Departments of Microbiology & Immunology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
1 Department of Chemistry, and
2 Department of Microbiology and Immunology, Sophie Davis School of Biomedical Education, City College of New York, New York, NY 10031, USA
Correspondence to: L. Spatz
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Abstract |
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Keywords: anergy, double-stranded DNA, systemic lupus erythematosus, tolerance
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Introduction |
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Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of IgG anti-double stranded (ds) DNA antibodies. The presence of these antibodies correlates with disease activity, especially glomerulonephritis. To understand the immunological defects in SLE and the regulation of anti-dsDNA B cells, we previously generated non-autoimmune, NZW mice transgenic for the 2b heavy chain of the R4A anti-dsDNA antibody (1). The R4A anti-dsDNA antibody is encoded by an S107, V11 heavy chain gene and a V
1 light chain gene, and has been shown to deposit in glomeruli of SCID mice (2,3). We have previously demonstrated that tolerance induction is intact in NZW R4A-C
2b transgenic mice and have characterized three populations of anti-DNA B cells that are differentially regulated in these mice (1,4). There is an anergic population that secretes high-affinity anti-dsDNA antibodies after in vitro stimulation with LPS. These B cells predominantly use V
1 light chain genes which are somatically mutated. There is a second subset of B cells with high affinity for dsDNA, that is targeted to deletion but can be rescued in autoimmune, NZB/W F1 mice transgenic for R4A-C
2b and in mice transgenic for both R4A-C
2b and the proto-oncogene, bcl-2 (4,5). This population utilizes non-V
1 light chain genes. The third population is not tolerized, and produces antibodies that display low-affinity for dsDNA and utilizes a spectrum of light chain genes (6).
Other laboratories have studied the regulation of IgM anti-DNA B cells using transgenic mouse models and have observed that several mechanisms of tolerance, including anergy, deletion and receptor editing, contribute to the regulation of dsDNA binding B cells. While B cells specific for single-stranded (ss) DNA have been shown to be targeted to anergy only, those specific for dsDNA have been shown to be targeted to receptor editing, anergy or deletion depending upon the affinity of the anti-dsDNA antibody for the autoantigen (711).
We and others have demonstrated that IgG transgenic heavy chains can promote normal B cell development in mice (12,13). However, we have been concerned that the mechanisms of tolerance induction in NZW mice transgenic for R4A-C2b may not be representative of tolerance mechanisms in non-transgenic animals since the expression in naive B cells of IgG heavy chains prior to IgM heavy chains is not physiological. This study was, therefore, undertaken to generate NZW mice transgenic for the R4A-Cµ heavy chain and to determine whether tolerance is maintained in these mice in a manner similar to that observed in R4A-C
2b mice. The R4A-Cµ heavy chain utilizes a VDJ region that is identical to that used by the R4A-C
2b transgene, previously described (1). The only difference between these transgenes is that the
2b constant region has been replaced by a µ constant region in the R4A-Cµ construct. In R4A-Cµ mice the transgenic µ heavy chain can pair with a variety of endogenous light chains to produce both dsDNA binding and non-dsDNA binding antibodies.
In this study, we observe that tolerance is maintained in the R4A-Cµ mice. Moderate- to high-affinity dsDNA binding B cells persist in the periphery of these mice and like their 2b counterparts they do not spontaneously secrete antibody. These B cells display characteristics of anergic B cell including reduced surface expression of the BCR, arrested development, shortened lifespan and inability to be activated by BCR cross-linking. However, unlike their
2b counterparts they can be easily rescued by hybridoma technology in the absence of prior stimulation with lipopolysaccharide, and can be activated to differentiate and secrete antibody in vitro by T cell-derived factors. Hence there appears to be a qualitative difference in the level of anergy induced in the R4A-Cµ and the R4A-C
2b B cells which may be a consequence of differences in expression levels of the transgenes or may be due to differences in heavy chain isotypes. Nevertheless, the observation that R4A-Cµ dsDNA binding B cells are responsive to T cell-derived factors but not BCR cross-linking suggests that these B cells are partially functional and that signaling pathways leading to their activation can be uncoupled.
