May 4, 1998 - Abstract. CD21, the complement receptor type 2 (CR2), binds the complement fragments iC3b, C3dg and. C3d, interacts with CD23 (the ...
International Immunology, Vol. 10, No. 8, pp. 1197–1202
© 1998 Oxford University Press
Human B and T lymphocytes have similar amounts of CD21 mRNA, but differ in surface expression of the CD21 glycoprotein Moritz Braun1, Inga Melchers1, Hans H. Peter2 and Harald Illges1,3 1Clinical
Research Unit on Rheumatology, and Division of Rheumatology and 2Clinical Immunology, Albert-Ludwigs-University, Medical Center, 79106 Freiburg, Germany
3Present
address: Faculty of Biology, University of Konstanz, 5560M662, 78457 Konstanz, Germany
Keywords: CD21, CR2, semi-quantitative PCR, soluble CD21, T lymphocyte CR2
Abstract CD21, the complement receptor type 2 (CR2), binds the complement fragments iC3b, C3dg and C3d, interacts with CD23 (the low-affinity receptor for IgE), and binds IFN-α. This 145 kDa glycoprotein merits particular interest because it plays a pivotal role in the activation and proliferation of B cells by lowering the signal threshold. In human disease CD21 is important as a receptor for Epstein–Barr virus and HIV. CD21 is primarily expressed on B lymphocytes and follicular dendritic cells, but has also been reported on T cells. We established a semi-quantitative PCR and compared the CD21 mRNA levels of B and T lymphocytes with the expression of the CD21 glycoprotein on the surface of the respective cells by flow cytometry. The B cell lines Raji and Ramos and the T cell lines Jurkat and Molt4 expressed equal amounts of CD21 mRNA, but differed in surface staining. To address the question to which extent primary human B and T lymphocytes express CD21 mRNA and membrane-bound CD21 glycoprotein, we separated B cells, CD4F and CD8F T cells from peripheral blood mononuclear cells of healthy donors. B lymphocytes and CD4F or CD8F T cells expressed similar amounts of CD21 mRNA. Nevertheless only B cells, but not CD4F or CD8F T cells, expressed detectable amounts of CD21 on their cell surface. Expression of the CD21 exon 11 has been reported being restricted to follicular dendritic cells only. To the contrary, we found that both purified B and T cell subpopulations expressed CD21 mRNA with and without exon 11. Introduction CD21, the complement receptor type 2 (CR2), belongs to the family of the regulators of complement activation (1). This 145 kDa glycoprotein is mainly expressed on B lymphocytes (2) and follicular dendritic cells (3), but to a lesser extent it is also found on thymocytes (4–6), a subpopulation of peripheral T lymphocytes (7,8), cervical and pharyngeal epithelial cells (9,10), basophils (11), natural killer cells (12), and astrocytes (13). It binds the complement fragments iC3b, C3dg and C3d (14), functions as receptor for Epstein–Barr virus (EBV) (15) and HIV (16,17), interacts with IFN-α (18) and CD23 (the lowaffinity receptor for IgE 5 FcεRII) (19) and is strongly involved in B cell responses to T cell-dependent antigens (20–23). By interactions with CD19, CD81 (TAPA-1) and Leu-13, CD21 forms a signal transduction complex on the surface of B lymphocytes (24,25) or associates with the complement
receptor type 1 (CD35) (26). Furthermore a soluble form of CD21 was detected in the supernatant of the B cell line Raji (27), the T-ALL cell line Molt4 (28), in sera of healthy donors (28) and at elevated levels in the sera of patients suffering from B-CLL (29), rubella virus infection or EBV-associated diseases (30). Inspired by our recent observation of reduced CD21 expression in synovial fluid lymphocytes, we found it interesting to study the expression of CD21 in human B and T lymphocytes by comparing mRNA and protein levels using semi-quantitative PCR and flow cytometry analysis. Moreover, we were interested in the expression of the previously described follicular dendritic cell-specific isoform of CD21 mRNA in primary B and T cells (31). Here we show that transformed B and T cell lines as well as purified B and T lymphocytes of
Correspondence to: H. Illges Transmitting editor: A. Radbruch
Received 18 February 1998, accepted 4 May 1998
1198 CD21 mRNA in B and T cells the peripheral blood of healthy volunteers contain similar amounts of CD21 mRNA, but differ in the surface expression of the CD21 glycoprotein. Furthermore, sorted primary B and T cells express both CD21 isoforms, i.e. the one with exon 11 and the splice variant lacking exon 11.
