels of human IL-6 in culture medium were quantified by ELISA (BD Pharmingen).

Flow cytometry

Aliquots of cells of 5 x 105 cells in 0.1 ml PBS were incubated with saturating concentrations of primary mAb for 30 min at 4°C followed by two washes. For indirect immunofluorescence, the cells were then incubated with saturating concentrations of FITC-conjugated goat anti-mouse IgG F(ab')2 at 4°C for another 30 min. After washing, the cells were analyzed immediately for immunofluorescence intensity using a FACScan (BD Biosciences).

Data analysis

Student’s t test, unpaired or paired according to experimental design, was used to analyze the data for degranulation, calcium influx, and cytokine production. The paired t test was used to compare means between conditions in matched pair design. The unpaired t test was used to compare means between samples of donors homozygous for the different alleles. The Mantel-Haenszel {chi}2 test was used to test for differences in the genotype and allele frequencies in affected donors and controls both in African Americans and in the combined genetic data from two ethnic groups (Caucasian and African American). The association between genotype and the human lupus phenotype in Caucasians was tested with Fisher’s exact test because of the low frequency number in one cell.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
SNP detection in Fc{alpha}RI coding region

Two sets of overlapping RT-PCRs were used to amplify the full length of the Fc{alpha}RI coding region and the purified PCR products were directly sequenced. As shown in Fig. 1A, we identified five SNPs located at nucleotide positions 363 (rs1865096), 376 (rs11666735), 679, 836, and 844 in the coding region of Fc{alpha}RI. The SNPs 363G->A and 679T->C are silent polymorphisms that change an arginine codon from AGA to AGG and a leucine codon from TTG to CTG, respectively (Table I). Nonsynonymous SNPs at 836 and at 376, which change a proline codon (CCG) to a leucine (CTG) and an aspartate (GAC) to asparagine (AAC), respectively, both have minor allele frequencies <0.10. More importantly for the purposes of this study, we confirmed that the SNP 844A->G transition that changes amino acid codon 248 from serine (AGC) to glycine (GGC) is a more common polymorphism located in the CYT domain of CD89 (Fig. 1) (39).


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  		Table I. The distribution of Fc{alpha}RI coding region SNPs in an ethnically diverse groupa

 
The Gly248 and Ser248 alleles of Fc{alpha}RI differ in their capacity for receptor-mediated calcium mobilization

Fc{alpha}RI can associate with the FcR {gamma}-chain that contains an ITAM (41). Upon Ab-mediated cross-linking of Fc{alpha}RI or binding of IgA immune complexes to Fc{alpha}RI, the tyrosines of the {gamma}-chain ITAM are phosphorylated by Src family kinases and the tyrosine-based signaling cascade is initiated (42). Because the CYT domain of Fc{gamma}RIII (CD16) influences receptor-mediated intracellular calcium mobilization (43), we hypothesized that the two Fc{alpha}RI CYT domain alleles might also affect the receptor signaling. Using stably transfected RBL cells sorted for the equivalent expression of each of the two Fc{alpha}RI alleles on the cell surface to ensure equivalent binding of the stimulant (Fig. 2A), we tested comparative receptor-initiated calcium signaling. RBL cells expressing Fc{alpha}RI were opsonized with anti-Fc{alpha}RI mAb A59 F(ab')2. When the goat anti-mouse IgG (H+L) F(ab')2 was added to cross-link the Fc{alpha}RI, cells expressing the Gly248-Fc{alpha}RI allele were more efficient in mediating intracellular calcium release than Ser248-Fc{alpha}RI allele (Fig. 2B).


Figure 2
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  		FIGURE 2. The Ser248/Gly248 polymorphism alters calcium mobilization and degranulation in RBL cells. A, There was no significant difference in expression of the Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI alleles in stably transfected RBL-2H3 cells (mean fluorescent intensity, 86.5 + 21.6 for the Ser248-Fc{alpha}RI allele and 91.2 + 11 for the Gly248-Fc{alpha}RI allele, p = not significant; n = 4). No detectable degranulation and intracellular calcium influx were induced by F(ab')2 of mIgG, A59, and 32.2 plus goat anti-mouse F(ab')2 in the parental RBL-2H3 cells. B, Anti-CD89 mAb A59 F(ab')2 induces a rise in intracellular Ca2+ levels in RBL cells expressing the Gly248-Fc{alpha}RI and Ser248-Fc{alpha}RI alleles. RBL cells were loaded with indo-1-AM, opsonized with mAb A59 F(ab')2 (5 µg/ml), and stimulated at 60 s with F(ab')2 goat anti-mouse (20 µg/ml). In all experiments, the rise in [Ca2+]i induced by Gly248-Fc{alpha}RI was greater than that by Ser248-Fc{alpha}RI (mean peak of [Ca2+]i (nM) above baseline: 280.4 ± 51.68 and 114.0 ± 20.8 for the Gly248-Fc{alpha}RI and Ser248-Fc{alpha}RI alleles, respectively; p < 0.01 from t test; n = 5). No calcium influx was seen when RBL cells expressing either the Gly248-Fc{alpha}RI and Ser248-Fc{alpha}RI alleles were stimulated with mIgG F(ab')2 plus goat anti-mouse F(ab')2. C, Anti-CD89 mAb A59 F(ab')2 induced degranulation in RBL cells expressing Gly248-Fc{alpha}RI and Ser248-Fc{alpha}RI alleles. RBL cells were prelabeled with mAb A59 F(ab')2 (5 µg/ml) in the tissue culture wells and stimulated with F(ab')2 goat anti-mouse (20 µg/ml). In all experiments the degranulation measured as the release of beta-hexosaminidase activity was significantly higher in the RBL cells with Gly248-Fc{alpha}RI than with Ser248-Fc{alpha}RI at all time points (*, p < 0.01 from t test; n = 4). No degranulation was induced by the irrelevant mIgG F(ab')2 plus goat anti-mouse F(ab')2 in the stable RBL cells expressing either Gly248-Fc{alpha}RI and Ser248-Fc{alpha}RI alleles. D, Anti-CD64 mAb 32.2 F(ab')2 induced degranulation in RBL cells expressing chimeric receptors of CD64EC-TM/CD89CYT with the Gly248 allele or Ser248 alleles. RBL cells were opsonized with mAb 32.2 F(ab')2 (2.5 µg/ml) in the tissue culture wells and stimulated with F(ab')2 goat anti-mouse (2.5 µg/ml). In all experiments degranulation measured by the release of beta-hexosaminidase activity through CD64 cross-linking was significantly higher in the RBL cells with the Gly248 allele than the Ser248 allele at all time points (*, p < 0.01 from t test; n = 4). No degranulation was induced by the irrelevant mIgG F(ab')2 plus goat anti-mouse F(ab')2 in the stable RBL cells expressing either Gly248 allele and Ser248 allele chimeric receptors.

