Published online before print September 10, 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RI on human plateletsDivision of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
2 Correspondence: Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, 202 Shelby Interdisciplinary Biomedical Science Building, 1825 University Boulevard, Birmingham, AL 35294-2182, USA. E-mail: jmwu{at}uab.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RI (CD89) is a human IgA FcR expressed on cells of myeloid lineage such as neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells. FcRI mediates cell activation through Src family kinases and downstream tyrosine-based phosphorylation pathways. However, the role of IgA and the expression and role of its cognate receptor FcRI (CD89) in platelet activation are undefined. In the current study, we demonstrate that human platelets express FcRI mRNAs and proteins. Furthermore, we show that the platelet FcRI is associated with the FcR 
-chain, and cross-linking of FcRI leads to Syk phosphorylation. Clustering of FcRI induces pre-mRNA splicing and protein production of tissue factor and IL-1β, suggesting novel roles for human platelet FcRI and serum IgA in thrombosis and inflammation.
Key Words: CD89 • pre-mRNA splicing • IL-1_oblique>β • tissue factor
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RI, a type I immune membrane receptor, is expressed on the cells of myeloid lineage including neutrophils, eosinophils, monocytes, tissue macrophages, and subpopulations of dendritic cells [1
]. FcRI binds monomeric and dimeric IgA, and clustering of FcRI by IgA immune complexes initiates endocytosis, phagocytosis, and other immune responses [2
]. The FcR for IgA (FCAR) gene is located in the leukocyte receptor cluster on human chromosome 19q13.4 along with platelet collagen receptor glycoprotein VI (GP-VI), NK cell immune receptors, and leukocyte Ig-like receptors [3
4
5
6
7
]. FcRI shares sequence similarity with platelet collagen receptor GP-VI [5
], killer cell inhibitory receptors, and other members of the leukocyte receptor cluster [3
, 4
]. FcRI orthologs have been identified in chimpanzees, macaques, cattle, horses, and rats [8
9
10
11
]. However, no equivalent gene has been identified in mice [11
].
The membrane distal extracellular domain (EC1) of FcRI is responsible for ligand-binding activity, and FcRI binds to each IgA CH2–CH3 domain interface by forming a 2:1 complex with a Fc dimer [12
, 13
]. FcRI can associate with the FcR -chain through its transmembrane domain. However, FcRI is often expressed in the absence of -chain pairing [14
]. FcRI mRNAs have at least 10 alternatively spliced variants, among which the splicing isoform lacking 22 residues (66 nucleotides) within EC2 is expressed on alveolar macrophages in vivo [1
]. The alternatively spliced FcRI isoforms might have physiologic relevance in IgA-mediated host defense [15
].
Human IgA (hIgA) has a central role in host defense on mucosal surfaces. In human serum, IgA is the second-most abundant Ig after IgG, and IgA accounts for one-fifth of the total Ig. hIgA is primarily of the IgA1 subclass and exists mainly in monomeric form (>95%) [16
]. In the intestinal lamina propria, the B lineage cells produce polymeric IgA, which exists almost exclusively as dimers. IgA dimers can be joined by a J-chain polypeptide and then linked to the secretory component to form a complex called secretory IgA (SIgA). Through FcRI, SIgA is able to initiate inflammatory responses, such as respiratory burst of polymorphonuclear leukocytes and degranulation of eosinophils [16
, 17
].
