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CD21) ectodomain shedding by its cytoplasmic domain
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Molecular Immunology 45 (2008) 2127–2137
Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain Melanie M. Hoefer a,1 , Annette Aichem a , Andrew M. Knight b , Harald Illges c,∗ a
Biotechnology Institute Thurgau, 8280 Kreuzlingen, Switzerland School of Surgical and Reproductive Sciences, Institute of Cellular Medicine, The Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom c University of Applied Sciences, Department of Natural Sciences, Immunology and Cell Biology, von-Liebig-Str. 20, 53359 Rheinbach, Germany b
Received 23 November 2006; received in revised form 6 November 2007; accepted 14 December 2007
Abstract Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation. © 2007 Elsevier Ltd. All rights reserved. Keywords: B cell activation; Complement receptor 2/CD21; Glutathione; Pervanadate; Shedding
1. Introduction Complement receptor 2 (CR2/CD21) is a type I transmembrane glycoprotein of about 145 kDa that binds complement fragments (C3d(g), iC3b) and interferon (IFN)-␣, and acts as the Epstein–Barr virus (EBV) receptor on human B lymphocytes (Asokan et al., 2006; Carroll, 2004; Rickert, 2005; Tedder et al., 2002). In mice, the complement receptors 1 and 2 (CR1/CR2, CD35/CD21) are encoded by the same locus (Cr2) and the two different products are generated by alternative splicing of the mRNA. CD21 is a part of the membrane protein complex comprising CD19/CD21/CD81, which enhances B cell antigen receptor (BCR) signaling in response to complement-coated
Abbreviations: GSH, glutathione; NAC, N-acetylcysteine; PV, pervanadate; MMP, matrix metalloproteinase; SCR, short consensus repeat. ∗ Corresponding author. Tel.: +49 2241 865570; fax: +49 2241 8658570. E-mail address: [email protected] (H. Illges). 1 Present address: The Burnham Institute for Medical Research, Infectious and Inflammatory Disease Center, La Jolla, CA 92037, USA. 0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.12.015
antigens by several orders of magnitude. CD21/CD35-deficient mice reveal decreased antibody production and impaired germinal center (GC) formation in response to T cell-dependent and -independent antigens (Carroll, 2004; Rickert, 2005). The controlled proteolytic cleavage of cell surface proteins and the resulting release of the ectodomain as a soluble protein define a process termed “shedding”. Several different types of proteins have been reported to be regulated by this mechanism. In fact, 2–4% of all cell surface proteins are shed by members of the well over 500 proteases comprising mammalian “degradome”. Importantly, the soluble forms of the respective proteins retain their biological activity and have regulatory functions (Arribas and Merlos-Su´arez, 2003; Dello Sbarba and Rovida, 2002; Overall and Blobel, 2007). This is also true for CD21 (Fremeaux-Bacchi et al., 1999). The major form of soluble (s)CD21 has been identified as a single molecular species of 126 kDa (Masilamani et al., 2002). It has also been described as a marker of B cell activation in humans and was shown to inhibit EBV-binding and -infection of B cells (Huemer et al., 1993; Nemerow et al., 1990). The immune
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modulatory properties of sCD21 were further revealed by its binding to the low-affinity IgE-receptor CD23 and sCD23, thereby regulating monocyte activation and differentiation, as well as IgE-responses (Fremeaux-Bacchi et al., 1998a,b). Moreover, recombinant sCD21 suppresses the antibody response to T cell-dependent antigens in mice (Hebell et al., 1991), and inhibition of B cell/follicular dendritic cell (FDC) interactions with sCD21 reduced antibody responses by 10–1000-fold (Qin et al., 1998). sCD21 levels are often altered in pathologic conditions including various lymphoproliferative leukemias, such as chronic B lymphocytic leukemia (B-CLL) (Lowe et al., 1989), acute EBV-infection and other virus-associated diseases (Huemer et al., 1993; Larcher et al., 1995), and autoimmune diseases (Masilamani et al., 2004a,b). While CD21 is constitutively shed, B cell activation through mitogen (PMA + calcium ionophore), anti-IgM + anti-CD40 cross-linking (Masilamani et al., 2003), or pervanadate (PV) stimulation (Aichem et al., 2006) leads to enhanced human CD21-shedding, whereas cell surface expression of another member of the B cell co-receptor complex, CD19, remained at a constant level (Hoefer and Illges, unpublished data). In addition, we could demonstrate that CD21-shedding might be a redoxregulated process that can be induced with the thiol antioxidants glutathione (GSH), N-acetylcysteine (NAC), and the oxidant PV in CD21-transfected HEK293 cells, human peripheral blood mononuclear cells (PBMC) and the Burkitt lymphoma line Daudi (Aichem et al., 2006). The process involves both a serine and a metalloprotease activity. The reducing agents GSH and NAC do not induce tyrosine phosphorylation and cell activation, but rather directly activate the metalloprotease or modify intramolecular disulfide bridges within the extracellular domain of CD21 or both, and thereby facilitate access to the CD21 cleavage site (Aichem et al., 2006). In contrast, the tyrosine-specific phosphatase inhibitor and oxidant PV mimics BCR signaling and operates through activation of a signaling pathway that comprises the B cell adaptor SLP-65 (SH2-domain-containing leukocyte protein of 65 kDa), the protein kinases Syk and Lyn (Wienands et al., 1996,1998), and further downstream, protein kinase C (PKC) (Aichem et al., 2006). PV was shown to induce protein tyrosine phosphorylation, cell activation, and shedding of members of very diverse protein families, such as the ErbB-family tyrosine kinase HER4/ErbB4, the heparan sulfate proteoglycan syndecan-1 (CD138), the cell adhesion molecule L1 (CD171), and the heparin-binding EGF-like growth factor (HB-EGF) (Blobel, 2005; Brummer et al., 2004; Dello Sbarba and Rovida, 2002; Reth, 2002). Recently, the involvement of the Erk1/2-mitogen-activated protein kinase pathway downstream of PKC was shown to regulate PV-induced neural cell adhesion molecule (NCAM)-shedding (Hinkle et al., 2006). The mechanism of how shedding is regulated is currently not well understood, however, several cell surface proteins require their cytoplasmic region to control their shedding rates, and an involvement of the actin–cytoskeleton has e.g. been shown for 160 pervanadate (PV) (Heiz et al., 2004), l-selectin (CD62L) (Smalley and Ley, 2005), and CD44 (Nagano and Saya, 2004) among others.
