Expression of recombination activating genes 1 and 2 in peripheral B cells of patients with systemic lupus erythematosus.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 5, May 2002, pp 1255–1263 DOI 10.1002/art.10264 © 2002, American College of Rheumatology Expression of Recombination Activating Genes 1 and 2 in Peripheral B Cells of Patients With Systemic Lupus Erythematosus Hermann J. Girschick,1 Amrie C. Grammer,2 Toshihiro Nanki,2 Eduardo Vazquez,2 and Peter E. Lipsky2 Objective. To analyze immunoregulatory abnormalities in patients with systemic lupus erythematosus (SLE) by assessing the expression of messenger RNA (mRNA) for types 1 and 2 recombination activating genes (RAG) in the peripheral blood of patients with active SLE. Methods. We examined B cell populations and also individual B cells from patients with SLE for the expression of RAG mRNA. Results. Analysis of bulk mRNA indicated that RAG1 and RAG2 mRNA were found routinely in peripheral B cells of patients with active SLE, but not in healthy subjects. When assessed on a single-cell basis, there was a 3-fold increase in the frequency of RAG1and RAG2-expressing B cells in SLE patients compared with healthy subjects. Notably, B cells expressing both RAG1 and RAG2 mRNA expressed only IgD mRNA, but not IgG mRNA. Fifty percent of RAG-expressing B cells also expressed VpreB mRNA, whereas all expressed CD154 mRNA. Phenotypic analysis indicated that RAGexpressing B cells were activated, mature B cells. Conclusion. These results indicate that RAG expression is up-regulated in peripheral IgDⴙ and VpreBⴙ B cells of patients with active SLE. These cells may contribute to the immunoregulatory abnormalities in patients with SLE. Numerous aberrations of the immune system have been reported in systemic lupus erythematosus (SLE) and have been implicated in the pathogenesis of this autoimmune disease (1,2). Included in these abnormalities are immune-cell signaling defects resulting in hyperactivity of B and T cells (3), hypomethylation of DNA leading to abnormal autoreactive B cells (4), and changes in the immunoglobulin repertoire of lupus B cells leading to an increased number of B cell–producing autoantibodies (5). In addition, enhanced mutational activity of immunoglobulin genes has been implicated in the pathogenesis of SLE (6–8). Although the causes of autoantibody production have not been completely delineated, it has been suggested that one contributing feature might be a failure of editing or revision of autoreactive B cell receptors (BCRs) (5,9). On the other hand, recent data suggest that receptor editing/revision might be enhanced in patients with SLE (10,11). V–D–J rearrangement of immune-receptor genes is a feature of developing lymphocytes in the bone marrow (12) and depends on recombination activating gene (RAG) enzymes (13). RAG enzymes are expressed at high levels during ontogeny, but their expression diminishes in immature B cells and is usually absent in recirculating, mature, naive B cells (14,15). However, we (16) and others have demonstrated a marked reexpression of the RAG-encoding endonucleases RAG1 and RAG2 during germinal center reactions in secondary lymphoid organs (17–28). This has led to the proposal that V–D–J editing/revision in secondary lymphoid organs might be a mechanism to rescue B cells whose antigen receptor avidity has been decreased as a result of somatic hypermutation or to rescue B cells in which somatic mutation has generated autoreactivity (8,21). In mice, in contrast to humans, peripheral transcription of Supported by NIH grant AI-31229. Dr. Girschick is a recipient of a Deutsche Forschungsgemeinschaft grant, Gi-295/1-1. Dr. Grammer’s work is supported by a grant from the Arthritis Foundation. 