Human platelets are anucleate cells that have hemostatic and inflammatory functions [18
, 19
]. As a rich source for cytokines and chemokines, platelets have major roles in the pathogenesis of human inflammatory diseases [20
21
22
23
24
25
]. Human platelets possess functional spliceosomes that are able to process pre-mRNAs into mature mRNAs, from which proteins can be translated and processed in platelets [26
, 27
]. Antiplatelet agents appear to protect renal function in IgA nephropathy patients [28
], suggesting that pathogenic IgA immune complexes may be involved in the activation of platelets and in the pathogenesis of IgA nephropathy. In the current study, we demonstrate that human platelets express a functional IgAR (FcRI). Cross-linking of FcRI leads to Syk phosphorylation. Furthermore, FcRI mediates tissue-factor, pre-mRNA splicing and proinflammatory cytokine (IL-1β) production in human platelets, suggesting novel roles for human platelet FcRI and serum IgA in thrombosis and inflammation.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RI mAb MIP8a [murine IgG1 (mIgG1)] was purchased from Serotec (Raleigh, NC, USA). Mouse anti-human CD62P (P-selectin) mAb was from eBioscience (San Diego, CA, USA). mAb A59 and A77 are mIgG1 specific for FcRI as described previously [29
]. Anti-FcR -chain hybridoma (clone 7D3, mIgG1) was generated and characterized at the Epitope Recognition and Immunoreagent Core Facility of the University of Alabama at Birmingham (UAB; Birmingham, AL, USA). The purified mAb (A59, A77, and 7D3) were produced at the Epitope Recognition and Immunoreagent Core Facility of UAB. The F(ab')2 preparations of mAb A59 and 7D3 were produced at Rockland Immunochemicals (Gilbertsville, PA, USA), and the purity of these antibody F(ab')2 preparations was confirmed by SDS gel staining and Western blotting assays. Human serum IgA, FITC-conjugated and nonconjugated goat anti-mouse IgG (H+L), mIgG F(ab')2, and FITC-conjugated goat anti-hIgA were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Goat anti-hIgA F(ab')2 was purchased from Southern Biotechnologies (Birmingham, AL, USA). Rabbit anti-CD89 (FcRI) cytoplasmic domain (CD89cyt) polyclonal antibody (pAb) was produced using GST-CD89cyt fusion protein at Cocalico Biologicals (Reamstown, PA, USA). Anti-Syk rabbit pAb, anti-Syk mAb, and anti-Syk pAb-conjugated agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiphosphotyrosine mAb (clone 4G10), anti-human tissue-factor mAb (clone GMA-320), and rabbit anti-FcR -chain pAb were obtained from Upstate Biotech (Waltham, MA, USA). AminoLink kit (cyanogen bromide-activated Sepharose 4B coupling gel and reagents) was purchased from Pierce (Rockford, IL, USA). Human IL-1β cytokine ELISA detection kits were obtained from BD PharMingen (San Jose, CA, USA). Human IgG, fibrinogen, thrombin, and PGE1 were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Preparation of human platelets
The human studies were reviewed and approved by the Institutional Review Board of UAB, and all donors provided written, informed consent. Venous blood (50 ml) was collected from healthy, normal donors in BD Vacutainer tubes containing Na2 EDTA (BD Biosciences, San Jose, CA, USA). Platelets were purified as described by Shiraki el al. [30
] with small modification. Briefly, platelet-rich plasma (PRP) was obtained by centrifugation (15 min, 175 g) at room temperature. Packed cells were discarded, and PGE1 (1 µM) was added to the PRP and maintained throughout the purification procedure. PRP was centrifuged again (10 min, 175 g) to remove residual leukocytes. To avoid the contamination of peripheral blood leukocytes, platelets were collected from the upper half to two-thirds of the PRP supernatant for experimental use. Platelets were isolated by centrifugation of the resulting PRP (15 min, 1000 g) at room temperature. Leukocyte contamination was evaluated by flow cytometry with anti-human CD45 and CD14 mAb (Invitrogen, Carlsbad, CA, USA). The contaminated leukocytes were assessed by positive staining for CD45 or CD14 in gating of the entire platelet population. In addition, monocyte contamination was evaluated by amplification of CD14 mRNA by RT-PCR. The purified platelets were resuspended in 10 ml Tyrode-HEPES buffer or platelet activation buffer (HBSS containing 5 mM glucose, 0.1% BSA, and 10 mM HEPES, pH 7.4).
Platelet FcRI RT-PCR and cDNA sequencing
Total RNA was isolated from 109 platelets by using TRIzolTM total RNA isolation reagent (Gibco-BRL, Grand Island, NY, USA). Total RNA (1 µg) was used to synthesize cDNA with the SuperScriptTM preamplification system (Gibco-BRL). RT-PCR was carried out with the forward primer (5'-ATT GAC CAC ATG GAC GCA AAC AAG G-3' anneals between nt 298 and nt 322) and the reverse primer (5'-CCA AGA GAG CCA CGA GGA CCA GTC-3' anneals between nt 743 and nt 766) to yield a FcRI cDNA fragment (469 bp; GenBank Accession Number for FcRI: X54150). For CD14 RT-PCR, the forward primer (5'-AAA GCA CTT CCA GAG CCT GC-3') and reverse primer (5'-TCG AGC GTC AGT TCC TTG AGG-3') were used as described by Panes et al. [31
]. The PCR reaction was carried out on a GeneAmp PCR System 9700 (Applied Biosystems Inc., Foster City, CA, USA) using 2 µl cDNA (synthesized with the SuperScriptTM preamplification system), 200 nM each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 2.5 U Taq DNA polymerase in a 50-µl reaction vol starting with 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. PCR products were purified with the QIAquick gel extraction kit (Qiagen Inc., Chatsworth, CA, USA). All PCR products were sequenced in both directions on an ABI 3700 sequencer using BigDye terminator cycle sequencing kit (Version 3.1; Applied Biosystems Inc.).