The cytoplasmic domain of CD21 has been implicated in antigen presentation (Barrault and Knight, 2004), B lymphocyte signaling (Bouillie et al., 1999), and may interact with the cytoskeleton through the formin homologue overexpressed in spleen FHOS/FHOD1 (Gill et al., 2004). FHOS/FHOD1 regulates gene transcription, actin–cytoskeleton structure, and cell migration (Koka et al., 2003). In the present study, we addressed the question whether B cell activation would trigger murine CD21-shedding, and whether the process involves redox regulation. For this, we stimulated B cells with PV and the thiol antioxidants GSH and NAC. We further examined the role of the cytoplasmic domain in CD21-shedding using mouse B cells expressing different CD21 cytoplasmic domain truncation mutants as well as CD21 containing point mutations where the three tyrosines in the cytoplasmic domain were altered to alanines or phenylalanines. 2. Material and methods 2.1. Cell purification and culture Splenocytes were prepared from 4- to 12-month-old male C57BL/6 mice. Spleens were homogenized, the resulting cell suspensions filtered and loaded onto a Ficoll gradient (Biochrom, Berlin, Germany). The isolated lymphocytes were washed twice with fresh serum-free medium (RPMI 1640) and then treated as described below. To exclude the possibility of measuring shedding of CD35 and soluble CD35 and to study the role of the CD21 cytoplasmic domain, we also used the CD21/CD35 negative murine B cell line CH27 (Molina et al., 1990), stably transfected with various CD21 constructs containing truncated or mutated cytoplasmic domains as previously described (Barrault and Knight, 2004) (see Table 1). Cells were maintained at 37 ◦ C with 5% CO2 in RPMI 1640 supplemented with UltraGlutamine 1, penicillin/streptomycin (100 U/ml per 100 g/ml), non-essential amino acids (all Cambrex, Verviers, Belgium), 50 M mercaptoethanol (Sigma, Buchs, Switzerland) and 10% fetal calf serum (Linaris, Wertheim-Bettingen, Germany). In addition, 0.75 mg/ml G418 (Calbiochem, Lucerne, Switzerland) was included in the culture of the transfected CH27 cells. For those experiments using CD21 KHR expressing cells, two independent clones (IB2 and IB4) were used. The results from these two clones were nearly identical and those from clone IB2 are depicted. 2.2. Induction of CD21-shedding Freshly isolated splenocytes, CH27 and the different CH27-CD21 transfectants were adjusted to a cell density of 2 × 106 ml−1 and incubated for 2 h at 37 ◦ C in serum-free RPMI 1640. After this period the medium was removed, and fresh RPMI 1640 was added together with various concentrations of reduced l-glutathione, N-acetylcysteine (both Sigma), or pervanadate and incubated for additional 4 h at 37 ◦ C unless indicated otherwise. PV was freshly prepared for each experiment by mixing one volume of sodium orthovanadate (Na3 VO4 ,
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Table 1 CD21 constructs used in this study (Barrault and Knight, 2004)
Residues in the murine CD21 cytoplasmic domain sequence are numbered according to the specifications of Molina et al. (1990), starting at the first isoleucine in SCR1. The humans sequence is shown for comparison. H.s., Homo sapiens; M.m., Mus musculus.
100 mM, pH 10) with one volume of H2 O2 (100 mM) and was used within 10 min of preparation. After incubation, cells were subjected to analysis of CD21 cell surface expression by flow cytometry and the cell culture supernatants were collected for ELISA analysis.
strate solution (Sigma) was added and incubated until blue color developed. Following the addition of 1 M H2 SO4 to stop the reaction, the absorption was measured at 450 nm (reference wavelength 690 nm) using a Tecan Sunrise microplate reader (Tecan, Maennedorf, Switzerland).
2.3. Determination of cell surface CD21 by flow cytometry
2.5. Immunofluorescence stainings and microscopy
For analysis of cell surface expression of CD21, cells were stained with anti-CD21/CD35-PE antibody, clone 7G6 (BD Biosciences, Basel, Switzerland) and subjected to flow cytometry analysis (BD LSR) using CellQuest software (both BD Biosciences). The viability of the cells was determined by TO-PRO-3-iodide exclusion (Molecular Probes, Leiden, Netherlands). The murine B cell marker B220 (anti-B220-FITC, BD Biosciences) was used for gating the B cell population in the splenocyte preparations of C57BL/6 mice. Only living gated cells were used for analysis, and viability of the cells did not decrease more than 5–10% upon stimulation.
Cells were pre-incubated with serum-free RPMI 1640 for 2 h as described above and then allowed to adhere on poly-llysine (Sigma) coated glass cover slips during the 4 h incubation time. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. The cells were stained with anti-CD21 (clone 7E9) and either Alexa Fluor 546- or Alexa Fluor 488-conjugated secondary goat anti-rat antibodies, and nuclei were stained with DAPI (all Molecular Probes). Stained cells were mounted with mowiol on microscope slides and examined with a Zeiss LSM 510 Meta laser scanning confocal microscope, using a Plan-Neofluar 40×/1.4 oil DIC and a Plan-Apochromat 63×/1.4 oil DIC objective (Zeiss, Jena, Germany).
2.4. Determination of sCD21 by sandwich ELISA 2.6. Statistical analysis and other calculations ELISA plates (TPP, Trasadingen, Switzerland) were coated overnight at 4 ◦ C with rat anti-mouse CD21/CD35 antibody, clone 7G6 (Kinoshita et al., 1988) (2 g/ml in PBS). After blocking with PBS/3% BSA for 2 h at room temperature (RT), plates were washed with PBS/0.05% Tween-20. Then, cell culture supernatants were added and incubated overnight at 4 ◦ C. After washing, biotinylated rat anti-mouse CD21/CD35 antibody, clone 7E9 (Kinoshita et al., 1988) was added (2 g/ml in PBS/3% BSA) and incubated for 1 h at RT. After another washing step, plates were incubated for 30 min at RT with streptavidin–peroxidase (Biosource, Nivelles, Belgium; 0.5 g/ml in PBS/3% BSA). After washing, TMB liquid sub-
All experiments were performed at least three times. Values in figures are given as means ± S.E.M. Statistical analysis was performed using GraphPad InStat (GraphPad Software, San Diego, CA, USA). Unpaired parametric testing with the Tukey–Kramer multiple comparisons test (Fig. 1A, 3, 4, 5A and B, 6A) or t-test (Fig. 2A and B) was performed. Significantly different values are indicated (*p < 0.05; **p < 0.01; ***p < 0.001). In Figs. 2C and 3B the amounts of sCD21 released from untreated cells were set to 100%, therefore, increased amounts of sCD21 from treated cells are reflected in greater percentage values.