 
The Gly248 and Ser248 alleles of Fc{alpha}RI differ in their capacity for receptor-mediated degranulation

Fc{alpha}RI is expressed on myeloid cells including neutrophils, eosinophils, monocytes, dendritic cells, and tissue macrophages. Fc{alpha}RI can initiate potent immune responses such as degranulation, endocytosis, phagocytosis, Ab-dependent cellular cytotoxicity, Ag presentation, and the release of inflammatory mediators. Because of the importance of receptor-mediated degranulation, we investigated whether the two alleles of Fc{alpha}RI mediate comparable degranulation in RBL cells. Despite comparable expression of Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI, cross-linking of the Fc{alpha}RI on the RBL cell induced significantly more degranulation with the Gly248-Fc{alpha}RI allele than with the Ser248 allele (Fig. 2C). To confirm that this effect was dependent on the alleles of the Fc{alpha}RI {alpha}-chain CYT domain, we created Fc{gamma}RI/Fc{alpha}RI chimeric receptors consisting of Fc{gamma}RIa (CD64) EC and TM domains fused with 41 residues of the Fc{alpha}RI CYT domain (CD64EC-TM/CD89CYT). Stably transfected RBL cells expressed comparable levels of CD64EC-TM/CD89CYT chimeric receptors on the cell surface (data not shown). Remarkably, similar to native Fc{alpha}RI receptors, cross-linking of the chimeric receptors induced significantly more degranulation in RBL cells expressing the CD64EC-TM/CD89CYT receptor carrying a Gly248-Fc{alpha}RI allele than in cells expressing CD64EC-TM/CD89CYT carrying a Ser248-Fc{alpha}RI allele (Fig. 2D). These data demonstrate that the Fc{alpha}RI (CD89) {alpha}-chain CYT domain influences the receptor signaling capacity. Furthermore, the two naturally occurring alleles of the Fc{alpha}RI {alpha}-chain CYT domain significantly alter the receptor’s degranulation function.

The Gly248 and Ser248 alleles of Fc{alpha}RI alter receptor-mediated cytokine release

Receptor-mediated cytokine release plays an important role in modulation of the immune system. To compare the effects of the Fc{alpha}RI alleles on cytokine release, we generated stable P388D1 cell lines with comparable expression of the different Fc{alpha}RI alleles (Ser248-Fc{alpha}RI or Gly248-Fc{alpha}RI) with or without mutations within the TM domain to alter {gamma}-chain association (Arg209 or Leu209; Fig. 3A). In contrast to transfectants with the wild-type Arg209 TM sequence of Fc{alpha}RI, the TM mutants carrying Leu209 lost their association with the endogenous FcR {gamma}-chain (Fig. 3B). Cross-linking of the Fc{alpha}RI on cells expressing the Gly248-Fc{alpha}RI allele induced significantly more IL-6 and TNF-{alpha} release than that induced with cells expressing the Ser248-Fc{alpha}RI allele (Fig. 3C, upper panel for IL-6 and lower panel for TNF-{alpha}). Because Fc{alpha}RI not paired with an FcR {gamma}-chain represents the major fraction of Fc{alpha}RI molecules expressed on the cell surfaces of human monocytes and neutrophils (26), we also determined whether the "{gamma}-chainless" Fc{alpha}RI might be able to mediate cytokine production and whether there might be a difference in cytokine release between the Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI alleles in the absence of the FcR {gamma}-chain. As shown in Fig. 3, A and B, cells expressing the Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI alleles with the TM Leu209 mutation showed comparable receptor expression. In the absence of FcR {gamma}-chain pairing, stimulation of the Ser248-Fc{alpha}RI allele receptor was unable to induce IL-6 and TNF-{alpha} release (two left bars in Fig. 3D, upper panel for IL-6 and lower panel for TNF-{alpha}, respectively). Surprisingly, however, stimulation of the Gly248-Fc{alpha}RI allele induces significant release of IL-6 and TNF-{alpha} in the absence of the FcR {gamma}-chain pairing (two right bars in Fig. 3D, upper panel for IL-6 and lower panel for TNF-{alpha}, respectively). These data demonstrate that the CD89 {alpha}-chain, and specifically the Gly248-Fc{alpha}RI allele of the receptor, maintains signaling capacity in the absence of the {gamma}-chain. The data further demonstrate that the alleles of the Fc{alpha}RI {alpha}-chain CYT domain do not require the presence of the FcR {gamma}-chain to significantly influence receptor functions and raise the possibility of another molecular partner mediating this function.