Platelet activation and platelet lysate preparation
For activation experiments, platelets were suspended at the density of 2 x 109 cells/ml in ice-cold platelet activation buffer and divided into 0.5 ml aliquots. mAb A59 F(ab')2 was added at a final concentration of 10 µg/ml, where required, and all aliquots were incubated on ice for 40 min. Platelets were then centrifuged at 4°C (1 min, 2655 g) in a Beckman GS-15R centrifuge. The supernatants containing unbound antibodies were removed by aspiration, and the platelet pellets were resuspended gently in 0.5 ml ice-cold platelet activation buffer. Platelets were activated at 37°C by thrombin (1 unit/ml) plus fibrinogen (100 µg/ml) or by clustering the bound A59 F(ab')2 with goat anti-mouse IgG F(ab')2 (30 µg/ml) for the predetermined time periods. Reactions were terminated by adding an equal volume of 2x Triton X-100 lysis buffer (1% Triton X-100, 10 mM EDTA, 10 mM HEPES, pH 7.4, 2 mM PMSF, 3 mM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and incubated on ice for 30 min to ensure the complete lysis of platelets. Platelet lysates were then centrifuged at 4°C (20 min, 12,000 g) to remove cell debris. Alternatively, platelet activations were terminated by the addition of 1 ml ice-cold PBS. The platelets were pelleted with a quick spin in a centrifuge at 4°C (1 min, 2700 g).
Immunoprecipitation and immunoblotting
Nontreated and activated platelet cell lysates were immunoprecipitated at 4°C for 2 h using 2 µg appropriate antibodies conjugated to Sepharose 4B beads. Immunoprecipitates were washed three times with ice-cold 1x Triton X-100 lysis buffer and once with ice-cold PBS. Immunoprecipitated proteins on the beads were dissociated in 30 µl 6x Laemmli sample buffer. For the detection of FcRI and tissue factor (TF) in human platelets, the platelet whole cell lysates were mixed directly with 6x Laemmli sample buffer. Proteins were denatured by heating (5 min, 100°C) and separated in 12% SDS-PAGE under reducing conditions. The proteins in gels were electrotransferred to nitrocellulose membranes (Sigma Chemical Co.), which were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 1 h at room temperature and probed overnight at 4°C with appropriate primary antibodies. Blots were washed three times with PBS-T and incubated with the appropriate HRP-conjugated secondary antibody for 60 min at room temperature. Blots were washed three more times and were developed using an ECL detecting system (Pierce). In some experiments, membranes were stripped, and immunoblotting was repeated with another antibody.
Flow cytometry
Aliquots of the washed platelets or cells (106) in 0.1 ml PBS were incubated with fluorescence-labeled or unlabeled, appropriate antibodies (anti-FcRI mAb at the final concentration of 5 µg/ml) for 45 min on ice and followed by two washes. For indirect immunofluorescence staining, the platelets or cells were then incubated with saturating concentrations of FITC-conjugated goat anti-mouse IgG F(ab')2 at 4°C for 45 min. After wash, the immunofluorescence intensity of cells was analyzed immediately using a FACScan (BD Biosciences).
For IgA-binding assays, washed platelets (106) in 0.1 ml PBS were pretreated with human IgG (final concentration: 10 µg/ml) for 45 min on ice for the blockade of FcRs. Then, human serum IgA (final concentration: 100 µg/ml) was added to the platelets, which were incubated on ice for 60 min. After washing with PBS-BSA buffer (PBS containing 1.0% BSA and 0.05% NaN3), platelets were incubated with FITC-conjugated goat anti-hIgA (final concentration: 100 µg/ml) for 45 min on ice. The platelets were washed twice and analyzed by a FACScan (BD Biosciences). To determine whether hIgA binding to platelets was inhibited competitively by anti-FcRI mAb, platelets (106) were pretreated with anti-FcRI mAb MIP8a (final concentration: 50 µg/ml) or mIgG1 isotype control (final concentration: 50 µg/ml) for 60 min on ice before incubation with human serum IgA.
To examine IgA-induced platelet activation, hIgA (final concentration: 100 µg/ml) was incubated with platelets. After wash, the surface-bound IgA on platelets was cross-linked with goat anti-hIgA antibody F(ab')2 (final concentration: 80 µg/ml; Southern Biotechnologies) at 37°C for 30 min. Platelets stimulated by thrombin (1 unit/ml) were used as positive control. The surface levels of platelet CD62P were assayed with flow cytometry using a FACScan.