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Methods |
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Southern and Northern blot analysis
Tail DNA was digested with SacI, transferred to nitrocellulose after electrophoresis and hybridized with a 570-bp BamHIPstI probe containing part of the CH2 and CH3 domains of the µ constant region (15). Total RNA was isolated from the lung, kidney, heart, spleen and bone marrow of a transgenic NZW mouse and from the spleen of a non-transgenic littermate using Tri-Reagent (MRC, Cincinnati, OH). RNA was electrophoresed on a denaturing formaldehyde gel, transferred to nitrocellulose and hybridized to the Cµ probe described above or a probe specific for the S107 VH gene family (16).
ELISAs
To detect IgMa anti-dsDNA antibodies, Immulon-2, 96-well plates (Dynatech, Chantilly, VA) were coated with salmon sperm dsDNA (Calbiochem, La Jolla, CA) or calf thymus dsDNA diluted to 100 µg/ml in PBS and filtered through a 0.45 µm nitrocellulose filter (Millipore, Bedford, MA) to remove ssDNA. Plates were dried overnight at 37°C and blocked in PBS, 1.0% BSA, pH 7.2 for 2 h at room temperature. Wells were then incubated with serum samples diluted 1:100 in PBS, 0.1% BSA, culture supernatants from in vitro activation assays or hybridoma supernatants normalized to 5 µg/ml, for 2 h at 37°C. Plates were washed 6 times in PBS, 0.05% Tween 20 and then incubated with biotinylated rat anti-mouse IgMa antibody (biotinanti-mouse IgMa; PharMingen, San Diego, CA) diluted 1:200 in PBS, 0.1% BSA for 1 h at 37°C. Plates were washed again and incubated with a 1:1000 dilution of alkaline phosphatase-conjugated streptavidin (streptavidinAP) (Southern Biotechnology, Birmingham, AL) for 1 h at 37°C and then developed with p-nitrophenyl disodium phosphate as substrate (Sigma, St Louis, MO). Plates were read at 405 nm using a Titertek Multiscan ELISA reader.
Total IgMa antibodies in mouse sera and in culture supernatants were detected by ELISA. Briefly, 96-well polystyrene, Falcon plates (Becton Dickinson, Franklin Lakes, NJ) were coated at 1.0 µg/well with goat anti-mouse IgM antibody (Southern Biotechnology). Plates were blocked and washed as described above and then incubated with mouse sera or culture supernatants for 1 h at 37°C. Wells were washed again and incubated with biotinanti-mouse IgMa followed by streptavidinAP. Plates were developed with substrate solution and read at 405 nm on a Titertek Multiscan ELISA reader. IgMa antibodies in mouse sera were quantitated by ELISA, as previously described using biotinanti-mouse IgMa antibody followed by streptavidinAP and an allotype-matched standard (3).
In vitro LPS stimulation
Splenocytes isolated from five, 8- to 10-week-old NZW mice, transgenic for the R4A-Cµ heavy chain, were cultured in triplicate at 2x106cells /ml in RPMI 1640 medium (Sigma) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mM L-glutamine (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma), 100 µM non-essential amino acids (Sigma) and 50 µM ß-mercaptoethanol, in 24-well tissue culture plates with and without 10 µg/ml lipopolysaccharide (LPS) (Escherichia coli. serotype 055:B5; Sigma). After incubation for 72 h at 37°C, 5% CO2, cell supernatants were collected and assayed by ELISA for the presence of IgMa antibodies to dsDNA and for total IgMa antibodies.