Figure 3
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  		FIGURE 3. The Ser248 and Gly248 alleles differ in receptor-mediated cytokine production. Tissue culture wells were coated with anti-Fc{alpha}RI mAb A59 F(ab')2 (20 µg/ml) overnight at room temperature. The washed cells expressing Fc{alpha}RI were incubated in mAb F(ab')2 coated wells and cultured for 24 h. The level of murine IL-6 or TNF-{alpha} in culture supernatants was quantified by ELISA. A, Stably transfected P388D1 cell lines express each of the Fc{alpha}RI (CD89) allele (Ser248 and Gly248), either with (Leu209) or without TM mutations (Arg209) at equivalent levels (Arg209Ser248-Fc{alpha}RI = Arg209Ser248; Leu209Ser248-Fc{alpha}RI = Leu209Ser248; Arg209Gly248-Fc{alpha}RI = Arg209Gly248; Leu209Gly248-Fc{alpha}RI = Leu209Gly248). B, Fc{alpha}RI (CD89) with Arg209 to Leu209 mutations within the TM region do not associate with the FcR {gamma}-chain. Fc{alpha}RI (CD89) immunoprecipitates (IP), immunoblotted with rabbit anti {gamma}-chain polyclonal Abs on unreduced SDS-PAGE, indicate that the receptors with wild type Arg209 maintain their ability to associate with the {gamma}-chain (lane 1, Arg209Ser248; lane 3, Arg209Gly248). Lane 2 (Leu209Ser248) and lane 4 (Leu209Gly248) show that the receptors with Leu209 mutations within the TM region have lost their ability to associate with the {gamma}-chain. C, Upper panel, IL-6 production induced through Fc{alpha}RI specific stimulation was significantly different between two Fc{alpha}RI (CD89) alleles in their native {gamma}-chain-associated Arg209 forms. In all experiments, the IL-6 production induced by the cross-linking of CD89 was significantly higher in the P388 cells carrying the Gly248 allele (Arg209Gly248) compared with the Ser248 allele (Arg209Ser248) (27.31 ± 1.37 pg/ml in Arg209Gly248 cells and 14.66 ± 0.79 pg/ml in Arg209Ser248 cells respectively; p < 0.01 from t test; n = 4). Lower panel, allelic differences in TNF-{alpha} production mediated by Fc{alpha}RI in the absence of {gamma}-chain association. In all experiments TNF-{alpha} production was significantly higher with the Gly248-Fc{alpha}RI allele (Arg209Gly248) than with the Ser248-Fc{alpha}RI allele (Arg209Ser248) (637.3 ± 8.85 pg/ml for Arg209Gly248 cells and 470.0 ± 27.41 pg/ml for Arg209Ser248 cells, respectively; *, p < 0.01; n = 4). D, Upper panel, Allelic differences in IL-6 production mediated by Fc{alpha}RI in the absence of {gamma}-chain association. The Ser248-Fc{alpha}RI (Leu209Ser248) was unable to induce IL-6 release through the receptor cross-linking in the absence of {gamma}-chain association. However, Gly248-Fc{alpha}RI (Leu209Gly248) maintained its ability to induce IL-6 release despite the absence of {gamma}-chain association (16.86 ± 1.4 pg/ml with anti-Fc{alpha}RI mAb F(ab')2 compared with 5.26 ± 0.15 pg/ml with mIg F(ab')2 control; *, p < 0.01 from t test, n = 4). Lower panel, Allelic differences in TNF-{alpha} production mediated by Fc{alpha}RI in the absence of FcR {gamma}-chain association. Although the Ser248-Fc{alpha}RI allele (Leu209Ser248) lost its ability to induce TNF-{alpha} production, the Gly248-Fc{alpha}RI allele (Leu209Gly248) maintained its capacity to induce TNF-{alpha} production in the absence of {gamma}-chain association (389.0 ± 16.7 pg/ml with Gly248-Fc{alpha}RI (Leu209Gly248) compared with mIg F(ab')2 control of 162.1 ± 24.9 pg/ml; *, p < 0.01 from t test; n = 4).

 
Lyn association with Fc{alpha}RI is allele sensitive

The cross-linking of Fc{alpha}R activates Src family kinase activity and induces tyrosine phosphorylation of several cellular proteins, including p72Syk, a major target of early protein tyrosine kinase activity. The Src family member Lyn coprecipitates with aggregated Fc{alpha}RI complexes and is one of the protein tyrosine kinases involved in receptor-mediated signaling (44). These observations prompted us to investigate whether Lyn might associate with Fc{alpha}R independent of the {gamma}-chain and provide an explanation for the activity of Gly248-Fc{alpha}RI. We used three independent assays to assess the association of Fc{alpha}RI with Lyn. First, we generated a GST-CD89 CYT tail fusion protein to confirm that Lyn is associated with the CYT tail of Fc{alpha}RI. Fig. 4A demonstrates that the Fc{alpha}RI CYT domain, fused with GST, is able to bind Lyn in P388D1 cell lysates. GST-CD89CYT was also able to pull down Lyn from 293 cells and U937 cells (data not shown), demonstrating that Lyn associates with Fc{alpha}RI through the Fc{alpha}RI CYT tail. Second, we used a column-based immunoprecipitation assay to establish the relationship between Lyn and Fc{alpha}RI. Lysates from cell lines expressing four different constructs of Fc{alpha}RI at similar levels (Fig. 3A) were precleared on irrelevant mIgG1 isotype control columns. The precleared cell lysates were then passed over columns packed with agarose beads conjugated with the anti-Fc{alpha}RI mAb A77. The proteins eluted from these affinity columns were probed with rabbit anti-Lyn Abs. The bottom panel of Fig. 4B shows that the levels of CD89 (Fc{alpha}RI) eluted from the columns were similar among four different cell lines. However, more Lyn was isolated from lysates with the Gly248-Fc{alpha}RI allele than with the Ser248-Fc{alpha}RI allele in the presence of {gamma}-chain association (upper panel of Fig. 4B, lanes 1 and 2). This difference was also pronounced in the TM mutants carrying Leu209 that are unable to pair with the FcR {gamma}-chain (upper panel of Fig. 4B, lanes 3 and 4). No CD89 or Lyn could be detected by Western blotting in eluates from the mIgG1 control columns (data not shown). These data establish the specificity of interaction between Lyn and Fc{alpha}RI and show the quantitative differences in the association between Lyn and two alleles of Fc{alpha}RI.