Methods
Cell lines and primary lymphocytes The B-cell lines Raji and Ramos as well as the T cell lines Molt4 and Jurkat (ATCC, Rockville, MD) were maintained in IMDM (Gibco/BRL, Eggenstein, Germany) supplemented with 10% FCS (PAA Laboratories, Linz, Austria), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco/BRL). Lymphocytes from peripheral blood (buffy coat and EDTAblood samples) were diluted 1:2 in PBS and purified by Ficoll density gradient centrifugation (Pharmacia, Uppsala, Sweden). The interphase containing the peripheral blood mononuclear cells (PBMC) was isolated and washed twice with PBS.
Separation of PBMC subpopulations by MACS and analysis by FACS Purified PBMC were separated with MACS into three fractions by sorting with anti-CD19, anti-CD4 and anti-CD8 mAb according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the sorted cells was verified by flow cytometry analysis using a FACScan (Becton Dickenson, Mountain View, CA). Surface expression of CD21, CD19, CD4 and CD8 was determined by flow cytometry. Anti-CD21 mAb BU-33–FITC was purchased from Harlan Sera-Lab (Oxford, UK), anti-CD21 mAb BL13–FITC from Immunotech (Hamburg, Germany), antiCD19–phycoerythrin (PE) and anti-CD4–PE mAb were from Dakopatts (Denmark), and anti-CD8–FITC was obtained from Caltag (San Francisco, USA).
RNA isolation and cDNA synthesis Cells (107) were lysed in 1 ml of denaturing solution containing 4 M guanidinium isothiocyanate, 25 mM sodium citrate (pH 7), 0.1 M β-mercaptoethanol and 0.5% sodium lauroyl sarcosinate. The cells were passed 7–10 times through a 2031.5 guage needle to achieve fragmentation of DNA as well as denaturation of proteins. The RNA was thereafter isolated by phenol extraction and isopropanol precipitation. The quality of the prepared RNA was verified by agarose gel electrophoresis and the optical density measured in a photometer (Beckmann, Fullerton, CA). Aliquots of 10 µg RNA were adjusted to 16 µl with DEPCH2O and possible tertiary structures were denatured by incubating the tubes for 10 min at 65°C. The RNA tubes were transferred on ice and the following reagents were added: 5 µl 53reaction buffer, 1 µl oligo(dT)12–18 primer 2, 2 µl DTT (all Gibco BRL), 1 µl (10 µmol) dNTP (Pharmacia), 0.5 µl RNase inhibitor (30 U/µl; MBI Fermentas, Lithuania) and 0.5 U/µl reverse transcriptase (superscript II RT; 200 U/µl; Gibco/ BRL). The reaction was incubated at 42°C for 60 min and the resulting cDNA diluted 1:2 with autoclaved deionized water.
The quality of the synthesized cDNA was controlled by PCR amplification of GAPDH.
Plasmid construction and semi-quantitative PCR In order to quantitate CD21 gene expression by RT-PCR we have constructed a plasmid as described elsewhere (32). In brief, an internal SspI–EcoRV fragment was deleted from the plasmid p21TM containing 599 bp of the membrane exon spanning region. The resultant 158 bp smaller insert (pQ21) spans 441 bp and therefore can easily be distinguished from cDNA on agarose gels. The optical density of the purified insert was measured in a photometer (Beckmann) and 1:2 dilution series were prepared from 100 pg/µl to 25 fg/µl. For the simultaneous amplification of cDNA and insert (pQ21) we used: 35.5 µl autoclaved deionized water, 5 µl 103reaction buffer, 3 µl MgCl2 (25 mM; MBI Fermentas), 1 µl (10 (µmol) dNTP (Pharmacia), 1.25 µl (250 ng/µl) 21S2761 59 primer, 1.25 µl (250 ng/µl) 21R3360 39 primer (Dr Igloi, Biologie III, Freiburg, Germany), 1 µl cDNA, 1 µl insert (pQ21), one drop mineral oil (Sigma, Deisenhofen, Germany) and 1 µl (0.5 U/µl) Taq polymerase (MBI, Lithuania; 4 U/µl). The sequences of the oligonucleotides were: 21S2761: 59-GGA ACC TGG AGC CAA CCT GCC-39; 21R3360: 59-CTG GGC TCC CAT CTT TAC cat-39. Optimized PCR-conditions were: 5 min 95°C for denaturation and hot start, 95°C 20 s, 57°C 1 min, 72°C 1 min, 30 cycles, 10 min 72°C. Exon 11 was amplified from cDNA using the following oligonucleotides: 21S1800: 59-AGA GCA CCA TCC GTT GTA CA-39, 21R12AB 59-CCA GTC AAC TGG TAC CCA TC-39. The PCR conditions were: 20 s 95°C, 1 min 66°C, 1 min 72°C, 35 cycles, 10 min 72°C. Results
B and T cell lines have equal amounts of CD21 mRNA, but differ in surface expression We examined established malignant tumor cell lines of B and T cell origin for the expression of CD21 mRNA and surface protein by semi-quantitative PCR and cytofluorometry. In the semi-quantitative PCR serial dilutions (1:2) of the insert (pQ21) were titrated into the cDNA of the B cell lines Raji and Ramos and the T cell lines Jurkat and Molt4 (Fig. 1). As can be seen in Fig. 1 the equivalence region was between 0.75 and 1.5 pg for the B-cell lines Raji and Ramos as well as for the T-ALL cell line Molt4, and ranged from 1.5 to 3 pg for the T-cell line Jurkat. Therefore all four cell lines examined expressed comparable amounts of CD21 mRNA. To address the question whether the mRNA levels of CD21 correlate with its surface expression we performed flow cytometry. The B cell lines Raji and Ramos (Fig. 2) and the T cell line Jurkat expressed CD21 on their cell surface, whereas the T cell line Molt4 (Fig. 2) was negative.