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Fig. 1. Murine CD21 cell surface expression is modulated by pervanadate. (A) The transfected murine CH27 B cell lines CD21 wt and CD21 KHR were stimulated for 4 h with various PV concentrations and analyzed by flow cytometry for CD21 cell surface expression. Data represent mean values of at least three experiments (±S.E.M.; untreated control = 100%). *p < 0.05, ***p < 0.001 vs. data of control cells. (B) Immunofluorescence of CD21 wt and CD21 KHR cells stimulated with 20 or 200 M PV for 4 h followed by staining for CD21 (original magnification 630×). Scale bar: 20 m.
3. Results 3.1. Pervanadate regulates the cell surface expression of murine CD21 The oxidant and tyrosine-specific phosphatase inhibitor PV is an established agent to induce shedding of diverse cell surface molecules (Blobel, 2005). In B cells, PV induces a similar signaling pathway as generated by antigen stimulation (Reth, 2002). Thus, we determined if PV was also able to induce murine CD21-shedding. For this, the CD21/CD35 negative B cell line CH27 that had been previously transfected with murine wildtype CD21 (CD21 wt), was used (Barrault and Knight,
2004). In addition, the role of the CD21 cytoplasmic domain was investigated using CH27 cells transfected with a truncated version, CD21 KHR, where the CD21 cytoplasmic domain consists only of the three transmembrane adjacent residues, lysine (K), histidine (H) and arginine (R) (Barrault and Knight, 2004) (see Table 1). After challenging the cells for 4 h with a range of PV concentrations, CD21 cell surface expression was measured by flow cytometry (Fig. 1A). Due to differences in CD21 cell surface staining of CD21 wt compared to the CD21 KHR expressing cells all results are depicted in relative amounts (%) (see Table 2). Whilst lower concentrations of PV initially caused an increase in wildtype CD21 surface expression, higher concentrations caused a significant decrease
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Fig. 2. Time dependency of pervanadate-induced shedding. (A) Splenocytes of 10 spleens from C57BL/6 mice were pooled and treated with 20 M or 200 M PV for 4 h. At the indicated time points, cells were analyzed for CD21 cell surface expression by flow cytometry, and gates were set to the B220+ B cells. (B) CD21 wt and CD21 KHR cells were stimulated with 200 M PV for 4 h (t0 = 100%). At the indicated time points, cells were analyzed by flow cytometry and the cell culture supernatants were used for ELISA analysis (C). Data represent the mean of four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 significance of differences between CD21 wt and CD21 KHR cells, calculated by unpaired t-test.
(Fig. 1A). In contrast, at higher concentrations the CD21 cell surface expression in cells expressing the CD21 KHR cytoplasmic domain mutant was not down-regulated to the same degree as compared to CD21 wt expressing cells. This difference of PV-induced CD21 regulation was also observed when the cells were examined by immunofluorescence stainings (Fig. 1B).
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Fig. 3. Influence of truncation and site-directed mutagenesis of the cytoplasmic domain of CD21 on its shedding properties upon pervanadate stimulation. (A) Various CH27 transfectants (see Table 1) were treated with 20 or 200 M PV for 4 h and subjected to analysis by flow cytometry for measurement of CD21 surface levels. Data represent mean values of four experiments (±S.E.M.; untreated control = 100%). (B) Quantitation of sCD21 present in the cell culture supernatants as determined by capture ELISA (±S.E.M., untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells. (C) From the raw data of B and A the ratios of soluble CD21 to cell surface expression of CD21 were calculated (±S.E.M.), showing that the CD21 KHR cells shed more CD21 both spontaneously and induced compared to all other cell lines. **p < 0.01, ***p < 0.001 significance of differences between CD21 wt and truncated or site-directed mutant cells with the respective treatment.
3.2. The reaction to pervanadate is time dependent In order to determine a time-dependent effect of PV treatment on CD21 levels on B cells we next performed kinetic studies. Splenocytes isolated from C57BL/6 mice were incubated with 20 or 200 M PV up to 4 h, and CD21 cell surface expression was measured at the indicated time points on the B220+ gated cells (Fig. 2A). After 15 min of stimulation, the B cells showed a slight peak of cell surface CD21 expression with both PV
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concentrations, which declined rapidly with 200 M PV, and stabilized after 2–3 h to a level around 70% of control. With the lower PV concentration, the cells showed a similar increase within the first 15 min of stimulation, followed by a drop to almost basal levels and finally increased again to approximately 10% above control levels. In parallel experiments, we also followed the kinetics of CD21 cell surface expression of CD21 wt and CD21 KHRtransfected CH27 B cells after treatment with 200 M PV (Fig. 2B). The initial peak of expression seen in ex vivo B cells was not detected with the CD21 expressing cell lines (Fig. 2B). However, a similar rate of surface CD21-reduction was observed in the cells expressing CD21 wt. Noticeably, this reduction was faster than in the CD21 KHR cells. After 4 h, the CD21 cell surface expression was reduced by 50% in both cell types. Although previous studies have failed to detect ligand-induced CD21 internalization, unless cross-linked to a functional BCR (Barrault and Knight, 2004), we were keen to confirm that this down-regulation was indeed a result of shedding rather than internalization. We therefore used an ELISA to assess the presence of soluble CD21 in the supernatant of the treated cells and determined that the relative amounts of sCD21 shed from the CD21 KHR expressing cells were only slightly elevated when compared to the levels obtained from CD21 wt expressing cells (Fig. 2C). Thus, PV stimulation of B cells induced an up-regulation of cell surface CD21 at lower concentrations and induced cytoplasmic domain-dependent CD21 ectodomain shedding at higher concentrations. 3.3. Deletion of the full CD21 cytoplasmic domain has an enhancing effect on CD21 basal and induced shedding rates To identify the regions of the CD21 cytoplasmic domain that may be responsible for the opposed effects of high and low PV concentrations, the CD21 levels in several additional transfected CH27 cell lines expressing various other cytoplasmic domain truncations or point mutations within this domain (see Table 1) were studied after treatment for 4 h with 20 or 200 M PV (Fig. 3A). To control for the differences seen in CD21 expresTable 2 Median fluorescence intensities of the different CD21-transfected CH27 B cell lines Cell line