Figure 4
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  		FIGURE 4. Association of Lyn with Fc{alpha}RI through the CYT domain. A, GST-CD89CYT binding to Lyn. The equal amounts (5 µg/lane) of the GST or GST-CD89CYT fusion protein (bottom panel) bound to glutathione-agarose beads were used to bind Lyn in cell lysates of 2 x 107 P388D1 cells. Lyn, eluted from beads and detected by immunoblotting, was evident in GST-CD89CY but not in GST only. B, Column immunoprecipitation (IP) of Fc{alpha}RI (CD89) and Lyn. Cell lysates from four different stably transfected cell lines were loaded onto the columns packed with agarose beads cross-linked with A77. The bottom panel shows similar levels of Fc{alpha}RI eluted from the columns for each of the four different cell lines. The Gly248-Fc{alpha}RI allele (lane 2) coimmunoprecipitates more Lyn than the Ser248-Fc{alpha}RI allele (lane 1) both in the presence and in the absence of {gamma}-chain association (lanes 3 and 4). C, Immunoprecipitation (IP) of Lyn with rabbit anti-Lyn Ab coimmunoprecipitates Fc{alpha}RI (CD89). Lyn was immunoprecipitated using rabbit anti-Lyn Abs, and coimmunoprecipitation of Fc{alpha}RI was detected with anti-Fc{alpha}RI immunoblots. Although both alleles coimmunoprecipitated with Lyn, more of the Gly248 allele (lane 2) compared with the Ser248 allele (lane 1) was evident. In the absence of the {gamma}-chain this differential was more pronounced, and the Ser248 allele essentially lost its association with Lyn (lane 3). Results shown are representative of at least four experiments for each study.

 
Finally, we immunoprecipitated Lyn using rabbit anti-Lyn Abs and performed anti-Fc{alpha}RI immunoblot analysis to examine the amount of receptor associated with Lyn. Although a comparable amount of Lyn was immunoprecipitated from each of the different cell lines expressing Fc{alpha}RI constructs (Fig. 4C, bottom panel), Lyn was able to pull down significantly more Gly248-Fc{alpha}RI protein (Fig. 4C, lane 2) than Ser248-Fc{alpha}RI protein (lane 1) in the presence of {gamma}-chain association. To further demonstrate the dependence of the association between Fc{alpha}RI and Lyn on the Fc{alpha}RI {alpha}-chain, cell lines expressing similar levels of Leu209 TM mutant Fc{alpha}RI were used for coimmunoprecipitation. Although the Leu209 TM mutant receptor of both Fc{alpha}RI alleles did not associate with endogenous {gamma}-chains (Fig. 3B, upper panel; Fig. 4B, middle panel), the Gly248-Fc{alpha}RI readily coimmunoprecipitated with Lyn (Fig. 4C, upper panel, lane 4). In contrast, very little if any of the Ser248-Fc{alpha}RI allele receptor (Fig. 4C, upper panel, lane 3) was evident.

The Ser248 and Gly248 alleles of Fc{alpha}RI affect receptor mediated IL-6 cytokine release by human neutrophils

Because human neutrophils are the primary cell population constitutively expressing high levels of Fc{alpha}RI, we compared the effects of the Fc{alpha}RI alleles on IL-6 release by neutrophils from the genotyped homozygous donors. Neutrophils from donors of both genotypes expressed comparable levels of Fc{alpha}RI (data not shown). Consistent with the results obtained from the transfected P388D1 cell lines, cross-linking of the Fc{alpha}RI with A59 F(ab')2 induced significantly more IL-6 release by neutrophils from Gly248-Fc{alpha}RI homozygous donors compared with the Ser248-Fc{alpha}RI homozygous donors (Fig. 5A; p < 0.05). Human IgA-mediated cross-linking of the receptors also induced significantly more IL-6 release by neutrophils from Gly248-Fc{alpha}RI allele homozygous donors compared with the Ser248-Fc{alpha}RI allele homozygous donors (Fig. 5B; p < 0.05). Interestingly, the cross-linking of Fc{alpha}RI in the Ser248-Fc{alpha}RI homozygous donors significantly inhibited IL-6 release in human IgA-stimulated neutrophils (p = 0.015; Fig. 5B). Taken together, our data demonstrate that the alleles of Fc{alpha}RI CYT domain significantly affect receptor mediated cytokine release by primary human neutrophils ex vivo.


Figure 5
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  		FIGURE 5. The Fc{alpha}RI alleles affect receptor mediated IL-6 cytokine release from human neutrophils. A, IL-6 release induced by anti-Fc{alpha}RI mAb A59 F(ab')2 on neutrophils from homozygous donors of either Ser248 or Gly248 alleles. The level of IL-6 release was significantly higher in A59 F(ab')2-treated neutrophils from the Gly248 homozygous donors compared with the Ser248 homozygous donors (8.66 ± 1.85 pg/ml for the Gly248 homozygous donors and 2.34 ± 1.32 pg/ml for the Ser248 homozygous donors respectively; p = 0.032 from t test; n = 4). The IL-6 release was not significantly different between Ser248 homozygous donors and Gly248 homozygous donors in mIgG F(ab')2-treated neutrophils (p = 0.598 from t test; n = 4). B, IL-6 release induced by the treatment of neutrophils with human IgA. The levels of IL-6 release by neutrophils from the Gly248 homozygous donors was significantly higher compared with the Ser248 homozygous donors (14.62 ± 4.81 pg/ml for the Gly248 homozygous donors and 0.2 ± 0.1 pg/ml for the Ser248 allele homozygous donors respectively; p = 0.040 from t test; n = 3). Additionally, human IgA significantly inhibited IL-6 release from neutrophils in all Ser248 homozygous donors (5.08 ± 0.66 pg/ml in the untreated neutrophils to 0.20 ± 0.10 pg/ml in the human IgA treated neutrophils; p = 0.015 from paired t test; n = 3), whereas all Gly248 homozygous donors showed an increase (4.19 ± 0.33 pg/ml in the untreated neutrophils to 14.62 ± 4.81 pg/ml; p = 0.177 from paired t test; n = 3).