TF and IL-1β pre-mRNA splicing assays
To examine the TF pre-mRNA splicing, primers that target sequences in TF gene exon four (5'-CTC GGA CAG CCA ACA ATT CAG-3') and exon five (5'-CGG GCT GTC TGT ACT CTT CC-3') were used to determine endogenous splicing of TF pre-mRNA in nonstimulated and activated platelets as described by Schwertz et al. [26
]. RT-PCR of the mature TF mRNA generates a 297-bp DNA fragment, and the amplification of the unspliced TF pre-mRNA produces a 904-bp DNA fragment. To detect human IL-1β pre-mRNA splicing in platelets, primers that anneal at human IL-1β gene exon one (5'-CGA GGC ACA AGG CAC AAC AG-3') and exon two (5'-TGT AAT AAG CCA TCA TTT CAC T-3') were used. These exonic primers allow us to detect the mature IL-1β mRNA (121 bp) and the unspliced IL-1β pre-mRNA (584 bp). The One-Step RT-PCR system (SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase) was used to amplify the pre-mRNA and mature mRNA of TF and IL-1β from total RNA purified from the nonstimulated and activated platelets, according to the manufacturer’s instruction (Invitrogen).
Human IL-1β cytokine assays
The purified platelets were resuspended at the density of 5 x 108 cells/ml in RPMI-1640 culture medium without FBS. The platelets were stimulated in 24-well tissue-culture plates (Corning) coated with anti-FcRI mAb A59 F(ab')2 (20 µg/ml) or mIgG F(ab')2 (20 µg/ml), hIgA (20 µg/ml), or thrombin (1 unit/ml) plus fibrinogen (100 µg/ml). The wells were washed twice with PBS and then blocked with 5% BSA prior to the addition of 2.5 x 108 platelets (in 0.5 ml culture medium) for each well. The platelets were incubated at 37°C in 5% CO2 for various periods of time. At each time-point, the platelets in culture media were collected, and platelets were pelleted by centrifugation. The culture supernatants were collected for IL-1β cytokine determination. The levels of human IL-1β in culture medium were quantified by ELISA according to the manufacturer’s instruction (BD PharMingen).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RI in human plateletsRI in human myeloid cells is well established, and the functions of FcRI in human myeloid cells have been characterized extensively [1
]. Interestingly, polymorphisms in the FCAR gene are associated with human myocardial infarction, suggesting that FcRI may be involved in platelet functions [32
]. Therefore, we investigated whether human platelets express FcRI gene products. To rule out the contamination of PBMCs, we carried out flow cytometry assays with the purified platelets. The contamination of PBMCs was less than 1/108, as assayed by flow cytometry with anti-human CD45 (marker for hematopoietic cells), CD14 (marker for monocytes), and CD61 (marker for human platelets; data not shown). The similar degree of purity was reported by others using the same preparation procedure [30
]. In addition, we examined the presence of CD14 mRNA (a specific marker of PBMCs) in our platelet preparations by RT-PCR. CD14 mRNA was absent in our platelet preparations (data not shown), indicating that the contamination of leukocytes in platelets was negligible. Total RNA was isolated from the purified platelets, and the platelet mRNAs were transcribed reversely into cDNAs. RT-PCR was carried out to detect FcRI cDNA. We were able to detect FCAR gene products (cDNAs) in platelets from all normal, healthy donors (Fig. 1A
). The identity of FCAR cDNAs was confirmed further by direct sequencing of the cDNA products (data not shown).
 View larger version (38K): [in a new window] |
Figure 1. Expression of FcRI gene products in human platelets. (A) RT-PCR analysis of FcRI mRNA in human platelets. Total RNA was isolated from the purified platelets from six individuals (Lanes 1, 2, and 4–7) or from the peripheral blood leukocytes of a healthy donor (Lane 3*, positive control for FcRI mRNA). Platelet RNA was reversely transcribed, and FcRI cDNAs were amplified by RT-PCR. The identity of FcRI cDNA products in human platelets was confirmed further by direct sequencing. (B) Detection of FcRI on the surface of human platelets. FcRI was detected using anti-FcRI mAb MIP8a (bold line) in flow cytometry analysis by comparing with the mIgG1 isotype control (dashed line). The inset shows the staining of FcRI (bold line) compared with mIgG1 isotype control (thin line) on human neutrophils. This is representative of four independent experiments with at least four normal, healthy donors shown.
|
RI proteinRI is expressed on the surface of human platelets. Anti-FcRI mAb MIP8a was used to stain the purified human platelets. As shown in Figure 1B
, staining of platelets with anti-FcRI mAb MIP8a caused a fluorescence-intensity shift compared with mIgG1 isotype control, indicating that FcRI is expressed on the cell surface of human platelets. However, the level of FcRI expression on human platelets was much lower than that on neutrophils (Fig. 1B
, inset). Similar results were obtained with anti-FcRI mAb A77 and A59 (data not shown), further confirming the expression of FcRI on platelets. Using Western blot analysis with anti-FcRI mAb (A77, A59, and MIP8a) and rabbit pAb against the FcRI cytoplasmic domain, we detected multiple FcRI protein species in human platelet cell lysates from all healthy donors (Fig. 2
), suggesting the human platelet may express multiple splicing forms of FcRI protein in human platelets.