1 Hermann J. Girschick, MD (current address: Children’s Hospital, University of Wuerzburg, Wuerzburg, Germany): University of Texas Southwestern Medical Center, Dallas; 2Amrie C. Grammer, PhD, Toshihiro Nanki, MD, Eduardo Vazquez, BS, Peter E. Lipsky, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland. Drs. Girschick and Grammer contributed equally to this work. Address correspondence and reprint requests to Peter E. Lipsky, MD, Scientific Director, NIAMS, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 9N228, Bethesda, MD 20892-1820. E-mail: firstname.lastname@example.org. Submitted for publication October 22, 2001; accepted in revised form January 11, 2002. 1255 1256 GIRSCHICK ET AL RAG genes is strictly limited and found only in B cell precursor cells and transitional cells that have left the bone marrow and were completing maturation in the spleen, but not in peripheral blood or other lymphoid organs (29,30). Thus, the potential for revision of a BCR seems to be tightly regulated in normal animals, and might be different in mice and humans. In humans, small populations of normal B cells in the peripheral blood have been reported to express RAG messenger RNA (mRNA) (16,31). We have reported that RAG-expressing peripheral B cells are postswitch IgG⫹ B cells (16), whereas others have reported that these B cells also express the surrogate light chain VpreB component (31). Some of these VpreB⫹ B cells might be recent bone marrow immigrants still expressing RAGs and VpreB, as has been demonstrated in the mouse (29,30). Alternatively, reexpression of the surrogate light chain has recently been demonstrated in tonsillar germinal center B cells, suggesting a physiologic role of VpreB in peripheral B cell development (16). Consistent with this conclusion is the recent report of VpreB expression in CD27⫹ memory B cells with mutated IgVH genes (31), consistent with the conclusion that this molecule might be up-regulated during activation of human memory B cells. RAG-mediated receptor editing/revision has been documented as an important means to avoid autoimmunity in transgenic animals (27,32–36). Similarly, enhanced peripheral receptor editing/revision has been reported to occur in patients with active SLE as an attempt to avoid autoimmunity (8,11). Whether overexpression of RAG is associated with enhanced peripheral receptor editing/revision in patients with active SLE remains to be determined. In order to investigate whether there is abnormal RAG expression in patients with active SLE, RAG mRNA expression in peripheral blood B cells of SLE patients was investigated. Compared with healthy subjects, a 3-fold increase of RAG1- and RAG2-mRNA– expressing B cells was documented in SLE patients. In contrast to healthy subjects, who expressed RAGs in postswitch memory B cells, SLE patients expressed RAGs in IgD⫹ preswitch, mature B cells that exhibited an activated, mature phenotype. PATIENTS AND METHODS Preparation of B cells from peripheral blood. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation from the heparinized peripheral blood of 9 patients with active SLE and 14 healthy donors. The patients fulfilled the revised criteria for classification of SLE (37). Because of the small amount of blood available from patients with active SLE, B cells were directly purified by flow cytometric cell sorting. To accomplish this, B cells from the SLE patients (n ⫽ 9) were stained for surface expression of CD19 using a biotinylated anti-human CD19 mononuclear antibody (mAb) (Coulter, Miami, FL) followed by Streptavidin-RED670 (Life Technologies, Gaithersburg, MD) or antigen-presenting cell (APC)–conjugated anti-CD19 mAb (Becton Dickinson, San Jose, CA), and were isolated from PBMCs with a FACStarPlus or FACS Vantage flow