In vitro activation and ELISpot
Splenocytes isolated from transgenic mice were enriched for B cells by depletion of T cells using anti-Thy-1.2 magnetic beads and MACS separation columns (Miltenyi Biotec, Auburn, CA). Enriched B cell preparations were diluted in RPMI 1640, 10% FCS, 50 µM ß-mercaptoethanol to a concentration of 2.0x106 cells /ml and incubated at 37°C, 5% CO2 in the presence of either 20 µg/ml of LPS, 10µg/ml F(ab')2 fragment of goat anti-mouse IgM (ICN, Cappel, Aurora, OH), 10 µg/ml of antibody to CD40 (PharMingen, San Diego, CA), 300 U of recombinant mouse IL-4 (rIL-4) (PharMingen), 10 µg/ml of anti-CD40 plus 300 U of rIL-4, 200 U of rIL-5 (PharMingen), 300 U of rIL-6 (PharMingen) or a cocktail of rIL-4, rIL-5 and r-IL-6. After 48 h in culture, cells were harvested and plated on Immulon-2 plates coated with 100 µg/ml of salmon sperm dsDNA or on Falcon plates coated with a 1:1000 dilution of anti-IgM antibody (Southern Biotechnology). After 6 h, plates were washed 5 times with 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.2 and then biotinanti-mouse IgMa diluted 1:300 in PBS, 0.1% BSA was added to wells and plates were incubated overnight at 4°C. Plates were washed again and then incubated with a 1:1000 dilution of streptavidinAP for 3 h at room temperature. 5-Bromo-4-chloro-3-indolyl phosphate (Sigma) was added as substrate and plates were allowed to develop at room temperature for 24 h. Antibody-secreting cells were counted under a dissecting microscope.
Flow cytometry
Single-cell suspensions were prepared from the bone marrow and spleen of transgenic mice and non-transgenic littermates, and were depleted of red blood cells by ammonium chloride lysis. Cells were resuspended at a concentration of 1.0x106 cells/0.1ml of PBS, 1% BSA and were treated with Fc receptor block (CD16/CD32) (PharMingen) for 5 min on ice. Cell suspensions were then surface stained by incubation with a combination of antibodies for 30 min at 4°C followed by 2 washes with PBS, 1% BSA. The following antibodies were used: CyChromeanti-B220, FITCanti-CD24 (PharMingen), FITCanti- (Southern Biotechnology), phycoerythrin (PE)anti-IgMa and FITCanti-IgMb (PharMingen). In some experiments biotinanti-mouse IgMa or biotinanti-mouse IgMb (PharMingen) followed by streptavidinPE (Southern Biotechnology) were used. All samples were analyzed on a Coulter dual laser cytometer using Elite software. Gating was set for live lymphocytes based on forward and side scatter, and 50,000 events were collected for each sample. Analysis was performed using WinMDI, version 2.8 (Scripps Institute). For in vitro activation studies, splenocytes were enriched for B cells by depleting T cells by magnetic bead cell sorting using anti-thy1.2 microbeads (Miltenyi Biotec, Auburn, CA).
5-Bromo-2'-deoxyuridine (BrdU) labeling
BrdU labeling was according to Mandik-Nayak et al. (17). Briefly, NZW mice transgenic for R4A-Cµ or R4A-C2b or wild-type control NZW mice were injected i.p. every 12 h for 8 days with 200 µl of 3 mg/ml BrdU (Sigma). Splenocytes were isolated on day 8 and surface stained with CyChromeanti-B220 and either biotinanti-IgMa or biotinanti-IgMb followed by streptavidinPE. Cells were then fixed and permeabilized in 1% paraformaldehyde plus 0.1% Tween 20, and DNA was denatured in 0.14M NaCl, 4.2 mM MgCl2 buffer containing 10 µM HCl and 100 U/ml of DNase I. Cells were then stained with FITC-labeled anti-BrdU (Becton Dickinson) in order to detect BrdU incorporation and analyzed by flow cytometry. Gates were set on B220+, IgMa or B220+, IgMb and percent FITC staining was displayed as a histogram.
Generation of hybridomas
Spleen cells derived from (six) 8- to 10-week-old transgenic R4A-Cµ, NZW mice were fused with NSO cells as described previously (12).
Analysis of V gene expression
Hybridomas were screened for expression of V1 genes by RNA dot-blot, as previously described (12). A probe specific for the mouse V
1 gene family was used. Sequencing the variable regions of V
light chain genes was according to Spatz et al. (4).