 
The Gly248-Fc{alpha}RI allele and SLE

Because Fc{alpha}RI can activate a variety of cell programs in myeloid cells and IgA autoantibodies have received increasing attention (45, 46), we speculated that the proinflammatory Fc{alpha}RI alleles with their impact on receptor signaling might be associated with inflammatory diseases. We compared the genotype frequencies in African American and European American controls and found a significant enrichment of Gly248-Fc{alpha}RI in African Americans compared with European Americans (Table II; p < 0.01 from Fisher’s exact test). Furthermore, in African Americans the Gly248-Fc{alpha}RI allele was significantly enriched in SLE patients compared with corresponding normal controls (Mantel-Haenszel {chi}2; p = 0.0334). A similar tendency toward an increased Gly248-Fc{alpha}RI allele frequency was evident in European American SLE patients compared with controls and was most evident in a dominant genetic model with enrichment of homozygosity of the Gly248-Fc{alpha}RI genotype (p < 0.05 from Fisher’s exact test). In a combined analysis to test the significance of enrichment of the Gly248-Fc{alpha}RI allele in both populations, we found this allele significantly enriched in SLE populations compared with the control populations (Mantel-Haenszel {chi}2; p = 0.0194). These data suggest that the Gly248-Fc{alpha}RI allele may be considered a candidate risk factor for autoimmune disease, such as SLE.


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  		Table II. The distribution of Fc{alpha}RI SNP 844 (Ser248/Gly248) alleles in SLE patients and non-SLE normal controlsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The two naturally occurring alleles of human Fc{alpha}RI, Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI, differ in their quantitative functions and, remarkably, unlike Ser248-Fc{alpha}RI, the Gly248 allele is capable of inducing cytokine release even in the absence of FcR {gamma}-chain association. This capacity is presumably due, at least in part, to its ability to bind Lyn. This association of a Lyn kinase with Fc{alpha}RI may be mediated by an unconventional Src homology 3 domain in the CYT domain of Fc{alpha}RI comprised of the peptide sequence Trp247(Ser248/Gly248)Gln249Gln250, which includes the polymorphic site. This sequence is similar to the unconventional Src homology 3 domain binding motif (WxxQF/Y) frequently found in peroxisomal membrane proteins (47, 48, 49). Thus, it seems that Gly248-Fc{alpha}RI may have a constant potential for an activating and perhaps proinflammatory role, whereas the Ser248-Fc{alpha}RI allele requires Fc{alpha}RI:FcR {gamma}-chain pairing for the induction of cytokine release. Given that the net biological activity of Fc{alpha}RI, therefore, is dependent on both the genotype of the donor and the extent of Fc{alpha}RI:FcR {gamma}-chain pairing, it is not surprising that investigators have drawn divergent conclusions about the activating potential of Fc{alpha}RI-initiated activity (33, 34).

The difference in the biological potential of the two alleles provides a tool for probing the idea that the balance of Fc{alpha}RI-initiated activating/inhibitory activities might reflect these naturally occurring alleles and be important in setting thresholds for inflammatory responses (36). Accordingly, we reasoned that Fc{alpha}RI alleles might play a role in the pathophysiology of disease. Somewhat to our surprise, the Gly248-Fc{alpha}RI allele is over represented in systemic lupus. Although the mean IgA1 and total IgA in samples from SLE patients are significantly higher than those from the normal population (50), IgA Abs have received little attention in SLE. IgA anti-Ro/SSA, anti-La/SSB, anti-cardiolipin, and anti-beta2-glycoprotein-I autoantibodies are present in the serum of patients with SLE, Sjogren’s syndrome, and anti-cardiolipin syndrome (45, 51, 52) and, very recently, reports indicate that not only EBV seropositivity but also a specific IgA Ab against EBV are associated with SLE patients (46, 53). The reports of IgA autoantibodies and IgA anti-EBV seropositivity suggest a provocative and perhaps underappreciated link in which a more activating host response to IgA might influence an autoimmune and inflammatory response. The differential biologies of the Ser248-Fc{alpha}RI and Gly248-Fc{alpha}RI alleles could also play a role in other phenotypes involving IgA, including dermatitis herpetiformis, celiac disease, periodontal disease, and possibly IgA nephropathy. Even the uptake and inactivation of virus, demonstrated in epithelial cells with anti-HIV IgA, might be impacted by CD89 alleles (54). Because mice do not have FCAR, the gene encoding CD89, clues will not be forthcoming from studies of spontaneous mouse phenotypes but will need to be done in humans or, possibly, in mice reconstituted with human immune systems (55, 56).

Despite abundant mucosal and serum IgA, the overall role of IgA and Fc{alpha}RI in host defense and immune regulation remains a matter of some controversy. As noted, some studies have suggested an anti-inflammatory function for IgA and Fc{alpha}RI, as demonstrated in the suppression of neutrophil chemotaxis, down-regulation of the generation of reactive oxygen species, and the induction of the anti-inflammatory cytokine IL-1ra (32, 34). Because IgA in the serum does not fix complement by the classical pathway, IgA is thought to act as a "discrete housekeeper" in which foreign Ags are incorporated into immune complexes by IgA and removed by the phagocytic system, with little or no resultant inflammation (57). However, Fc{alpha}RI is a multichain immune recognition receptor that can associate with the ITAM-containing FcR {gamma}-chain subunit. The resultant trimeric Fc{alpha}RI/(FcR {gamma}-chain)2 initiates established phosphotyrosine-based signaling cascades, and this model is compatible with an activating, proinflammatory receptor (30, 41). The observation that the majority of Fc{alpha}RI expressed on human cells are not associated with {gamma}-chain (26), however, makes Fc{alpha}RI distinct from Fc{gamma}RI and Fc{gamma}RIII, and the apparent association of Gly248-Fc{alpha}RI with Lyn provides an alternative mechanism for initiating cell programs in an allele-sensitive fashion. The intensity of cytokine production stimulated through Gly248-Fc{alpha}RI alone is less than that through the Fc{alpha}RI:FcR {gamma}-chain, and these two signaling assemblies may have qualitative as well as quantitative differences in their capacities. Our data also suggest that IgA may inhibit IL6 production by neutrophils from donors carry the Ser248 allele of Fc{alpha}RI. This inhibitory role may be related to the inhibitory functions of the FcR {gamma}-chain recently described by Pasquier et al. (36) and by Olas et al. (58). Although the positively charged arginine in the TM domain of Fc{alpha}RI is required for the pairing with the FcR {gamma}-chain, little is known about the mechanisms regulating the pairing of Fc{alpha}RI:FcR {gamma}-chain. This step may be an important element in the understanding of overall Fc{alpha}RI biology.