 View larger version (27K): [in a new window] |
Figure 2. Detection of FcRI proteins in human platelets, which were purified as described in Materials and Methods. The purified platelets (109) from three normal, healthy donors (Lanes 1–3) were lysed, and cell lysates were separated in 12% SDS-PAGE under reducing conditions. FcRI proteins were detected on four separate blots with three anti-FcRI mAb (A77, A59, and MIP8a) and rabbit pAb against the FcRI cytoplasmic domain in Western blot analysis. FcRI proteins were shown as multiple protein bands with a molecular weight (Mr) between 50 and 70 kDa. IB, Immunoblot.
|
RIRI is able to bind hIgA, we performed flow cytometry analysis. Human platelets were incubated with hIgA, and the platelet-bound hIgA was detected with FITC-conjugated goat anti-hIgA. As shown (see Fig. 3
), hIgA was able to bind platelets, and treatment of platelets with anti-FcRI mAb MIP8a completely blocked the binding of hIgA to platelets. Treatment of platelets with mIgG1 isotype control decreased (but not completely) the binding of hIgA to platelets, indicating that platelets may bind some IgA nonspecifically or may express another IgA-binding receptor (or receptors) that could be blocked by mIgG1. Taken together, our data demonstrate that FcRI is a platelet IgA receptor capable of binding hIgA. To examine whether clustering of FcRI leads to platelet activation, we carried out a stimulation experiment. Human platelets were incubated with hIgA, and hIgA on platelets was cross-linked with goat anti-hIgA. As shown in Figure 3B
, cross-linking of the IgA receptor significantly increased the platelet CD62P expression, which was comparable with the thrombin stimulation, suggesting that FcRI can serve as an activation receptor on human platelets.
 View larger version (33K): [in a new window] |
Figure 3. hIgA binds and activates human platelets. (A) Binding of hIgA to platelets, which were preincubated with anti-FcRI-blocking mAb MIP8a (final concentration: 50 µg/ml; thin line, MIP8a+hIgA), mIgG1 isotype control (final concentration: 50 µg/ml; dashed line), or without any treatment (bold line, hIgA). After washing, human platelets were incubated with hIgA (final concentration: 100 µg/ml) and followed by FITC-conjugated goat anti-hIgA antibody. Platelets stained with FITC-conjugated goat anti-hIgA antibody alone were used as autofluorescence control (shaded histogram). (B) Induction of platelet activation by hIgA. Human platelets were stimulated as described in Materials and Methods. Platelet activation was defined as an increase of CD62P expression. The results were representative of four independent experiments.
|
RI leads to Syk kinase phosphorylationRI is associated with the FcR -chain and can mediate activation in human myeloid cells [29
, 33
]. The FcR -chain is present in human platelets, where the FcR -chain is physically and stably associated with the platelet collagen receptor GP-VI and serves as a critical signaling adaptor [34
35
36
]. To determine whether the platelet FcRI is associated with the FcR -chain, we performed immunoprecipitation with anti-FcRI mAb. As shown in Figure 4A
, the FcR -chain was coprecipitated with FcRI in platelet lysates from all five normal, healthy donors, confirming that FcRI is constitutively associated with the endogenous FcR -chain in human platelets.
 View larger version (67K): [in a new window] |
Figure 4. Induction of Syk phosphorylation by clustering of platelet FcRI on human platelets. (A) Physical association between the FcRI and FcR -chain in human platelets. Freshly isolated platelets were lysed in 1x Trition X-100 lysis buffer as described in Materials and Methods. Whole platelet cell lysates were immunoprecipitated (IP) with anti-FcRI mAb A59 F(ab')2-conjugated agarose beads from five donors (Lanes 1–5) or from a donor with mIgG F(ab')2-conjugated agarose beads as control (Lane 6). Immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted with mAb MIP8a (upper panel) or anti-FcR -chain pAb (lower panel), followed by HRP-conjugated second antibody. The blots were developed using an ECL-detecting system. The results were representative of three independent experiments. (B) Clustering of FcRI induced Syk phosphorylation. Purified human platelets (109) were stimulated with anti-FcRI mAb A59 F(ab')2 plus goat anti-mouse IgG F(ab')2 for the various periods of time (in seconds) as indicated. Reactions were terminated by adding an equal volume of 2x lysis buffer, and the FcR -chain was immunoprecipitated from platelet lysates with anti-FcR -chain mAb 7D3 F(ab')2-conjugated agarose beads. The immunoprecipitated proteins were resolved in SDS-PAGE and immunoblotted with anti-Syk pAb (top panel), antiphosphotyrosine antibody 4G10 (middle panel), or anti-FcR -chain pAb (bottom panel). The results were representative of three independent experiments.