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Results |
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Assays for serum Ig
Serum from 10, 7- to 12-week-old transgenic NZW mice and 10 non-transgenic NZW littermates were assayed for IgMa anti-dsDNA binding by ELISA (Fig. 2A). Since the transgene is of the a allotype and endogenous IgM in the NZW mouse strain is of the b allotype, transgenic IgM could be distinguished from endogenous IgM using an anti-IgM antibody specific for the a allotype. A purified monoclonal IgMa anti-dsDNA antibody (4D4) that utilizes the R4A heavy chain was used at a concentration of 1 µg/ml, as a positive control for dsDNA binding.
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Characterization of transgenic B cells by flow cytometry
Bone marrow and splenic B cells from transgenic mice and non-transgenic littermates were examined by flow cytometry. By staining bone marrow and splenic cells with CyChromeanti-B220 only or in combination with PEanti-IgMa and FITCanti-IgMb we determined the frequency of total B cells, B cells expressing the transgene (IgMa) and B cells expressing endogenous IgM (IgMb). Analysis of B220+ gated B cells revealed that the majority of transgene-expressing B cells in the bone marrow and spleen express IgMa only and not endogenous IgMb. This demonstrates that the transgene maintains intact allelic exclusion (Fig. 3A). The splenic B cells that fail to surface stain with PEanti-IgMa or FITCanti-IgMb express IgG heavy chains and none of the IgG B cells co-express IgMa (data not shown).
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Density of receptor
Studies by Goodnow et al. demonstrated that in mice transgenic for soluble hen egg lysozyme (HEL) and antibody to HEL, tolerant HEL binding B cells exhibited an altered phenotype characterized by a reduction in membrane IgM (20). To determine whether a subset of B cells in our transgenic mice also exhibited this receptor down-modulation, we stained splenocytes from transgenic and non-transgenic littermates with CyChromeanti-B220, FITCanti- and either biotinanti-mouse IgMa or biotinanti-mouse IgMb respectively, followed by streptavidinPE. Cells were gated on B220+, IgMa cells (transgenic mice) or B220+, IgMb cells (non-transgenic mice), and mean fluorescent intensity of
staining was compared for IgMa and IgMb B cells (Fig. 4
). A subset of transgene-expressing B cells displayed reduced expression of IgMa relative to IgMb, while another subset displayed a level of expression of IgMa comparable to that of IgMb. We speculate that the transgenic B cells expressing a reduced level of IgM are dsDNA binders while those expressing levels of IgM similar to endogenous B cells are non-dsDNA binders.
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It has been reported in some transgenic mouse models that anergic B cells can be induced to differentiate and secrete antibody in response to CD40L plus IL-4 while others have reported that B cells can proliferate but not secrete antibody under the same conditions (22,2527). We wished to determine whether anti-CD40 plus IL-4 could induce antibody secretion by dsDNA binding B cells from our transgenic mice. We, therefore, cultured T cell-depleted splenocytes for 48 h in the presence of antibody to CD40 and rIL-4. We observed by ELISpot a significant increase in the number of transgenic B cells secreting anti-dsDNA antibody, comparable to that observed with LPS (Fig. 6). Based on these results we also sought to determine the affect of incubating splenic B cells with a cocktail of T cell cytokines. We observed that a combination of rIL-4, rIL-5 and rIL-6 could also activate IgMa dsDNA binding B cells to differentiate and secrete anti-dsDNA antibody (Fig. 6C
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Lifespan measurement
Anergic B cells have been shown to have a shortened lifespan (17,28). We assessed the lifespan of B cells expressing the R4A-Cµ transgene relative to B cells expressing endogenous IgM. B cells from transgenic mice and non-transgenic littermates were continuously labeled with BrdU for 8 days, and the incorporation of BrdU was measured by flow cytometry. Populations that are rapidly turning over and have a reduced lifespan are replaced more rapidly by labeled cells from the bone marrow, and this is reflected as a higher percentage of cells that have incorporated BrdU. In three out of three experiments we observed that IgMa B cells have a slightly higher incorporation of BrdU relative to non-transgenic B cells, suggesting a reduced lifespan (Table 2).