cytometer (Becton Dickinson). Postsort analysis of isolated B cell populations revealed a purity of ⬎98%. Single-cell sorting. Individual CD19⫹ B cells of SLE patients 4, 6, 7, and 8 were sorted into 96-well polymerase chain reaction (PCR) plates (Robbins Scientific, Sunnyvale, CA) using a FACStarPlus flow cytometer (Becton Dickinson) outfitted with a single-cell deposition unit, as described previously (38). In some cases, cells were sorted in bulk and then deposited manually by limiting dilution. Preparation of RNA and complementary DNA (cDNA) from sorted bulk populations. Total RNA was extracted using the RNeasy RNA isolation kit (Qiagen, Chatsworth, CA). From each patient, 0.1 ⫻ 106 sorted B cells were used for RNA preparation. Contaminating genomic DNA was removed using RQ1 RNase-free DNAse according to the manufacturer’s instructions (Promega, Madison, WI). For conversion of mRNA into cDNA, Superscript II RNase H–reverse transcriptase (RT) (Life Technologies) was used according to the manufacturer’s instructions. One sample of each RNA was prepared by omitting the RT, and this was used as a control. In addition, RNA preparations from synovial fibroblasts were used as negative controls. The latter did not yield a product when amplified for RAG mRNA. Preparation of RNA and cDNA from sorted single cells. Five microliters of lysis solution (1 l of 5⫻ first-strand buffer B [Life Technologies], 0.01M dithiotureitol [Life Technologies], 1% Nonidet-P40 [Sigma, St. Louis, MO], 5 l of recombinant RNasin ribonuclease inhibitor [Promega], 800 M each of dATP, dCTP, dGTP, and dTTP [Sigma], 0.05 g oligod(T)12-18 [Pharmacia Biotech, Piscataway, NJ]) were added into each well of the PCR plate before sorting individual cells into the wells. The conversion of mRNA from a single cell was carried out with Superscript II RNase H–RT (Life Technologies) as described previously (16,39). PCR amplification. The relative amount of RAG1-, RAG2-, TdT-, IgD-, IgG-, VpreB-, 14.1-, and CD154-encoding cDNA in the different samples was determined by amplification of ␤-actin–adjusted samples using the PCR Southern technique (16). The primer pairs used are listed in Table 1. In the first round of PCR, 35 cycles of 30 seconds at 94°C, 1 minute at 60°C, and 2 minutes at 72°C with a final 5-minute extension at 72°C were carried out. A second nested step of PCR was performed in the analysis of cDNA generated from single cells. Five microliters of the first PCR product was subjected to nested PCR amplification, using primers as indicated in Table 1. For analysis of human RAG2 mRNA, two different 5⬘ primers for the alternative exon 1A and exon 1B RAG1 AND RAG2 EXPRESSION IN SLE Table 1. DNA 1257 Sequences of oligonucleotides used as primers for the amplification of complementary ␤-actin Sense Antisense Nested sense Nested antisense RAG1* Sense Antisense Nested sense Nested antisense RAG2* Exon 1A sense Exon 1B sense Antisense Nested sense Nested antisense TdT Sense Antisense VpreB Sense Antisense Nested sense Nested antisense 14.1 Sense Antisense Total IgD Sense Antisense Nested sense Nested antisense Total IgG Sense Antisense Nested sense Nested antisense 5⬘GTCCTCTCCCAAGTCCACACA3⬘ 5⬘CTGGTCTCAAGTCAGTGTACAGGTAA3⬘ 5⬘GTCCTCTCCCAAGTCCACACA3⬘ 5⬘CTCAAGTTGGGGGACAAAAAG3⬘ 5⬘GAGCAAGGTACCTCAGCCAG3⬘ 5⬘AACAATGGCTGAGTTGGGAC3⬘ 5⬘TTCTGCCCCAGATGAAATTC3⬘ 5⬘TGACCATCAGCCTTGTCCAG3⬘ 5⬘GCAGCCCCTCTGGCCTTC3⬘ 5⬘GCGGTCTCCAGACAAAAATC3⬘ 5⬘TTTCAGACTCCAAGCTGCCT3⬘ 5⬘TCTCTGCAGATGGTAACAGTCAG3⬘ 5⬘AGCGAAGAGGAGGGAGGTAG3⬘ 5⬘ACTTGAGCCCTCGGAAGAAG3⬘ 5⬘TTCCCTGCTCCTATGCATTC3⬘ 5⬘TGCACAGTTGTGGTCCTCAG3⬘ 5⬘TCTCCCTCTCCTCCTTCTCC3⬘ 5⬘AGTTGTGGTCCTCAGCCG3⬘ 5⬘GATGTCATGGTCGTTCCTCA3⬘ 5⬘GTAACCCATGGCCTGCTG3⬘ 5⬘CGCGTACTTGTTGTTGCTCT3⬘ 5⬘GGTACATGGGGACACAGAGC3⬘ 5⬘CTGCAGGGGTTAGCAGGTAG3⬘ 