Peripheral blood B and T lymphocytes express similar amounts of CD21 mRNA, but only the B cells have CD21 on their surface We next asked whether primary B and T lymphocytes of peripheral blood behave similarly. Buffy coat and EDTA-blood samples from healthy donors were treated as described in
CD21 mRNA in B and T cells 1199 Methods to obtain PBMC. These cells were isolated and separated into CD191, CD41 and CD81 lymphocytes by MACS. The purity of the three fractions was verified by flow cytometry (Fig. 3). B lymphocytes, characterized by the panB cell antigen CD19, and T lymphocytes, defined by either one of the T cell-specific antigens CD4 or CD8, were then examined by semi-quantitative PCR and FACS analysis. As depicted in Fig. 4 the equivalence region of the sorted B and T lymphocytes was in the range of 3 and 1.5 pg respectively, indicating that the B cells expressed higher amounts of CD21 mRNA. In contrast to the mRNA expression pattern, only B lymphocytes showed intense CD21 staining, whereas purified CD41 and CD81 T lymphocytes were negative for CD21 on their cell surface (Fig. 3). There was no difference in this regard between CD41 and CD81 T lymphocytes.
Purified CD41 and CD81 T cells and B cells express both exon 11-containing and exon 11-lacking CD21 mRNA Fig. 1. Semi-quantitative PCR of CD21 mRNA isolated from the B cell lines Raji and Ramos and the T cell lines Molt4 and Jurkat. B and T cell lines expressed comparable amounts of CD21 mRNA. PCRproducts were separated by agarose gel electrophoresis. The upper band of ~600 bp represents the cDNA, the lower bands the 160 bp shorter plasmid encoded fragment (pQ21). The amount of insert per tube is depicted on the top. In the equivalence region cDNA and insert have equal concentrations. The 1 kb ladder (Gibco/BRL) was applied as a size standard.
Recently Liu et al. (31) reported that B cells selectively express the small isoform (lacking exon 11) while follicular dendritic cells would express only the exon 11-containing long isoform of the CD21 mRNA. In a previous report we showed that unsorted PBMC from human tissues such as spleen, tonsil and blood, and a large collection of B and T cell lines always expressed both versions of CD21 mRNA (33). Now we examined purified B cells, CD41 and CD81 T cells from peripheral blood for the presence of exon 11 containing CD21 mRNA using RT-PCR. The results (Fig. 5) show that the sorted B and T cell subpopulations express both isoforms of CD21 mRNA. Although our analysis is not quantitative, the very similar intensity of the bands indicates that both isoforms are expressed in peripheral B and T cells at high amounts. Discussion
Fig. 2. Cytofluorometric analysis of the surface expression of CD21 on the B cell lines Raji and Ramos and the T cell lines Molt4 and Jurkat; the cell number (ordinate) is depicted against intensity of staining (abscissa); dotted line, control staining; bold line, anti-CD21stained cells. The staining depicted was done with mAB BU33–FITC.