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Surprisingly, we were also able to generate hybridomas from unstimulated, naive splenocytes from R4A-Cµ transgenic mice. Approximately 560 wells from six fusions showed growth of hybridomas. Seventeen of these clones (3%) produced high-affinity IgMa anti-dsDNA antibody. Ten of them were cloned for further analysis. These results indicate that the high-affinity anti-dsDNA B cells present in the R4A-Cµ transgenic mice can form viable hybridomas even without prior LPS activation. Thus, they differ from R4A-C2b anti-DNA B cells which can only be immortalized as hybridomas following activation with LPS. By ELISA we demonstrated that anti-dsDNA antibodies produced by hybridomas obtained from either unstimulated or LPS-stimulated splenocytes show a similar binding to dsDNA (Fig. 7
). We also measured affinities of anti-dsDNA antibodies obtained from both LPS-stimulated and unstimulated R4A-Cµ B cells by inhibition ELISA, and observed that all antibodies have similar apparent affinities for dsDNA ranging from 107 to 108.
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Ten of 11 antibodies produced by LPS-activated B cells utilize the V1-A gene and one utilizes the V
1-C gene. Only two V
1-A genes are mutated; one contains a somatic mutation in FR3, resulting in the substitution of a phenylalanine for a serine and one contains a substitution at the VJ joining region resulting in the replacement of a leucine with a proline. This latter change may not be the result of mutation in the periphery, but a consequence of rearrangement of the V J regions in the bone marrow. The V
1-C gene is unmutated. Seven of nine light chains sequenced from the antibodies obtained from unstimulated anti-dsDNA B cells utilize the V
1A light chain gene. One of the V
1A genes contains a substitution in the VJ joining region which results in the replacement of a tryptophan with a proline. Two of the light chains utilize non-V
1 genes (V
21-E and V
OX-1), neither of which is mutated. All sequences have been reported to GenBank.
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Discussion |
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It is intriguing that high-affinity, IgMa V1 dsDNA binding B cells can be rescued as hybridomas from unstimulated splenocytes since earlier studies demonstrated that high-affinity, IgG2b, V
1 dsDNA binding B cells could only be rescued if B cells were activated with LPS prior to fusion (4,12). It is also interesting that IgM dsDNA binding B cells can be activated to secrete antibody following in vitro stimulation with anti-CD40 plus IL-4 since recent observations revealed that IgG2b dsDNA binding B cells from R4A-C
2b mice cannot be activated to secrete antibody under these conditions (data not shown). The IgM dsDNA binding B cells may be partially functional relative to their IgG2b counterparts and in a weaker stage of anergy, making them more conducive to fusion and more responsive to T cell-derived factors. While qualitative differences in anergy in dsDNA bindng R4A-Cµ and R4A-
2b B cells may be due to differences in the expression level of the transgenes, we speculate that differences in heavy chain isotype may play a role as well and
heavy chains may transduce qualitatively different signals leading to tolerance induction than µ heavy chains.
Although anergy is defined as functional unresponsiveness to antigen, the characteristics of anergic B cells seem to vary in different transgenic models. Goodnow et al. first demonstrated that in mice transgenic for soluble HEL and antibody to HEL, anergic HEL binding B cells display reduced levels of surface Ig known as receptor down-modulation and have a shortened lifespan but are not arrested in development (28,29). They also showed that anergic HEL B cells can be induced to proliferate and differentiate into antibody-secreting cells, although at suboptimal levels following in vitro activation with LPS or CD40L plus IL-4 and IL-5 as well as with an antisera to CD40 plus IL-4 (22,25,30).