5⬘ACCGCCAGCAAGAGTAAGAA3⬘ 5⬘CTGCAGGGGTTAGCAGGTAG3⬘ 5⬘GCTGCCTGGTCAAGGACTAC3⬘ 5⬘CATCACGGAGCATGAGAAGA3⬘ 5⬘TTCCCCCCAAAACCCAAGGA3⬘ 5⬘CATCACGGAGCATGAGAAGA3⬘ * RAG ⫽ recombination activating gene. were used. The RAG1 sense primer was partly spanning the intron, since 7 nucleotides lay in exon 1 and 15 nucleotides in exon 2. The RAG2-1A sense primer completely spanned the intron, and the RAG2-1B primer partly spanned the intron, since 15 nucleotides lay in exon 1 and 8 nucleotides in exon 2. The same primers have been used previously and their specificity for RAG mRNA documented (16). Control samples of each preparation prepared without RT were run in parallel and were negative for the presence of contaminating DNA. PCR reactions without added cDNA were also run in parallel and did not yield a product. In addition, analysis of cDNA from individual synovial fibroblasts did not yield a product with RAG-specific primers. Thus, RAG amplification from genomic DNA was excluded. Detection of amplified cDNA by PCR Southern analysis. Quantification of cDNA in the different bulk samples was carried out as previously described (16). Initially, PCR amplification for the housekeeping gene ␤-actin was carried out in triplicate using 20, 25, 30, and 35 PCR cycles. RT-PCR products were analyzed on a 1.8% agarose gel and transferred to a nylon membrane by alkaline vacuum transfer (Bio-Rad, Hercules, CA). PCR Southern blots were incubated in hybridization buffer containing ␥32P-labeled probes for ␤-actin PCR products, as listed in Table 2. The amount of hybridized probe for the housekeeping gene was then quantified by PhosphorImager. The linear range of PCR was determined by plotting the amount of PhosphorImager counts (above background levels) of the hybridized probe as a function of the number of Table 2. Sequences of oligonucleotides used for the detection of polymerase chain reaction products by Southern blotting ␤-actin RAG1* RAG2* TdT VpreB 14.1 IgD IgG CD154 5⬘CTCAAGTTGGGGGACAAAAAG3⬘ 5⬘TCTCTGGAGCAATCTCCAGCA3⬘ 5⬘TTCCATCTGGATGTAAAGCAT3⬘ 5⬘TCCAAAATGAAGACGACCAA3⬘ 5⬘CTTGGAACCACAATCCGC3⬘ 5⬘AAGGCTACGCTGGTGTGTCT3⬘ 5⬘CCTGATGTGGCTGGAGGACCA3⬘ 5⬘GGGTGTACACCTGTGGTTCT3⬘ 5⬘TTATGAGGAGTGGGCAGGCTCAG3⬘ * RAG ⫽ recombination activating gene. 1258 GIRSCHICK ET AL cycles. The mean counts of these triplicates in the linear range of PCR amplification were calculated and were used to calculate an amount of cDNA (in l) of each preparation that contained the same relative content of housekeeping gene cDNA. This calculated volume of cDNA was subjected to further PCR amplification for specific cDNA (40). The amplified PCR products were again analyzed by agarose gel electrophoresis and transferred to a nylon membrane and blotted. The number of PCR cycles in which the PCR product was amplified in a linear range was again determined for each primer set, as described above for housekeeping genes. After hybridization using specific probes (Table 2), the amount of hybridized probe was quantified by PhosphorImager and visualized by exposure of the hybridized nylon membrane to a photographic emulsion. Flow cytometric analysis. Subsets of peripheral blood B cells were delineated by staining PBMCs for 30 minutes on ice with mAb specific for CD19 (phycoerythrin [PE]– labeled [Becton Dickinson]; Tri color–labeled [Caltag, South San Francisco, CA]), IgD (fluoroscein isothiocyanate–labeled [Caltag]; biotinylated [PharMingen, San Diego, CA]; followed by streptavidin PE [Becton Dickinson]), IgM (PE-labeled [Caltag]), CD154 (PE-labeled [Becton Dickinson]) (41), CD69 (PE-labeled [Becton Dickinson]), and CD38 (APC-labeled [Becton Dickinson]). Isotype-matched antibodies were used as controls. Analysis was performed with a FACS Calibur flow cytometer using CellQuest and Paint-a-Gate