|
R on human myeloid cells mediates activation signals via the FcR -chain and induces tyrosine phosphorylation of several cellular proteins, including p72Syk, which is a major target of early protein tyrosine kinase activity [1
]. Therefore, we analyzed the Syk recruitment after cross-linking of FcRI on human platelets. The FcR -chain was immunoprecipitated with anti-FcR -chain mAb 7D3 from lysates of the stimulated or nonstimulated platelets. The FcR -chain-associated Syk was detected with anti-Syk by Western blot analysis. No increase of Syk association (with the FcR -chain) and phosphorylation was observed in the platelets stimulated with the control mIgG (data not shown). We found that cross-linking of FcRI significantly increased association of the FcR -chain with Syk (Fig. 4B
, top panel). In addition, Syk tyrosine phosphorylation was increased significantly after FcRI clustering (Fig. 4B
, middle panel), suggesting platelet FcRI could mediate activation signals through the FcR -chain.
FcRI-induced platelet TF pre-mRNA splicing and protein production
Platelet TF is a 45-kDa transmembrane cell-surface glycoprotein essential for blood clotting. TF also plays an important role in a variety of biological processes such as inflammation, angiogenesis, metastasis, and cell migration [37
]. Resting human platelets express TF pre-mRNA that can be processed (or spliced) into mature TF mRNA in response to activation [26
]. The process of TF pre-mRNA splicing is required for platelet TF protein production [26
]. To investigate whether cross-linking of platelet FcRI is able to induce TF pre-mRNA splicing, we performed RT-PCR assays. Platelet FcRI was cross-linked with anti-FcRI mAb A59 F(ab')2 plus goat anti-mouse IgG F(ab')2. As shown in Figure 5A
, there was no mature TF mRNA (297 bp) in the nonstimulated platelets. Thirty minutes after cross-linking of FcRI, mature TF mRNA was produced in the platelets along with the decreased pool of unspliced TF pre-mRNA (904 bp). Sixty minutes after the stimulation of FcRI, almost all of the TF pre-mRNA was processed into the mature TF mRNA, suggesting that cross-linking of the IgA receptor (FcRI) on platelets is a potent signal for TF pre-mRNA splicing. Similarly, cross-linking of FcRI with hIgA plus goat anti-hIgA F(ab')2 also induced TF pre-mRNA splicing (Fig. 5A)
.
 View larger version (48K): [in a new window] |
Figure 5. FcRI-mediated TF pre-mRNA splicing and protein synthesis in platelets. (A) Cross-linking of platelet FcRI induced mature TF mRNA production. Purified human platelets (5x108) were stimulated with anti-FcRI mAb A59 F(ab')2 plus goat anti-mouse IgG F(ab')2 (Anti FcRI-marked lanes), with fibrinogen plus thrombin (Fib+Thr-marked lanes), or with hIgA plus goat anti-hIgA F(ab')2 (hIgA-marked lanes) for various periods of time (in min). Nonstimulated platelets were labeled as Non. Total RNA was isolated from platelet pellets and used for RT-PCR. The lower panel shows RT-PCR results of GAPDH (internal controls for equal amount of total RNA) at each time-point. The results were representative of at least four independent experiments. (B) Induction of TF protein production by clustering of FcRI. The platelets (5x108) were stimulated in 24-well tissue-culture plates coated with hIgA, anti-FcRI mAb A59 F(ab')2 (Anti FcRI), and fibrinogen plus thrombin, respectively. The platelets were incubated at 37°C in 5% CO2 for various periods of time (hours). Platelets in culture media were collected by centrifugation at various points of time (0, 1, 5, and 20 h) and lysed in 1x radioimmunoprecipitation assay buffer. Proteins in platelet lysates were separated by SDS-PAGE, and the production of TF was analyzed on Western blots. The results were representative of three independent experiments.
|
RI leads to TF protein production. Platelet FcRI was cross-linked with plate-bound anti-FcRI mAb F(ab')2 or hIgA. As shown in Figure 5B
, stimulation of FcRI for 1 h induced the production of the TF protein in platelets. Stimulation with anti-FcRI mAb F(ab')2 (Anti FcRI) induced more platelet TF protein production as compared with the stimulation with fibrinogen plus thrombin (Fib+Thr) at 5 h and 25 h time-points (Fig. 5B)
. Similarly, stimulation of platelets with immobilized hIgA also induced TF protein production (Fig. 5B)
.