CD21 median fluorescence intensities (mean ± S.E.M. (n))
CH27 CD21 wt CD21 YSI CD21 LET CD21 YYTK CD21 KHR (IB2) CD21 KHR (IB4) CD21 YYFF CD21 YYAA CD21 YF CD21 YA
24 205 185 169 160 114 141 172 216 123 195
± ± ± ± ± ± ± ± ± ± ±
3 (9) 19 (18) 18 (8) 22 (8) 34 (7) 12 (16) 22 (7) 22 (7) 39 (7) 12 (7) 24 (7)
sion levels in the different untreated transfectants (see Table 2), the respective untreated CD21 levels were converted to 100% CD21 cell surface expression. In addition, sCD21 in the cell culture supernatants was also measured by ELISA (Fig. 3B) and the values calculated in percentages, with sCD21 levels released from untreated cells set to 100%. All transfectants up-regulated CD21 upon 20 M PV stimulation, except for the CD21 KHR expressing cells. With 200 M PV treatment, CD21 cell surface staining was lowered in all lines as well, although to a lesser degree from the CD21 KHR cells and the site-directed mutants. The sCD21 levels were in a comparable range for all cell lines. In order to compare the actual shedding rates of all cell lines, ratios of the cell surface expression and the respective ssCD21 data measured in ELISA were calculated from the raw data of this experiment (Fig. 3C). From this calculation it was evident that the CD21 KHR transfectant was unique in its shedding properties, showing higher basal shedding as well as induced shedding rates as compared to all other cell lines. 3.4. Shedding of murine CD21 can be induced by thiol antioxidants To determine whether murine B cells also shed CD21 upon stimulation with the thiol antioxidants GSH and NAC, we compared CD21 levels on primary B cells obtained from spleens of C57BL/6 mice with CH27-CD21 wt cells. After challenging the cells for 4 h with the respective stimuli the cell surface expression of CD21 was measured by flow cytometry. Upon GSH and NAC treatment, CD21 cell surface expression was down-modulated in both B220+ gated splenocytes (Fig. 4A) and CH27-CD21 wt cells (Fig. 4B) in a concentration-dependent manner. These findings confirm our results from human CD21-expressing cells (Aichem et al., 2006). 3.5. Influence of the cytoplasmic domain of CD21 on thiol antioxidant induced CD21-shedding We were interested if the CD21 KHR transfectant would also show enhanced shedding rates after thiol antioxidant treatment. Therefore, CD21 wt and CD21 KHR expressing CH27 cells were challenged for 4 h with a range of GSH and NAC concentrations and subjected to analysis by flow cytometry. Shedding of cell surface CD21 from CD21 KHR expressing cells occurred at lower GSH or NAC concentrations in comparison to CD21 wt expressing cells (Fig. 5A and B). Even at GSH or NAC concentrations as low as 0.1 mM, CD21 KHR expressing cells showed a marked reduction of surface CD21 levels, whereas CD21 wt expressing cells required a concentration of at least 5 mM GSH for significant CD21 cell surface reduction (p < 0.001). Moreover, CD21 levels decreased to a higher extent from the cell surface of CD21 KHR cells, leaving less than 25% of CD21 molecules on the cell surface when treated with 10 mM GSH. In parallel to these experiments, immunofluorescent stainings followed by microscopic analysis of the two transfectants were also performed. Fig. 5C shows that after treatment of the cells with 5 mM GSH for 4 h, both CD21 wt and CD21 KHR cell lines showed a noticeable decrease of CD21 cell surface staining.
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sion was reduced to 40%, and after 4 h, only 20% of the untreated control levels remained. In contrast, all other cell lines reduced CD21 cell surface expression to approximately 75% compared to control levels after 4 h incubation with GSH. Thus, the absence of the cytoplasmic domain resulted in both faster and increased levels of CD21-shedding, indicating a pivotal regulatory role for the cytoplasmic domain in this process. 4. Discussion
Fig. 4. CD21 on mouse B cells is shed upon stimulation with thiol antioxidants. (A) C57BL/6 mouse splenocytes were treated with different concentrations of GSH or NAC for 4 h and subjected to analysis of CD21 cell surface expression by flow cytometry; the B lymphocyte population was analyzed by gating of B220+ cells. The mean of three independent experiments is shown (±S.E.M.; untreated control = 100%). (B) The CD21-transfected CH27 B cell line (CH27CD21 wt) was treated as described in A. Data represent the mean of at least four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells.
As deficiency of the cytoplasmic domain of CD21 had an enhancing effect on CD21-shedding induced by thiol antioxidants as well, we studied the CD21 levels in the other transfected CH27 cell lines (see Table 1) after treatment with 0.5 or 5 mM GSH (Fig. 6A). Similarly, the CD21 KHR cells showed a significant (p < 0.001) decrease of CD21 cell surface staining with 0.5 mM GSH treatment, which was even more pronounced with 5 mM GSH. However, the CD21-shedding observed in the additional transfectants was similar to those in CD21 wt cells. Hence, it appears that the three membrane proximal residues KHR present in the CD21 cytoplasmic domain are sufficient for this effect. Following, the CD21-shedding kinetics were compared from the transfectants expressing the various CD21 cytoplasmic domain truncation mutants by challenging the cells for up to 4 h and measuring CD21 surface staining at different time points. As discerned in Fig. 6B, all cell lines shed CD21 in a time-dependent manner. Remarkably, the CD21 KHR expressing cells lost CD21 expression much faster. After 1 h the CD21 cell surface expres-