software (Becton Dickinson). RESULTS RAG1, RAG2, TdT, VpreB, 14.1, and CD154 mRNA expression in bulk cDNA from peripheral B cells of SLE patients. RAG1 mRNA was uniformly expressed in B cell populations from all patients with active SLE (Figures 1A and B). RAG2 mRNA was found in 5 of 9 patients with active SLE. Using the same PCR technique, RAG1 mRNA expression was not detectable in peripheral blood B cells from 14 normal subjects, whereas RAG2 mRNA was found in only 3 of 14 normal donors (16). One of 9 SLE patients exhibited TdT mRNA expression, whereas 6 of 9 SLE mRNA preparations were positive for VpreB mRNA, and 4 of 9 SLE samples were positive for the surrogate () light chain constant region encoding human gene 14.1. By comparison, normal B cell populations were rarely positive for TdT, VpreB, and 14.1 mRNA (Table 3). CD154 expression has been demonstrated by normal tonsil B cells, B cell lines, and activated peripheral blood B cells and has been implicated in homotypic B cell activation during germinal center B cell development (41–42). Moreover, CD154 has been reported to be expressed by circulating B cells of patients with active SLE (43,44). Therefore, we analyzed the current group of patients with active SLE for B cell Figure 1. A, Expression of types 1 and 2 recombination activating genes (RAG1 and RAG2), TdT, surrogate light chain (VpreB and 14.1), and CD154 mRNA in peripheral B cells of 9 patients with active systemic lupus erythematosus (SLE). CD19⫹ peripheral B cells from the peripheral blood of 9 patients with active SLE and from 14 healthy donors were sorted with a FACS Vantage flow cytometer. Specific mRNA for RAG1, RAG2, TdT, surrogate light chain (VpreB and 14.1), and CD154 were amplified as described in Patients and Methods, and reverse transcriptase–polymerase chain reaction (RT-PCR) products were detected by Southern hybridization. As positive controls, extractions of mononuclear cell mRNA were isolated from the thymus (lane A), from the peripheral blood of a preB-leukemia patient (lane B), and from a tonsil (lane C). RT-PCR using “RT⫺ only” controls (lanes D and E) and a “no cDNA” control (lane F) was performed for all samples and did not yield a product. B, The amount of hybridized RAG1 and RAG2 probe in samples from SLE patients and healthy individuals was quantified by PhosphorImager and normalized against the amount of probe detected in human tonsil, with results shown as the percentage of expression. expression of CD154. Notably, 7 of 9 SLE B cell samples exhibited CD154 mRNA expression. RAG1, RAG2, IgG, and IgD mRNA expression by individual peripheral B cells of SLE patients. Analysis of individual peripheral blood B cells from 3 SLE RAG1 AND RAG2 EXPRESSION IN SLE 1259 patients (patients 4, 6, and 8) indicated that variable percentages (mean 23%) of cells in each patient expressed RAG1 mRNA (Figure 2). The frequency of RAG2 mRNA–expressing B cells ranged from 17% to 48% (mean 32%). Only the coordinate action of RAG1 and RAG2 together can lead to recombination of the BCR (45). Therefore, the frequency of B cells expressing RAG1 plus RAG2 mRNA was analyzed. As shown in Figure 2, 3–10% of individual SLE B cells manifested coordinate RAG1 and RAG2 mRNA expression (mean 8%), a frequency that was higher than in CD19⫹ B cells of healthy individuals (⬍3%). To assess the stage of maturation of the B cells expressing RAG1 plus RAG2 mRNA, IgG and IgD mRNA were examined in these individual cells to determine whether they were pre- or postswitch B cells. Few IgG mRNA–expressing cells were detected in SLE patients 4, 6, and 8, in contrast to an abundance of IgD mRNA–expressing cells (mean 44% of ␤-actin– positive cells). This predominance of IgD mRNA– expressing B cells was consistent with