Our genotyping data suggest that the proinflammatory 844G allele (Gly248-Fc{alpha}RI) may be associated with SLE in African American and Caucasian populations, raising the possibility that IgA Ab production may play an important role in modulating the immune responses. This association needs to be extended in independent case control and family-based association studies, and linkage disequilibrium with other susceptibility gene(s) in this region must also be evaluated. However, based on the reports that Fc{gamma}Rs contribute to the susceptibility to human autoimmune diseases beyond SLE, genetic associations of Fc{alpha}RI variants with other human inflammatory diseases, perhaps conditioned on other genetic effects and stratified by different ethnic groups, are likely to be identified.


   Acknowledgments
 
We thank Drs. Max Cooper and Hiromi Kubagawa for helpful discussions and Dr. Kubagawa for anti-Fc{alpha}RI mAbs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01-AR33062 (to R.P.K.), N01-AI40068, M01-RR00032, and P01-AR49084 (to R.P.K.). The FACS Core Facility of the University of Alabama at Birmingham Arthritis and Musculoskeletal Center was supported by Rheumatic Diseases Core Center (National Institutes of Health Grant P30-AR48311). Back

2 Address correspondence and reprint requests to Dr. Robert P. Kimberly, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, 172 Shelby Interdisciplinary Biomedical Science Building, 1825 University Boulevard, Birmingham, AL 35294. E-mail address: rpk{at}uab.edu Back

3 Abbreviations used in this paper: TM, transmembrane; EC, extracellular; AM, acetoxymethylester; [Ca2+]i, intracellular CA2+ concentration; CYT, cytoplasmic; mIgG, murine IgG; RBL, rat basophilic leukemia; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism. Back