FcRI-mediated IL-1β production in human platelets
IL-1 is a potent, proinflammatory cytokine implicated in inflammation, septic shock, wound healing, and the growth of certain leukemia [38
39
40
]. Activated platelets are a rich resource of IL-1β, which constitutes a link between the coagulation and inflammatory cascades [41
]. However, platelets contain predominantly IL-1β pre-mRNA that is needed to be spliced into mature IL-1β mRNA in response to platelet activation [27
]. To examine whether cross-linking of platelet FcRI induces IL-1β pre-mRNA splicing, we carried out RT-PCR assay. Purified human platelets were stimulated with anti-FcRI mAb A59 F(ab')2 plus goat anti-mouse IgG F(ab')2 (Anti FcRI-marked lanes) or without any treatment (nonstimulated platelets). Total RNA was used for quantitative RT-PCR (Fig. 6A
). We observed that the mature IL-1β mRNA (121 bp) was increased significantly 30 min and 60 min after clustering of FcRI along with the decrease of IL-1β pre-mRNA (584 bp) compared with those in the resting platelets. Similarly, cross-linking of FcRI with hIgA plus goat anti-hIgA F(ab')2 also induced IL-1β pre-mRNA splicing (Fig. 6A)
. To learn whether clustering of platelet FcRI induces IL-1β production, we carried out ELISA assays. The platelets were stimulated with plate-bound mIgG F(ab')2, thrombin plus fibrinogen, anti-FcRI mAb A59 F(ab')2, and hIgA, respectively. As shown in Figure 6B
, immobilized anti-FcRI mAb A59 F(ab')2 and hIgA induced significantly more platelet IL-1β protein production than the immobilized mIgG F(ab')2 or fibrinogen plus thrombin (P<0.0001). Taken together, these data demonstrate that human platelet FcRI is a functional IgA receptor and that hIgA is able to activate platelets.
 View larger version (23K): [in a new window] |
Figure 6. FcRI-mediated IL-1β pre-mRNA splicing and protein synthesis in human platelets. (A) Cross-linking of platelet FcRI induced mature IL-1β mRNA production. Purified human platelets (5x108) were stimulated with anti-FcRI mAb A59 F(ab')2 plus goat anti-mouse IgG F(ab')2 (Anti FcR-marked lanes), with hIgA plus goat anti-hIgA F(ab')2 (hIgA-marked lanes) or without stimulation (lane labeled as Non). Total RNA was isolated from platelets pellets and used for RT-PCR. The results were representative of at least four independent experiments. (B) Induction of IL-1β release by clustering platelet FcRI. The platelets (2.5x108) were stimulated with plate-bound mIgG F(ab')2, thrombin plus fibrinogen, anti-FcRI mAb A59 F(ab')2, and hIgA, respectively. IL-1β in culture supernatants was quantified with ELISA at 5 h and 20 h time-points. The data represent the mean ± SEM of four independent experiments. *, Significant differences between platelets stimulated with plate-bound A59 F(ab')2 or hIgA and platelets stimulated with fibrinogen plus thrombin or mIgG F(ab')2 (P<0.0001).
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The platelets are produced by cytoplasmic budding of the bone marrow megakaryocytes. Although platelets do not contain any genomic DNA, they inherited much of cytoplasmic contents from megakaryocytes and contain large amounts of mRNA with a great capacity for protein synthesis. Platelets contain pre-mRNA for many platelet proteins including TF and IL-1β [26 , 27 , 41 ]. Activated platelets are the major source of a number of proinflammatory mediators, and platelets are considered as immune cells that play important roles in the pathogenesis of human inflammatory diseases [20 , 23 ].
Platelets express FcRs for IgG and IgE [49
, 50
]. As the sole IgG FcR expressed on human platelets, FcRIIA contributes to the pathophysiology of diseases such as heparin-induced thrombocytopenia and antibody-mediated thrombocytopenia [51
52
53
]. In the current study, we demonstrated that human platelets also express the IgA receptor FcRI, which is capable of mediating platelet activation. To our knowledge, this is the first study to document the existence of a functional IgA receptor on human platelets. Therefore, our findings fill the knowledge gap regarding the ability of hIgA to activate platelets and certainly will extend our understanding of FcRI functions in humans.
FcRI, as a multichain immune recognition receptor, can associate with the ITAM-containing FcR -chain subunit. The trimeric FcRI/(FcR -chain)2 initiates phosphotyrosine-based signaling cascades. Therefore, FcRI could serve as a typical activating and proinflammatory receptor [33
]. In this study, we observed that FcRI expressed on human platelets is constitutively associated with the FcR -chain and that clustering of FcRI induced Syk phosphorylation, suggesting that platelet FcRI may serve as an activation receptor in human platelets. Stimulation of FcRI induced TF pre-mRNA splicing and production, suggestive of a role for FcRI in thrombosis. Furthermore, clustering of platelet FcRI by hIgA and anti-FcRI mAb induced IL-1β production, signifying that FcRI is a potent proinflammatory receptor in platelets. Interestingly, cross-linking of platelet FcRI induced more TF and IL-1β protein production than the established platelet activators (fibrinogen plus thrombin; Figs. 5B
and 6B
), suggesting that IgA immune complexes may be more effective in the initiation of a proinflammation response than fibrinogen and thrombin.