the expression of surface immunoglobulins analyzed by immunostaining and fluorescence-activated cell sorter analysis (mean ⫾ SEM 73 ⫾ 14% IgM⫹,IgD⫹ and 21 ⫾ 1% IgM⫹,IgD⫺). From 33% to 66% of RAG1 plus RAG2 mRNA–positive B cells expressed IgD mRNA (mean 50%). No coordinate RAG1 and RAG2 mRNA expression was found in IgG mRNA–positive cells (Figure 2). Table 3. Comparison of RAG1, RAG2, TdT, VpreB, 14.1, and CD154 mRNA expression by B cell preparations from healthy individuals and systemic lupus erythematosus (SLE) patients, as defined by semiquantitative polymerase chain reaction and Southern blotting* mRNA type Healthy individuals (n ⫽ 14) SLE patients (n ⫽ 9) RAG1 RAG2 TdT VpreB 14.1 CD154 0 (0) 3 (21) 0 (0) 2 (14) 0 (0) ND 9 (100) 5 (56) 1 (11) 6 (67) 4 (44) 7 (77) * CD19⫹ peripheral B cells from the SLE patients were sorted with a FACS Vantage flow cytometer. Specific mRNA for types 1 and 2 recombination activating genes (RAG1 and RAG2), TdT, surrogate light chain, and CD154 were amplified as described in Patients and Methods, using comparable amounts of total RNA. Reverse transcriptase–polymerase chain reaction products were detected by Southern blot hybridization. The number (%) of mRNA-positive B cell preparations is shown. The results from the healthy individuals were previously reported (16). ND ⫽ not done. Figure 2. Expression of RAG1, RAG2, IgG, and IgD mRNA in individual peripheral B cells of SLE patients. Individual CD19⫹ B cells from 3 patients with active SLE were analyzed. Only ␤-actin mRNA–positive cells were considered for analysis of the frequency of RAG1- and RAG2-mRNA–containing cells. The frequency of RAG1⫹, RAG2⫹, RAG1⫹ and RAG2⫹, IgD⫹, and IgG⫹ cells, as well as the frequency of IgD⫹ or IgG⫹ cells in the RAG1- and RAG2-expressing cells, were determined in the various subsets. The frequency of IgD⫹ and IgG⫹ cells in the RAG1⫹,RAG2⫹ cells was calculated as (RAG1⫹ ⫹ RAG2⫹ ⫹ Ig⫹)/(RAG1⫹ ⫹ RAG2⫹) ⫻ 100. See Figure 1 for definitions. RAG1, RAG2, VpreB, and CD154 mRNA expression by individual peripheral B cells of an SLE patient. In patient 7, we investigated the expression of RAG1, RAG2, VpreB, and CD154 mRNA in individual peripheral B cells (Figure 3). Ten percent of B cells were positive for RAG1 and RAG2 mRNA. Fifty percent of these cells were VpreB mRNA positive. Notably, all double-RAG–positive cells were positive for CD154. 1260 GIRSCHICK ET AL Figure 3. Expression of RAG1, RAG2, VpreB, and CD154 mRNA by individual peripheral B cells of an SLE patient. Individual CD19⫹ B cells from 1 patient with active SLE were analyzed. Only ␤-actin mRNA–positive cells were considered in the analysis of the frequency of RAG1- and RAG2-mRNA–containing cells. The frequency of RAG1⫹, RAG2⫹, RAG1⫹ and RAG2⫹, VpreB⫹, and CD154⫹ B cells and the frequency of VpreB⫹ or CD154⫹ cells in the RAG1- and RAG2-expressing B cells was determined in the various subsets. The frequency of VpreB⫹ or CD154⫹ cells in the RAG1⫹,RAG2⫹ cells was calculated as (RAG1⫹ ⫹ RAG2⫹ ⫹ VpreB⫹ or CD154⫹)/ (RAG1⫹ ⫹ RAG2⫹) ⫻ 100. See Figure 1 for definitions. Identification of RAG1ⴙ, RAG2ⴙ, CD19ⴙ, IgDⴙ cells in SLE as activated, mature B cells. As shown in representative samples in Figure 4, the majority of IgM⫹,IgD⫹ cells in SLE expressed CD154 (mean ⫾ SEM 84 ⫾ 21%), whereas few normal B cells expressed this marker (mean ⫾ SEM 7 ⫾ 2%). In SLE, CD154⫹, preswitch IgD⫹ SLE B cells that expressed mRNA for both RAG1 and RAG2 coexpressed IgM and markers of activation (Figure 4). IgM⫹,IgD⫹ SLE CD19⫹ B cells coexpressed CD69 (mean ⫾ SEM 76 ⫾ 22%) and CD38 (69 ⫾ 10%), whereas only a few cells of this subset from healthy individuals expressed CD69 (mean ⫾ SEM 9 ⫾ 3%) or CD38 (8 ⫾ 3%). Although differing in activation status, the percentage