To our surprise we found that B and T cell lines as well as primary CD191, CD41 and CD81 lymphocytes isolated from healthy volunteers expressed similar amounts of CD21 mRNA respectively. B cells express up to 2-fold more CD21 mRNA compared to T cells. In contrast to the mRNA levels we found that along the route of biosynthesis the fate of the CD21 glycoprotein is strikingly different between B and T cells. While we observed high surface expression in cell lines and cells belonging to the B lineage, membrane-bound CD21 could neither be detected on primary T lymphocytes nor on the T cell lines Molt4, CEM and Hut78 (Fig. 2 and data not shown). Only the T cell line Jurkat was weakly stained. We have stained the cells with directly labeled antibodies and cannot exclude the fact that T cells do not express CD21 very weakly on their surface. Whereas our results concerning the expression of CD21 on B lymphocytes are in accordance with previous reports (2,15), its expression on T lymphocytes is controversial. About 20 years ago EBV receptors were detected on the surface of the T cell line Molt4 allowing virus binding but not virus penetration (34). Later on CD21 was identified on the surface of the T cell lines HPB-ALL, Jurkat and Molt3, whereas in the same report the T cell line CEM and CD41 and CD81 cells from peripheral blood were CD21– (7). In one patient suffering from systemic lupus erythematosus up to 89% of the CD31 T cells displayed CD21 on their
1200 CD21 mRNA in B and T cells
Fig. 3. PBMC were isolated and separated into CD41, CD81 and CD191 cells by MACS. The purity of the fractions was verified by FACS analysis. In the subpopulation of T lymphocytes, defined by the antigens CD41 or CD81, no CD191 or CD211 cells could be detected and vice versa. Only the B lymphocytes, characterized by the pan-B cell antigen CD19, expressed CD21 on their surface as revealed by staining with BU33–FITC.
Fig. 5. RT-PCR from purified CD41, CD81 and CD191 peripheral blood lymphocytes demonstrated the simultaneous expression of exon 11-containing and -lacking CD21 mRNA in both primary B and T cells.
Fig. 4. Purified CD41, CD81 and CD191 peripheral blood lymphocytes were analyzed for CD21 mRNA levels by semiquantitative PCR. B and T lymphocytes expressed similar amounts of CD21 mRNA (these results were obtained and confirmed by analyzing two different buffy coats and PBMC of a healthy donor; each PCR was done at least twice).
cell surface (35). Others reported that 30–40% of normal peripheral blood T lymphocytes show CD21 surface expression 10-fold lower than that of peripheral blood B lymphocytes (8). Using flow cytometry we were unable to detect CD21 on the surface of primary normal T lymphocytes or on the T cell line Molt4. Nevertheless, we found that normal T lymphocytes have similar amounts of CD21 mRNA when compared to B lymphocytes. This striking difference between CD21 mRNA and CD21 protein expression in B and T cells could be due to selective shedding of CD21 from the T cell surface by an as yet unidentified protease, a mechanism which has been suggested before (36,37). We suggest that B cells primarily express membrane-bound CD21, whereas mainly T cells
CD21 mRNA in B and T cells 1201 contribute to the serum pool of soluble CD21. In addition to our observation described here, there are at least two further lines of evidence for this hypothesis. CD21 was detected by means of immunohistology on the surface of B lymphocytes, but only in the cytoplasm of T lymphocytes (38). Two brothers suffering from Bruton’s X-linked agammaglobulinaemia and therefore lacking B lymphocytes had normal serum levels of soluble CD21 (28). Because of the interference of recombinant soluble CD21 with T-dependent immune responses (39) it is tempting to speculate that CD21 released from normal T cells is involved in the regulation of immune effector functions. The simultaneous expression of both exon 11-containing and -lacking CD21 transcripts in B and T cells from the peripheral blood conflicts with a report by Liu et al. who used sorted tonsilar B lymphocytes from the mantel zone of germinal centers and found expression of the short, exon 11-lacking, CD21 mRNA only (31). Because follicular mantel zone B cells (IgD1, CD38–) are equivalent to peripheral blood B cells we find these observations in contradiction to our results. The specific staining with the mAb 7D6, DRC-1 and KiM4 (directed against follicular dendritic cells) was shown to exclusively recognize COS7 cells transfected with the exon 11-containing CD21 construct. It will be interesting to analyze in peripheral blood B and T cells whether exon 11-containing transcripts are translated into protein and whether this protein is expressed on the cell surface. Acknowledgements We thank Drs H. Eibel, K. Warnatz and C. Scha¨tzlein for critical reading of the manuscript and discussion. This work was supported by DFG grant Me 604/4 and a fellowship of the Hans-Hench-Stiftung to M. B.
Abbreviations CR2 EBV PBMC
complement receptor type 2 Epstein–Barr virus peripheral blood mononuclear cells
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