hIgA plays important roles in the pathogenesis of various human inflammatory diseases. IgA autoantibodies anticardiolipin and anti-β2-glycoprotein I were the most prevalent isotypes in African American systemic lupus erythematosus (SLE) patients [54 ]. IgA anti-Ro/SSA, anti-La/SSB, anticardiolipin, and anti-β2-glycoprotein-I autoantibodies were found frequently in serum of patients with SLE and Sjogren’s syndrome [55 , 56 ]. IgA anti-β2-glycoprotein-I autoantibodies, as the most important isotype in the antiphospholipid syndrome, were associated strongly with acute myocardial infarction and unexplained, recurrent abortions in women [57 58 59 60 61 62 ]. Furthermore, a significantly increased IgA rheumatoid factor (IgA-RF; IgA autoantibodies) was found in rheumatoid arthritis (RA) patients, and the appearance of IgA-RF is a good indicator for the development of RA [63 ]. hIgA also plays important roles in the pathogenesis of other phenotypes, including dermatitis herpetiformis, celiac disease, periodontal disease, and IgA nephropathy. Recently, an association was established between FCAR gene polymorphism and myocardial infarction [32 ]. The presence of an IgA antibody against β2-glycoprotein I has been associated significantly with strokes, extensive and recurrent venous thrombosis, and coincident arterial and venous thrombosis in cancer patients [64 ]. Taken together, IgA and the receptors for IgA appear to play critical roles in the pathogenesis of inflammatory and thrombotic diseases.
Antiplatelet agents are widely used in the treatment of IgA nephropathy patients in the Japanese population, and these agents protect renal function in IgA nephropathy patients [28
]. It has even been proposed that antiplatelet agents should be used as the standard therapy for IgA nephropathy. However, the mechanism of antiplatelet agents on IgA nephropathy remains unknown [28
]. FcRI-mediated platelet activation may be one of the mechanisms behind the effectiveness of antiplatelet therapy in IgA nephropathy patients. Circulating IgA immune complexes or IgA immune complex deposits in kidneys could directly activate the platelet IgA receptor and induce proinflammatory cytokine (such as IL-1β) production in IgA nephropathy patients. As the absolute number of platelets in the circulation (
1012) is enormous, the activated platelets may produce more cytokines than other immune cells combined [65
]. Therefore, FcRI-mediated platelet activation may be a reasonable explanation for IgA-mediated human inflammatory and thrombotic diseases. One can envision that antiplatelet agents may inhibit IgA-mediated platelet activation via the FcRI in IgA nephropathy patients.
Overall, our data demonstrate that human platelets can be activated via FcRI. Therefore, platelet FcRI may play critical roles in the process of thrombosis and in the pathogenesis of human inflammatory diseases. Furthermore, our data suggest that human platelets may also express multiple FcRI isoforms (Fig. 2)
. A significant change of FcRI isoform profiles has been observed in neutrophils from pneumonia patients compared with those from healthy individuals, suggesting that the alternatively spliced FcRI isoform might have physiologic relevance in IgA-mediated host defense [15
, 66
]. It is possible that the different FcRI isoforms may have distinct functions in human platelets. The identification of platelet FcRI isoforms and the detailed signaling pathways of FcRI in human platelets are currently under the investigation.
Received May 28, 2008; revised August 14, 2008; accepted August 25, 2008.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
R and the natural killer receptors J. Biol. Chem. 274,29019-29024 receptor essential for interaction with IgA J. Immunol. 162,2146-2153RI and its complex with IgA1-Fc Nature 423,614-620[Medline] association and protects against degradation of bound ligand J. Biol. Chem. 274,7216-7225 R, CD89) transcripts in inflammatory responses FEBS Lett. 535,205-209[CrossRef][Medline]RI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity Nat. Med. 6,680-685[CrossRef][Medline]RI (CD89) alleles determine the proinflammatory potential of serum IgA J. Immunol. 178,3973-3982 chain. Molecular basis for CD89/FcR chain association J. Biol. Chem. 270,29781-29787-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen EMBO J. 16,2333-2341[CrossRef][Medline]-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets J. Biol. Chem. 272,23528-23531-chain in response to the snake venom convulxin but not to collagen J. Biol. Chem. 276,12999-13006 receptor induces noncovalent association with the tyrosine kinase p72syk J. Biol. Chem. 269,32435-32440
RI) in human platelets and its intracellular expression in human megakaryocytes Blood 93,2543-2551 RIIA in the immune clearance of platelets: a transgenic mouse model J. Immunol. 162,4311-4318RIIA Blood 98,2442-2447 receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages J. Immunol. 156,4442-4448[Abstract]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