The study of animal models, particularly of mice, has also permitted a better understanding of the human immune system. However, the immune systems of mice and men differ in several aspects (Mestas and Hughes, 2004), including the organization of the CD21 locus (Kurtz et al., 1989). We have previously demonstrated that human CD21 is shed under various conditions. In this current study we provide evidence that mouse CD21 is also shed constitutively as well as upon B cell activation through PV and upon thiol antioxidant stimulation in a concentration- and time-dependent manner. We also define, that the cytoplasmic domain is potent modulator of CD21-shedding. There are contradictory reports about whether the cytoplasmic tails of the substrates of metalloproteases are involved in the shedding process (Schlondorff and Blobel, 1999). For example, transforming growth factor (TGF)-␣ and -amyloid precursor protein (APP) require only a juxtamembrane stalk region and no transmembrane or cytoplasmic tail sequences for efficient shedding (Arribas et al., 1997). In contrast, for L1 (Heiz et al., 2004), l-selectin (Smalley and Ley, 2005), and CD44 (Nagano and Saya, 2004) susceptibility to shedding seems to be regulated by their own cytoplasmic domain. We demonstrated earlier that the CD21 SCR16, the extracellular domain adjacent to the transmembrane domain is necessary for induction of shedding (Aichem et al., 2006). Here, we have examined the role of the CD21 cytoplasmic domain and found that removal of this region significantly enhanced shedding. Several possibilities could account for this finding. A negative feed-back loop could be accomplished, e.g. through interaction with the cytoplasmic domain of a membrane-type metalloprotease (Dello Sbarba and Rovida, 2002) or by interaction with another protein such as the formin FHOS/FHOD1 (Gill et al., 2004), thus linking to the cytoskeleton. The CD21 cytoplasmic domain could, as a result, act as an anchor. It was already shown that changes occur in the cytoskeleton upon PV stimulation (Faruki et al., 2000), and upon EBV-activation the CD21 cytoplasmic domain interacts with FHOS/FHOD1 (Gill et al., 2004). In a similar fashion to CD21, truncation of the cytoplasmic domain of L1 led to strongly increased basal shedding rates, and cytochalasin D treatment reduced basal wildtype L1-shedding, implying a direct involvement of the actin–cytoskeleton (Heiz et al., 2004). L1 is also released upon stimulation with PV and it was suggested that ectodomain shedding would occur (i) at the cell surface (mostly basal shedding and PMA-induced shedding, through ADAM17/TACE), and (ii) through so-called exosomes (methyl--cyclodextrin (MCD)-induced). Exosomes are small microvesicles (40–80 nm) that are released from the plasma membrane but originate from late endosomal compartments
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Fig. 5. Cells lacking the CD21 cytoplasmic domain shed CD21 at lower thiol antioxidant concentrations. The two transfected CH27 B cell lines, CD21 wt and CD21 KHR, were stimulated with different concentrations of GSH (A) or NAC (B) for 4 h, and analyzed for CD21 cell surface expression by flow cytometry. Data represent mean values of at least three experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells. (C) Immunofluorescence stainings of CD21 wt and CD21 KHR cells. Cells were treated with 5 mM GSH for 4 h (original magnification 400× for CD21 wt and 630× for CD21 KHR). Scale bar: 20 m.
and may serve as platform for ADAM10-mediated L1-cleavage (Gutwein et al., 2003; Stoeck et al., 2006). Of note, B cell derived exosomes appear to be enriched in MHC class I and II molecules indicating that they are produced in a later phase of B cell activation (Mignot et al., 2006), and complement receptor 1/CD35 is mainly released via ectosomes, another type of membranevesicles, which in contrast to exosomes do not require prior internalization (Pilzer et al., 2005). While our data indicate that CD21 may be shed in the initial phase of B cell activation, it will be interesting to test the hypothesis whether CD21 is also involved in later stages of B cell activation and perhaps released via exosomes or ectosomes as well. Differential regulation of the putatively involved matrix metalloproteinases (MMPs) and serine proteases, or by-passing the serine-dependent proteolysis step (Aichem et al., 2006), may additionally account for the observed differences in the CD21 wt and CD21 KHR shed-
ding and the differences observed using the different types of shedding induction. PV is a phosphotyrosine-specific phosphatase inhibitor and thus changes the intracellular balance between dephosphorylation and phosphorylation. It is also known to mimic BCR signaling and to activate NF-B through tyrosine phosphorylation and degradation of IB␣ (Brummer et al., 2004; Mukhopadhyay et al., 2000). Furthermore, PV acts as oxidant and may regulate MMP function through oxidation of the MMP pro-domain thiol (Ra and Parks, 2007). In order to compare the different cell lines with each other upon PV-stimulation, we calculated the ratio of soluble CD21 to its cell surface expression and thus revealed that removal of the CD21 cytoplasmic domain increased the spontaneous/basal as well as the PV-induced shedding rates of CD21. We found that lower PV concentrations (20 M) up-regulated wildtype CD21 expression, while shed-
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Fig. 6. Influence of truncation and site-directed mutagenesis of the cytoplasmic domain of CD21 on its shedding properties upon thiol antioxidant stimulation. (A) The transfected CH27 cells (see Table 1) were challenged with 0.5 or 5 mM GSH for 4 h and subjected to CD21 cell surface expression analysis by flow cytometry. Data represent mean values of at least four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, ***p < 0.001 vs. data of control cells. (B) The CD21 truncation mutants were treated with 5 mM GSH over a period of 4 h. At the indicated time points cells were analyzed by flow cytometry for CD21 cell surface expression. One representative experiment out of three is shown.