Received for publication December 21, 2005. Accepted for publication January 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Stewart, W. W., M. A. Kerr. 1990. The specificity of the human neutrophil IgA receptor (Fc{alpha}R) determined by measurement of chemiluminescence induced by serum or secretory IgA1 or IgA2. Immunology 71: 328-334. [Medline]
  2. van Egmond, M., E. van Garderen, A. B. van Spriel, C. A. Damen, E. S. van Amersfoort, G. van Zandbergen, J. van Hattum, J. Kuiper, J. G. van de Winkel. 2000. Fc{alpha}RI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity. Nat. Med. 6: 680-685. [Medline]
  3. Motegi, Y., H. Kita. 1998. Interaction with secretory component stimulates effector functions of human eosinophils but not of neutrophils. J. Immunol. 161: 4340-4346. [Abstract/Free Full Text]
  4. van Egmond, M., C. A. Damen, A. B. van Spriel, G. Vidarsson, E. van Garderen, J. G. van de Winkel. 2001. IgA and the IgA Fc receptor. Trends Immunol. 22: 205-211. [Medline]
  5. Kilian, M., J. Reinholdt, H. Lomholt, K. Poulsen, E. V. Frandsen. 1996. Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS 104: 321-338. [Medline]
  6. Monteiro, R. C., J. G. Van De Winkel. 2003. IgA Fc receptors. Annu. Rev. Immunol. 21: 177-204. [Medline]
  7. Fanger, M. W., L. Shen, J. Pugh, G. M. Bernier. 1980. Subpopulations of human peripheral granulocyes and monocytes express receptors for IgA. Proc. Natl. Acad. Sci. USA 77: 3640-3644. [Abstract/Free Full Text]
  8. Wende, H., M. Colonna, A. Ziegler, A. Volz. 1999. Organization of the leukocyte receptor cluster (LRC) on human chromosome 19q13.4. Mamm. Genome 10: 154-160. [Medline]
  9. Wagtmann, N., S. Rojo, E. Eichler, H. Mohrenweiser, E. O. Long. 1997. A new human gene complex encoding the killer cell inhibitory receptors and related monocyte/macrophage receptors. Curr. Biol. 7: 615-618. [Medline]
  10. de Wit, T. P., H. C. Morton, P. J. Capel, J. G. van de Winkel. 1995. Structure of the gene for the human myeloid IgA Fc receptor (CD89). J. Immunol. 155: 1203-1209. [Abstract]
  11. van Dijk, T. B., M. Bracke, E. Caldenhoven, J. A. Raaijmakers, J. W. Lammers, L. Koenderman, R. P. de Groot. 1996. Cloning and characterization of Fc{alpha}Rb, a novel Fc{alpha} receptor (CD89) isoform expressed in eosinophils and neutrophils. Blood 88: 4229-4238. [Abstract/Free Full Text]
  12. Morton, H. C., A. E. Schiel, S. W. Janssen, J. G. van de Winkel. 1996. Alternatively spliced forms of the human myeloid Fc{alpha} receptor (CD89) in neutrophils. Immunogenetics 43: 246-247. [Medline]
  13. Reterink, T. J., C. L. Verweij, L. A. van Es, M. R. Daha. 1996. Alternative splicing of IgA Fc receptor (CD89) transcripts. Gene 175: 279-280. [Medline]
  14. Pleass, R. J., P. D. Andrews, M. A. Kerr, J. M. Woof. 1996. Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils. Biochem. J. 318: 771-777.
  15. Toyabe, S., Y. Kuwano, K. Takeda, M. Uchiyama, T. Abo. 1997. IgA nephropathy-specific expression of the IgA Fc receptors (CD89) on blood phagocytic cells. Clin. Exp. Immunol. 110: 226-232. [Medline]
  16. Togo, S., T. Shimokawa, Y. Fukuchi, C. Ra. 2003. Alternative splicing of myeloid IgA Fc receptor (Fc{alpha}R, CD89) transcripts in inflammatory responses. FEBS Lett. 535: 205-209. [Medline]
  17. Patry, C., Y. Sibille, A. Lehuen, R. C. Monteiro. 1996. Identification of Fc{alpha} receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages. J. Immunol. 156: 4442-4448. [Abstract]
  18. Sondermann, P., R. Huber, V. Oosthuizen, U. Jacob. 2000. The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc{gamma}RIII complex. Nature 406: 267-273. [Medline]
  19. Garman, S. C., B. A. Wurzburg, S. S. Tarchevskaya, J. P. Kinet, T. S. Jardetzky. 2000. Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc{epsilon}RI {alpha}. Nature 406: 259-266. [Medline]
  20. Wines, B. D., M. D. Hulett, G. P. Jamieson, H. M. Trist, J. M. Spratt, P. M. Hogarth. 1999. Identification of residues in the first domain of human Fc{alpha} receptor essential for interaction with IgA. J. Immunol. 162: 2146-2153. [Abstract/Free Full Text]
  21. Morton, H. C., G. van Zandbergen, C. van Kooten, C. J. Howard, J. G. van de Winkel, P. Brandtzaeg. 1999. Immunoglobulin-binding sites of human Fc{alpha}RI (CD89) and bovine Fc{gamma}2R are located in their membrane-distal extracellular domains. J. Exp. Med. 189: 1715-1722. [Abstract/Free Full Text]
  22. Pleass, R. J., J. I. Dunlop, C. M. Anderson, J. M. Woof. 1999. Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fc{alpha} receptor (Fc{alpha}R) CD89. J. Biol. Chem. 274: 23508-23514. [Abstract/Free Full Text]
  23. Carayannopoulos, L., J. M. Hexham, J. D. Capra. 1996. Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between C{alpha}2 and C{alpha}3 in human IgA1. J. Exp. Med. 183: 1579-1586. [Abstract/Free Full Text]
  24. Herr, A. B., E. R. Ballister, P. J. Bjorkman. 2003. Insights into IgA-mediated immune responses from the crystal structures of human Fc{alpha}RI and its complex with IgA1-Fc. Nature 423: 614-620. [Medline]
  25. Herr, A. B., C. L. White, C. Milburn, C. Wu, P. J. Bjorkman. 2003. Bivalent binding of IgA1 to Fc{alpha}RI suggests a mechanism for cytokine activation of IgA phagocytosis. J. Mol. Biol. 327: 645-657. [Medline]
  26. Launay, P., C. Patry, A. Lehuen, B. Pasquier, U. Blank, R. C. Monteiro. 1999. Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcR{gamma} association and protects against degradation of bound ligand. J. Biol. Chem. 274: 7216-7225. [Abstract/Free Full Text]
  27. Honorio-Franca, A. C., P. Launay, M. M. Carneiro-Sampaio, R. C. Monteiro. 2001. Colostral neutrophils express Fc{alpha} receptors (CD89) lacking {gamma} chain association and mediate noninflammatory properties of secretory IgA. J. Leukocyte Biol. 69: 289-296. [Abstract/Free Full Text]
  28. Launay, P., A. Lehuen, T. Kawakami, U. Blank, R. C. Monteiro. 1998. IgA Fc receptor (CD89) activation enables coupling to syk and Btk tyrosine kinase pathways: differential signaling after IFN-{gamma} or phorbol ester stimulation. J. Leukocyte Biol. 63: 636-642. [Abstract]
  29. Lang, M. L., Y. W. Chen, L. Shen, H. Gao, G. A. Lang, T. K. Wade, W. F. Wade. 2002. IgA Fc receptor (Fc{alpha}R) cross-linking recruits tyrosine kinases, phosphoinositide kinases and serine/threonine kinases to glycolipid rafts. Biochem. J. 364: 517-525. [Medline]
  30. Morton, H. C., I. E. van den Herik-Oudijk, P. Vossebeld, A. Snijders, A. J. Verhoeven, P. J. Capel, J. G. van de Winkel. 1995. Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR {gamma} chain: molecular basis for CD89/FcR {gamma} chain association. J. Biol. Chem. 270: 29781-29787. [Abstract/Free Full Text]