of IgM⫹,IgD⫹ B cells in peripheral blood CD19⫹ B cells was similar for healthy volunteers (mean ⫾ SEM 82 ⫾ 5%) and SLE patients (73 ⫾ 14%). DISCUSSION By using a combination of flow cytometric cell sorting and RT-PCR analysis, the expression of RAG mRNA by peripheral blood B cells of SLE patients was examined. When bulk preparations of mRNA were analyzed, RAG1 transcripts were routinely detected in all patients, whereas RAG2 transcripts were detected in 5 of 9 patients. Using the same technique, none of the 14 normal B cell mRNA preparations expressed RAG1 and only 3 of 14 were positive for RAG2 mRNA. In addition, VpreB expression was up-regulated in SLE B cells. Comparable differences were noted when individual B cells were analyzed. Three times more SLE B cells than normal peripheral blood B cells expressed both types of RAG mRNA, and they were exclusively IgD mRNA positive. Further analysis in another SLE patient demonstrated that 50% of double-RAG–positive cells were VpreB mRNA positive, and 100% were CD154 mRNA positive. Flow cytometric analysis of IgD⫹ SLE B cells revealed that the majority of these cells were CD154 positive (for review, see ref. 46). Moreover, the IgD⫹ B cell subset in SLE coexpressed the activation markers CD69 and CD38. These data suggest that RAG1⫹, RAG2⫹,IgD⫹ SLE B cells are mature B cells that have undergone activation in vivo. Moreover, additional detailed phenotyping of IgM⫹,IgD⫹ B cells in these patients indicated that nearly all were CD27⫹ memory cells (data not shown). The previous finding that more than 95% of the Ig VH genes expressed by SLE B cells in the IgD⫹,CD27⫹ subset were somatically mutated (47) is further evidence that this subpopulation contains mature memory B cells. Although peripheral blood B cells expressing mRNA for VpreB have been detected in healthy donors (31), the frequency of these cells (0.5–1%) is much lower than that observed in SLE patients (8%). It has been demonstrated that this peripheral VpreB⫹ B cell population is positive for RAG1 and RAG2 mRNA. However, the frequency of RAG1⫹,RAG2⫹ B cells in this population is unknown (31). Recent work has demonstrated coordinate RAG mRNA expression in up to 3% of IgG⫹ individual normal peripheral B cells, but not in IgD⫹ B cells from healthy donors (16). This observation is in contrast to our finding in SLE patients, in which IgD⫹, but not IgG⫹, peripheral blood B cells coexpressed RAG1, RAG2, and VpreB. The suggestion has been made that VpreB⫹,RAG1⫹,RAG2⫹ B cells observed in the periphery or in secondary lymphoid tissues of mice are immature B cells that have left the bone marrow prematurely in response to inflammation or a dysfunction of the bone marrow–blood barrier (48). The latter could occur in SLE following induction of endothelial cell apoptosis by lupus anticoagulant autoantibodies (49) or a disruption of endothelial integrity by autoantibodies to vascular antigens (50,51). In contrast to the induction of increased numbers of immature B cells in the periphery following adjuvant challenge in mice (48), however, the current data clearly show that RAG1 AND RAG2 EXPRESSION IN SLE 1261 Figure 4. Abnormal expression of activation antigens in subsets of CD19⫹, IgM⫹, IgD⫹ peripheral blood B cells from systemic lupus erythematosus (SLE) patients. Freshly isolated peripheral blood mononuclear cells from SLE patients (B, C, and D) and a normal volunteer (A) were assessed for expression of CD19, IgM, and IgD as well as the activation antigens CD154, CD69, and CD38. Following staining with fluorochrome-conjugated monoclonal antibodies, CD19⫹, IgM⫹, IgD⫹ cells were gated and were assessed for expression of the various activation antigens. Shown are the patterns of IgM and IgD expression as well as activation