ding was induced with higher concentrations (200 M). Results from PV-treated primary B cells from C57BL/6 mice essentially confirmed our findings with the CH27-CD21 wt cells. CD21 is a known NF-B target gene and it was shown that PMA and LPS, both recognized inducers of NF-B similar to PV, lead to a rapid but only slight upregulation of CD21 (Tolnay et al., 2002). As PV is known to activate B cells (Brummer et al., 2004), we assumed that the induced up-regulation of CD21 expression could mimic the phenotype of marginal zone (MZ-) B cells (CD21hi , CD23− , IgMhi , IgDlow ), which represent a pre-activated cell type that is equipped to rapidly proliferate and to terminally differentiate into antibody-secreting plasma cells within hours. These cells reveal a much lower activation threshold compared to conventional, follicular B cells. This is due to their unique high CD21 expression levels that allow them to respond in a C3d-dependent fashion to T-independent type 2 antigens such as blood borne antigens, with relatively low-avid B cell receptors (Zandvoort and Timens, 2002). Other pre-activated CD21hi cells are represented by human EBV-activated B cells, which also leads to increased sCD21 levels in patients with EBV-associated diseases (Larcher et al., 1995). Furthermore, mitogenic stimulation of murine splenic B cells up-regulates CD21 expression (Axcrona
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et al., 1996), and allergens induce CD21 expression on B cells in patients with allergic asthma (Bonnefoy et al., 1995). Upon terminal B cell differentiation following the GC reaction, CD21 expression is declined (Arens et al., 2004). We therefore hypothesize, that B cell activation may be (at least) a two-step process fine-regulated by CD21 expression and shedding via NF-B as downstream regulator. With the first (or weak; low PV concentrations) signal, a rapid up-regulation of CD21 on the cell surface of na¨ıve B cells occurs to augment the signal, i.e. to pre-activate B cells, and after a second (or stronger; high PV concentrations) signal, CD21 is shed to prevent continued generation of ligand-induced signals after the initial stimulus has been transmitted. This theory is supported by the two-phase model of B cell activation proposed by Baumgarth (2000). Redox-regulation is important for multiple cellular functions, signal transduction and gene expression, especially of immune cells (Pastore et al., 2003). Here, we demonstrated that murine CD21 is also shed upon NAC and GSH stimulation and that truncation of the cytoplasmic domain led to a strong increase in CD21-shedding. As extracellularly applied GSH is not able to enter intact cells (De Flora et al., 2001), NAC does not change the GSH-levels when used on healthy cells (Nishinaka et al., 2001), and we obtained the same results with both agents, we assume that in our system, GSH and NAC act only extracellularly on CD21 or the unknown sheddase or both. Moreover, as neither GSH nor NAC activated the cells (Aichem et al., 2006), we did not expect any transcriptional activation of CD21 as suggested for the treatment with PV. By-passing the need for primary cleavage by a regulatory serine protease might explain the observed faster and increased shedding rates with the CD21 KHR mutant upon thiol antioxidant stimulation. To conclude, we have demonstrated that murine B cells shed CD21 spontaneously as well as upon stimulation and activation. Our data provide further evidence that the thiol antioxidants GSH and NAC and the oxidant PV activate different pathways, perhaps even different proteases, leading to CD21-shedding. A regulatory role of the cytoplasmic domain in CD21-shedding in this process was evident, and we thus suggest that CD21shedding might be a mechanism for the fine-tuning of early stages in B cell activation. Since changes in sCD21 levels in sera are associated with several pathologic conditions (Huemer et al., 1993; Larcher et al., 1995; Lowe et al., 1989; Masilamani et al., 2004a,b) it will be of considerable interest to study those in different knockout or transgenic mouse models. Ultimately, it is of primary importance to identify the yet unidentified CD21 sheddase(s) and to further elucidate the mechanistic details of CD21-shedding. Acknowledgments We would like to thank S. Grotegut for critical comments on the manuscript and helpful advice for the work on the confocal microscope at the Institute of Biochemistry and Genetics, Department of Clinical and Biological Sciences (DKBW), University of Basel, Switzerland. This work was supported by the Thurgauische Stiftung fuer Wissenschaft und Forschung and the EU through grant LSHM-CT-2004-005264.
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Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain Melanie M. Hoefer a,1 , Annette Aichem a , Andrew M. Knight b , Harald Illges c,∗ a
Biotechnology Institute Thurgau, 8280 Kreuzlingen, Switzerland School of Surgical and Reproductive Sciences, Institute of Cellular Medicine, The Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom c University of Applied Sciences, Department of Natural Sciences, Immunology and Cell Biology, von-Liebig-Str. 20, 53359 Rheinbach, Germany b
Received 23 November 2006; received in revised form 6 November 2007; accepted 14 December 2007
Abstract Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation. © 2007 Elsevier Ltd. All rights reserved. Keywords: B cell activation; Complement receptor 2/CD21; Glutathione; Pervanadate; Shedding
1. Introduction Complement receptor 2 (CR2/CD21) is a type I transmembrane glycoprotein of about 145 kDa that binds complement fragments (C3d(g), iC3b) and interferon (IFN)-␣, and acts as the Epstein–Barr virus (EBV) receptor on human B lymphocytes (Asokan et al., 2006; Carroll, 2004; Rickert, 2005; Tedder et al., 2002). In mice, the complement receptors 1 and 2 (CR1/CR2, CD35/CD21) are encoded by the same locus (Cr2) and the two different products are generated by alternative splicing of the mRNA. CD21 is a part of the membrane protein complex comprising CD19/CD21/CD81, which enhances B cell antigen receptor (BCR) signaling in response to complement-coated
Abbreviations: GSH, glutathione; NAC, N-acetylcysteine; PV, pervanadate; MMP, matrix metalloproteinase; SCR, short consensus repeat. ∗ Corresponding author. Tel.: +49 2241 865570; fax: +49 2241 8658570. E-mail address: [email protected] (H. Illges). 1 Present address: The Burnham Institute for Medical Research, Infectious and Inflammatory Disease Center, La Jolla, CA 92037, USA. 0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.12.015
antigens by several orders of magnitude. CD21/CD35-deficient mice reveal decreased antibody production and impaired germinal center (GC) formation in response to T cell-dependent and -independent antigens (Carroll, 2004; Rickert, 2005). The controlled proteolytic cleavage of cell surface proteins and the resulting release of the ectodomain as a soluble protein define a process termed “shedding”. Several different types of proteins have been reported to be regulated by this mechanism. In fact, 2–4% of all cell surface proteins are shed by members of the well over 500 proteases comprising mammalian “degradome”. Importantly, the soluble forms of the respective proteins retain their biological activity and have regulatory functions (Arribas and Merlos-Su´arez, 2003; Dello Sbarba and Rovida, 2002; Overall and Blobel, 2007). This is also true for CD21 (Fremeaux-Bacchi et al., 1999). The major form of soluble (s)CD21 has been identified as a single molecular species of 126 kDa (Masilamani et al., 2002). It has also been described as a marker of B cell activation in humans and was shown to inhibit EBV-binding and -infection of B cells (Huemer et al., 1993; Nemerow et al., 1990). The immune