Erikson and colleagues observed differences in the characteristics of anergic ssDNA and dsDNA binding B cells (17,18,26,27). They observed that anergic ssDNA binding B cells cannot be induced to secrete antibody following BCR cross-linking; however, they can be induced to proliferate following LPS stimulation or cross-linkage with CD40L plus IL-4. Furthermore, they observed that these B cells have a normal lifespan and are not arrested in development. In contrast, they observed that dsDNA binding B cells have a shortened lifespan and are arrested in development, and display an antigen-experienced phenotype. In addition, they do not proliferate in response to LPS or BCR cross-linking and do not differentiate into antibody secreting cells in response to CD40L plus IL-4, although they do proliferate in response to these T cell factors (17,26,27).
Our studies demonstrate a phenotype that differs from that described above for anergic ssDNA and dsDNA binding B cells. While the dsDNA binding B cells in our transgenic mice show evidence of receptor down-modulation, arrested development, shortened lifespan and inability to be activated to secrete antibody following BCR cross-linking, they can be activated to secrete antibody following incubation with LPS or T cell-derived factors. These results are significant because the vulnerability of some `anergic' B cells to non-antigenic stimuli may provide a pathway to a breakdown in tolerance.
Differences in the avidity of interaction between autoantibodies and their autoantigen which reflects receptor affinity, surface density, and concentration of the autoantigen and differences in fine specificity, and the nature of the autoantigen may account for the range of phenotypes observed in anergic B cells. Just as the extent of receptor cross-linking determines whether autoreactive B cells are anergized, deleted or ignored, the strength and/or nature of antigenantibody interactions may determine qualitative differences in anergy (31,32).
The observation that R4A-Cµ dsDNA binding B cells are partially functional and can respond by antibody secretion to T cell-derived factors and LPS but not BCR cross-linking suggests an uncoupling of signaling cascades. Signaling pathways transduced via the BCR, CD40 ligation or LPS activates the transcription factor, NF-B (33 ,34). It is not clear whether these signaling pathways are distinct or have common intermediates upstream of NF-
B. Protein kinase C (PKC) has been shown to be necessary for BCR but not CD40 or LPS-induced activation of NF-
B while phosphoinositide 3' kinase (PI-3 kinase) has been shown to be necessary for both BCR- and LPS-induced activation of NF-
B (3335). Depletion or inhibition of a molecule unique to one pathway may have little affect on the other pathways. This could explain why a block in BCR signaling could be overcome by signaling through CD40 ligation or LPS. Inhibition of a factor common to all three pathways would prevent signaling following BCR cross-linking as well as CD40 ligation and LPS. Therefore, qualitative differences in anergy may be the consequence of blocking one or more signaling pathways. A paradigm for the uncoupling of signaling pathways comes from recent studies in B and T cells. Okkenhaug et al. demonstrated that a point mutation in the co-stimulatory molecule CD28 uncouples signals required for T cell proliferation and survival (36). Bone and Williams demonstrated that an inhibitor of PKC uncouples BCR signaling from signaling through LPS by blocking a PI-3 kinase-dependent pathway to NF-
B activation that is distinct for BCR signaling (35). Future studies will be aimed at determining mechanisms by which BCR uncoupling may be occurring in anergic R4A-Cµ dsDNA binding B cells.
In summary, this study describes a novel phenotype for dsDNA binding B cells; one in which B cells can be activated to differentiate and secrete antibody in response to T cell-derived factors or LPS, but are unresponsive to signaling through the BCR. The persistence of these `partially functional' B cells due to the uncoupling of the BCR signaling pathway from other signaling pathways may offer the immune system a protective advantage by enabling B cells cross-reactive to both self and foreign antigen to be down-regulated in response to autoantigen while still being available for recruitment in an inflammatory response.
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Acknowledgments |
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Abbreviations |
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AP alkaline phosphatase |
BrdU 5-bromo-2'-deoxyuridine |
CD40L CD40 ligand |
ds double stranded |
HEL hen egg lysozyme |
LPS lipopolysaccharide |
PE phycoerythrin |
PI-3 kinase phosphoinositide 3' kinase |
PKC protein kinase C |
SLE systemic lupus erythematosus |
ss single stranded |
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Notes |
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Transmitting editor: J. V. Ravetch
Received 6 July 2001, accepted 3 October 2001.
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References |
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