  31. Van Epps, D. E., S. L. Brown. 1981. Inhibition of formylmethionyl-leucyl-phenylalanine-stimulated neutrophil chemiluminescence by human immunoglobulin A paraproteins. Infect. Immun. 34: 864-870. [Abstract/Free Full Text]
  32. Van Epps, D. E., R. C. Williams, Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. Exp. Med. 144: 1227-1242. [Abstract/Free Full Text]
  33. Wolf, H. M., M. B. Fischer, H. Puhringer, A. Samstag, E. Vogel, M. M. Eibl. 1994. Human serum IgA downregulates the release of inflammatory cytokines (tumor necrosis factor-{alpha}, interleukin-6) in human monocytes. Blood 83: 1278-1288. [Abstract/Free Full Text]
  34. Wolf, H. M., I. Hauber, H. Gulle, A. Samstag, M. B. Fischer, R. U. Ahmad, M. M. Eibl. 1996. Anti-inflammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc{alpha}R (CD89)-mediated down-regulation of tumour necrosis factor-{alpha} (TNF-{alpha}) and IL-6 in human monocytes. Clin. Exp. Immunol. 105: 537-543. [Medline]
  35. Wolf, H. M., E. Vogel, M. B. Fischer, H. Rengs, H. P. Schwarz, M. M. Eibl. 1994. Inhibition of receptor-dependent and receptor-independent generation of the respiratory burst in human neutrophils and monocytes by human serum IgA. Pediatr. Res. 36: 235-243. [Medline]
  36. Pasquier, B., P. Launay, Y. Kanamaru, I. C. Moura, S. Pfirsch, C. Ruffie, D. Henin, M. Benhamou, M. Pretolani, U. Blank, R. C. Monteiro. 2005. Identification of Fc{alpha}RI as an inhibitory receptor that controls inflammation: dual role of FcR{gamma} ITAM. Immunity 22: 31-42. [Medline]
  37. Wu, J., J. C. Edberg, P. B. Redecha, V. Bansal, P. M. Guyre, K. Coleman, J. E. Salmon, R. P. Kimberly. 1997. A novel polymorphism of Fc{gamma}RIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100: 1059-1070. [Medline]
  38. Li, X., J. Wu, R. H. Carter, J. C. Edberg, K. Su, G. S. Cooper, R. P. Kimberly. 2003. A novel polymorphism in the Fc{gamma} receptor IIB (CD32B) transmembrane region alters receptor signaling. Arthritis Rheum. 48: 3242-3252. [Medline]
  39. Jasek, M., M. Manczak, A. Sawaryn, A. Obojski, A. Wisniewski, W. Luszczek, P. Kusnierczyk. 2004. A novel polymorphism in the cytoplasmic region of the human immunoglobulin A Fc receptor gene. Eur. J. Immunogenet. 31: 59-62. [Medline]
  40. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield, J. G. Schaller, N. Talal, R. J. Winchester. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25: 1271-1277. [Medline]
  41. Pfefferkorn, L. C., G. R. Yeaman. 1994. Association of IgA-Fc receptors (Fc{alpha}R) with Fc{epsilon}RI{gamma} 2 subunits in U937 cells: aggregation induces the tyrosine phosphorylation of {gamma} 2. J. Immunol. 153: 3228-3236. [Abstract]
  42. Morton, H. C., M. van Egmond, J. G. van de Winkel. 1996. Structure and function of human IgA Fc receptors (Fc{alpha}R). Crit. Rev. Immunol. 16: 423-440. [Medline]
  43. Hou, X., J. Dietrich, N. O. Geisler. 1996. The cytoplasmic tail of Fc{gamma}RIIIA{alpha} is involved in signaling by the low affinity receptor for immunoglobulin G. J. Biol. Chem. 271: 22815-22822. [Abstract/Free Full Text]
  44. Gulle, H., A. Samstag, M. M. Eibl, H. M. Wolf. 1998. Physical and functional association of Fc{alpha}R with protein tyrosine kinase Lyn. Blood 91: 383-391. [Abstract/Free Full Text]
  45. Cucurull, E., A. E. Gharavi, E. Diri, E. Mendez, D. Kapoor, L. R. Espinoza. 1999. IgA anticardiolipin and anti-beta2-glycoprotein I are the most prevalent isotypes in African American patients with systemic lupus erythematosus. Am. J. Med. Sci. 318: 55-60. [Medline]
  46. Chen, C. J., K. H. Lin, S. C. Lin, W. C. Tsai, J. H. Yen, S. J. Chang, S. N. Lu, H. W. Liu. 2005. High prevalence of immunoglobulin A antibody against Epstein-Barr virus capsid antigen in adult patients with lupus with disease flare: case control studies. J. Rheumatol. 32: 44-47. [Abstract/Free Full Text]
  47. Barnett, P., G. Bottger, A. T. Klein, H. F. Tabak, B. Distel. 2000. The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction. EMBO J. 19: 6382-6391. [Medline]
  48. Bottger, G., P. Barnett, A. T. Klein, A. Kragt, H. F. Tabak, B. Distel. 2000. Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner. Mol. Biol. Cell 11: 3963-3976. [Abstract/Free Full Text]
  49. Schliebs, W., J. Saidowsky, B. Agianian, G. Dodt, F. W. Herberg, W. H. Kunau. 1999. Recombinant human peroxisomal targeting signal receptor PEX5: structural basis for interaction of PEX5 with PEX14. J. Biol. Chem. 274: 5666-5673. [Abstract/Free Full Text]
  50. Conley, M. E., W. J. Koopman. 1983. Serum IgA1 and IgA2 in normal adults and patients with systemic lupus erythematosus and hepatic disease. Clin. Immunol. Immunopathol. 26: 390-397. [Medline]
  51. Diri, E., E. Cucurull, A. E. Gharavi, D. Kapoor, E. A. Mendez, E. Scopelitis, W. A. Wilson. 1999. Antiphospholipid (Hughes’) syndrome in African-Americans: IgA aCL and abeta2 glycoprotein-I is the most frequent isotype. Lupus 8: 263-268. [Abstract/Free Full Text]
  52. Greco, T. P., M. D. Amos, A. M. Conti-Kelly, J. D. Naranjo, J. W. Ijdo. 2000. Testing for the antiphospholipid syndrome: importance of IgA anti-beta2-glycoprotein I. Lupus 9: 33-41. [Abstract/Free Full Text]
  53. Parks, C. G., G. S. Cooper, L. L. Hudson, M. A. Dooley, E. L. Treadwell, E. W. St Clair, G. S. Gilkeson, J. P. Pandey. 2005. Association of Epstein-Barr virus with systemic lupus erythematosus: effect modification by race, age, and cytotoxic T lymphocyte-associated antigen 4 genotype. Arthritis Rheum. 52: 1148-1159. [Medline]
  54. Huang, Y. T., A. Wright, X. Gao, L. Kulick, H. Yan, M. E. Lamm. 2005. Intraepithelial cell neutralization of HIV-1 replication by IgA. J. Immunol. 174: 4828-4835. [Abstract/Free Full Text]
  55. Shultz, L. D., B. L. Lyons, L. M. Burzenski, B. Gott, X. Chen, S. Chaleff, M. Kotb, S. D. Gillies, M. King, J. Mangada, et al 2005. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R {gamma} null mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174: 6477-6489. [Abstract/Free Full Text]
  56. Traggiai, E., L. Chicha, L. Mazzucchelli, L. Bronz, J. C. Piffaretti, A. Lanzavecchia, M. G. Manz. 2004. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304: 104-107. [Abstract/Free Full Text]
  57. Conley, M. E., D. L. Delacroix. 1987. Intravascular and mucosal immunoglobulin A: two separate but related systems of immune defense?. Ann. Intern. Med. 106: 892-899. [Abstract/Free Full Text]
  58. Olas, K., H. Butterweck, W. Teschner, H. P. Schwarz, B. Reipert. 2005. Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells. Clin. Exp. Immunol. 140: 478-490. [Medline]



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