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modulatory properties of sCD21 were further revealed by its binding to the low-affinity IgE-receptor CD23 and sCD23, thereby regulating monocyte activation and differentiation, as well as IgE-responses (Fremeaux-Bacchi et al., 1998a,b). Moreover, recombinant sCD21 suppresses the antibody response to T cell-dependent antigens in mice (Hebell et al., 1991), and inhibition of B cell/follicular dendritic cell (FDC) interactions with sCD21 reduced antibody responses by 10–1000-fold (Qin et al., 1998). sCD21 levels are often altered in pathologic conditions including various lymphoproliferative leukemias, such as chronic B lymphocytic leukemia (B-CLL) (Lowe et al., 1989), acute EBV-infection and other virus-associated diseases (Huemer et al., 1993; Larcher et al., 1995), and autoimmune diseases (Masilamani et al., 2004a,b). While CD21 is constitutively shed, B cell activation through mitogen (PMA + calcium ionophore), anti-IgM + anti-CD40 cross-linking (Masilamani et al., 2003), or pervanadate (PV) stimulation (Aichem et al., 2006) leads to enhanced human CD21-shedding, whereas cell surface expression of another member of the B cell co-receptor complex, CD19, remained at a constant level (Hoefer and Illges, unpublished data). In addition, we could demonstrate that CD21-shedding might be a redoxregulated process that can be induced with the thiol antioxidants glutathione (GSH), N-acetylcysteine (NAC), and the oxidant PV in CD21-transfected HEK293 cells, human peripheral blood mononuclear cells (PBMC) and the Burkitt lymphoma line Daudi (Aichem et al., 2006). The process involves both a serine and a metalloprotease activity. The reducing agents GSH and NAC do not induce tyrosine phosphorylation and cell activation, but rather directly activate the metalloprotease or modify intramolecular disulfide bridges within the extracellular domain of CD21 or both, and thereby facilitate access to the CD21 cleavage site (Aichem et al., 2006). In contrast, the tyrosine-specific phosphatase inhibitor and oxidant PV mimics BCR signaling and operates through activation of a signaling pathway that comprises the B cell adaptor SLP-65 (SH2-domain-containing leukocyte protein of 65 kDa), the protein kinases Syk and Lyn (Wienands et al., 1996,1998), and further downstream, protein kinase C (PKC) (Aichem et al., 2006). PV was shown to induce protein tyrosine phosphorylation, cell activation, and shedding of members of very diverse protein families, such as the ErbB-family tyrosine kinase HER4/ErbB4, the heparan sulfate proteoglycan syndecan-1 (CD138), the cell adhesion molecule L1 (CD171), and the heparin-binding EGF-like growth factor (HB-EGF) (Blobel, 2005; Brummer et al., 2004; Dello Sbarba and Rovida, 2002; Reth, 2002). Recently, the involvement of the Erk1/2-mitogen-activated protein kinase pathway downstream of PKC was shown to regulate PV-induced neural cell adhesion molecule (NCAM)-shedding (Hinkle et al., 2006). The mechanism of how shedding is regulated is currently not well understood, however, several cell surface proteins require their cytoplasmic region to control their shedding rates, and an involvement of the actin–cytoskeleton has e.g. been shown for 160 pervanadate (PV) (Heiz et al., 2004), l-selectin (CD62L) (Smalley and Ley, 2005), and CD44 (Nagano and Saya, 2004) among others.
The cytoplasmic domain of CD21 has been implicated in antigen presentation (Barrault and Knight, 2004), B lymphocyte signaling (Bouillie et al., 1999), and may interact with the cytoskeleton through the formin homologue overexpressed in spleen FHOS/FHOD1 (Gill et al., 2004). FHOS/FHOD1 regulates gene transcription, actin–cytoskeleton structure, and cell migration (Koka et al., 2003). In the present study, we addressed the question whether B cell activation would trigger murine CD21-shedding, and whether the process involves redox regulation. For this, we stimulated B cells with PV and the thiol antioxidants GSH and NAC. We further examined the role of the cytoplasmic domain in CD21-shedding using mouse B cells expressing different CD21 cytoplasmic domain truncation mutants as well as CD21 containing point mutations where the three tyrosines in the cytoplasmic domain were altered to alanines or phenylalanines. 2. Material and methods 2.1. Cell purification and culture Splenocytes were prepared from 4- to 12-month-old male C57BL/6 mice. Spleens were homogenized, the resulting cell suspensions filtered and loaded onto a Ficoll gradient (Biochrom, Berlin, Germany). The isolated lymphocytes were washed twice with fresh serum-free medium (RPMI 1640) and then treated as described below. To exclude the possibility of measuring shedding of CD35 and soluble CD35 and to study the role of the CD21 cytoplasmic domain, we also used the CD21/CD35 negative murine B cell line CH27 (Molina et al., 1990), stably transfected with various CD21 constructs containing truncated or mutated cytoplasmic domains as previously described (Barrault and Knight, 2004) (see Table 1). Cells were maintained at 37 ◦ C with 5% CO2 in RPMI 1640 supplemented with UltraGlutamine 1, penicillin/streptomycin (100 U/ml per 100 g/ml), non-essential amino acids (all Cambrex, Verviers, Belgium), 50 M mercaptoethanol (Sigma, Buchs, Switzerland) and 10% fetal calf serum (Linaris, Wertheim-Bettingen, Germany). In addition, 0.75 mg/ml G418 (Calbiochem, Lucerne, Switzerland) was included in the culture of the transfected CH27 cells. For those experiments using CD21 KHR expressing cells, two independent clones (IB2 and IB4) were used. The results from these two clones were nearly identical and those from clone IB2 are depicted. 2.2. Induction of CD21-shedding Freshly isolated splenocytes, CH27 and the different CH27-CD21 transfectants were adjusted to a cell density of 2 × 106 ml−1 and incubated for 2 h at 37 ◦ C in serum-free RPMI 1640. After this period the medium was removed, and fresh RPMI 1640 was added together with various concentrations of reduced l-glutathione, N-acetylcysteine (both Sigma), or pervanadate and incubated for additional 4 h at 37 ◦ C unless indicated otherwise. PV was freshly prepared for each experiment by mixing one volume of sodium orthovanadate (Na3 VO4 ,
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Table 1 CD21 constructs used in this study (Barrault and Knight, 2004)
Residues in the murine CD21 cytoplasmic domain sequence are numbered according to the specifications of Molina et al. (1990), starting at the first isoleucine in SCR1. The humans sequence is shown for comparison. H.s., Homo sapiens; M.m., Mus musculus.
100 mM, pH 10) with one volume of H2 O2 (100 mM) and was used within 10 min of preparation. After incubation, cells were subjected to analysis of CD21 cell surface expression by flow cytometry and the cell culture supernatants were collected for ELISA analysis.
strate solution (Sigma) was added and incubated until blue color developed. Following the addition of 1 M H2 SO4 to stop the reaction, the absorption was measured at 450 nm (reference wavelength 690 nm) using a Tecan Sunrise microplate reader (Tecan, Maennedorf, Switzerland).
2.3. Determination of cell surface CD21 by flow cytometry
2.5. Immunofluorescence stainings and microscopy
For analysis of cell surface expression of CD21, cells were stained with anti-CD21/CD35-PE antibody, clone 7G6 (BD Biosciences, Basel, Switzerland) and subjected to flow cytometry analysis (BD LSR) using CellQuest software (both BD Biosciences). The viability of the cells was determined by TO-PRO-3-iodide exclusion (Molecular Probes, Leiden, Netherlands). The murine B cell marker B220 (anti-B220-FITC, BD Biosciences) was used for gating the B cell population in the splenocyte preparations of C57BL/6 mice. Only living gated cells were used for analysis, and viability of the cells did not decrease more than 5–10% upon stimulation.