antigen expression by the gated population. Bars on histograms from the gated CD19⫹, IgM⫹, IgD⫹ B cell population delineate positive staining for IgM, IgD, and each activation antigen using isotype-matched antibodies as controls. The mean fluorescence intensity of the cells expressing the activation antigen and the percentage of CD19⫹, IgM⫹, IgD⫹ B cells that are positive are listed to the right of each histogram. Histograms for activation antigens expressed by normal adult CD19⫹, IgM⫹, IgD⫹ B cells are representative of samples from 2 different normal, healthy volunteers. the SLE patients examined in this study have activated, mature B cells in their blood that coexpress RAG1, RAG2, and surrogate light chain. The current data also demonstrate that expression of RAG-encoded proteins is up-regulated in SLE B cells, although it remains to be determined whether these proteins are enzymatically active. Functional expression of RAG genes has been shown to lead to receptor editing/replacement of surface immunoglobulin. In this regard, previously published studies have observed evidence of receptor editing/replacement in both V and V chains from patients with active SLE (10,11). Although the signals required for RAG expression have not been completely delineated, induction of RAG expression in B cells is initiated by engaging surface immunoglobulin (sIg) (52–54) and maintained by engaging CD40 (18,31) possibly by a B cell lineage– specific activator protein/paired box gene 5 (BSAP)– dependent mechanism (55). This mechanism of RAG induction may be a critical factor in SLE B cells that may have been stimulated through sIg by autoantigens and through CD40 by abnormally expressed CD154 on adjacent B cells or on activated T cells (43,44,56). Of note, engaging sIg on B cells that are already positive for RAGs, such as immature B cells or activated B cells, results in a down-regulation of RAG expression (26). SLE B cells may not have received this down-regulatory signal or may be resistant to it. Recent data have demonstrated that the human RAG2 promoter, which, in contrast to the mouse pro- 1262 GIRSCHICK ET AL moter, regulates the expression of both RAG1 and RAG2, is regulated in both lymphoid and nonlymphoid cells and is differentially regulated in T and B cells (57). This B cell regulatory region is homologous to the mouse RAG2 promoter. The T cell–specific factors that directly regulate RAG2 are c-myb (58) and GATA-3 (59). B cell–specific regulation of RAG2 involves BSAP through a transactivation mechanism (50,57,60). RAG2 protein expression is restricted to cells in G1/G0 because of posttranscriptional degradation in the S/G2/M phase of mitosis (60). Importantly, and perhaps explaining the coordinate regulation of RAGs and VpreB, BSAP is also involved in regulation of the VpreB promoter (61). As an additional regulatory region, the RAG1 promoter, which regulates only RAG1 expression, is activated by nuclear transcription factor Y in a manner that is not lymphoid specific (62). Differential activity of RAG1 and RAG2 promoter regions may contribute to the uncoordinated expression of the RAG enzymes in healthy subjects (16) and SLE patients. In summary, the use of standard and single-cell RT-PCR analysis and flow cytometric evaluation has demonstrated a significant increase in the frequency of RAG1- and RAG2-mRNA⫹ B cells in the peripheral blood of SLE patients. RAG-expressing B cells also contained IgD, VpreB, and CD154 mRNA. The phenotype of these cells indicates that they are activated, mature B cells. Circulating mature B cells of patients with SLE, therefore, reexpress RAG enzymes after cellular activation, potentially becoming capable of receptor revision. 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