Cells were pre-incubated with serum-free RPMI 1640 for 2 h as described above and then allowed to adhere on poly-llysine (Sigma) coated glass cover slips during the 4 h incubation time. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. The cells were stained with anti-CD21 (clone 7E9) and either Alexa Fluor 546- or Alexa Fluor 488-conjugated secondary goat anti-rat antibodies, and nuclei were stained with DAPI (all Molecular Probes). Stained cells were mounted with mowiol on microscope slides and examined with a Zeiss LSM 510 Meta laser scanning confocal microscope, using a Plan-Neofluar 40×/1.4 oil DIC and a Plan-Apochromat 63×/1.4 oil DIC objective (Zeiss, Jena, Germany).
2.4. Determination of sCD21 by sandwich ELISA 2.6. Statistical analysis and other calculations ELISA plates (TPP, Trasadingen, Switzerland) were coated overnight at 4 ◦ C with rat anti-mouse CD21/CD35 antibody, clone 7G6 (Kinoshita et al., 1988) (2 g/ml in PBS). After blocking with PBS/3% BSA for 2 h at room temperature (RT), plates were washed with PBS/0.05% Tween-20. Then, cell culture supernatants were added and incubated overnight at 4 ◦ C. After washing, biotinylated rat anti-mouse CD21/CD35 antibody, clone 7E9 (Kinoshita et al., 1988) was added (2 g/ml in PBS/3% BSA) and incubated for 1 h at RT. After another washing step, plates were incubated for 30 min at RT with streptavidin–peroxidase (Biosource, Nivelles, Belgium; 0.5 g/ml in PBS/3% BSA). After washing, TMB liquid sub-
All experiments were performed at least three times. Values in figures are given as means ± S.E.M. Statistical analysis was performed using GraphPad InStat (GraphPad Software, San Diego, CA, USA). Unpaired parametric testing with the Tukey–Kramer multiple comparisons test (Fig. 1A, 3, 4, 5A and B, 6A) or t-test (Fig. 2A and B) was performed. Significantly different values are indicated (*p < 0.05; **p < 0.01; ***p < 0.001). In Figs. 2C and 3B the amounts of sCD21 released from untreated cells were set to 100%, therefore, increased amounts of sCD21 from treated cells are reflected in greater percentage values.
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Fig. 1. Murine CD21 cell surface expression is modulated by pervanadate. (A) The transfected murine CH27 B cell lines CD21 wt and CD21 KHR were stimulated for 4 h with various PV concentrations and analyzed by flow cytometry for CD21 cell surface expression. Data represent mean values of at least three experiments (±S.E.M.; untreated control = 100%). *p < 0.05, ***p < 0.001 vs. data of control cells. (B) Immunofluorescence of CD21 wt and CD21 KHR cells stimulated with 20 or 200 M PV for 4 h followed by staining for CD21 (original magnification 630×). Scale bar: 20 m.
3. Results 3.1. Pervanadate regulates the cell surface expression of murine CD21 The oxidant and tyrosine-specific phosphatase inhibitor PV is an established agent to induce shedding of diverse cell surface molecules (Blobel, 2005). In B cells, PV induces a similar signaling pathway as generated by antigen stimulation (Reth, 2002). Thus, we determined if PV was also able to induce murine CD21-shedding. For this, the CD21/CD35 negative B cell line CH27 that had been previously transfected with murine wildtype CD21 (CD21 wt), was used (Barrault and Knight,
2004). In addition, the role of the CD21 cytoplasmic domain was investigated using CH27 cells transfected with a truncated version, CD21 KHR, where the CD21 cytoplasmic domain consists only of the three transmembrane adjacent residues, lysine (K), histidine (H) and arginine (R) (Barrault and Knight, 2004) (see Table 1). After challenging the cells for 4 h with a range of PV concentrations, CD21 cell surface expression was measured by flow cytometry (Fig. 1A). Due to differences in CD21 cell surface staining of CD21 wt compared to the CD21 KHR expressing cells all results are depicted in relative amounts (%) (see Table 2). Whilst lower concentrations of PV initially caused an increase in wildtype CD21 surface expression, higher concentrations caused a significant decrease
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Fig. 2. Time dependency of pervanadate-induced shedding. (A) Splenocytes of 10 spleens from C57BL/6 mice were pooled and treated with 20 M or 200 M PV for 4 h. At the indicated time points, cells were analyzed for CD21 cell surface expression by flow cytometry, and gates were set to the B220+ B cells. (B) CD21 wt and CD21 KHR cells were stimulated with 200 M PV for 4 h (t0 = 100%). At the indicated time points, cells were analyzed by flow cytometry and the cell culture supernatants were used for ELISA analysis (C). Data represent the mean of four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 significance of differences between CD21 wt and CD21 KHR cells, calculated by unpaired t-test.
(Fig. 1A). In contrast, at higher concentrations the CD21 cell surface expression in cells expressing the CD21 KHR cytoplasmic domain mutant was not down-regulated to the same degree as compared to CD21 wt expressing cells. This difference of PV-induced CD21 regulation was also observed when the cells were examined by immunofluorescence stainings (Fig. 1B).
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Fig. 3. Influence of truncation and site-directed mutagenesis of the cytoplasmic domain of CD21 on its shedding properties upon pervanadate stimulation. (A) Various CH27 transfectants (see Table 1) were treated with 20 or 200 M PV for 4 h and subjected to analysis by flow cytometry for measurement of CD21 surface levels. Data represent mean values of four experiments (±S.E.M.; untreated control = 100%). (B) Quantitation of sCD21 present in the cell culture supernatants as determined by capture ELISA (±S.E.M., untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells. (C) From the raw data of B and A the ratios of soluble CD21 to cell surface expression of CD21 were calculated (±S.E.M.), showing that the CD21 KHR cells shed more CD21 both spontaneously and induced compared to all other cell lines. **p < 0.01, ***p < 0.001 significance of differences between CD21 wt and truncated or site-directed mutant cells with the respective treatment.
3.2. The reaction to pervanadate is time dependent In order to determine a time-dependent effect of PV treatment on CD21 levels on B cells we next performed kinetic studies. Splenocytes isolated from C57BL/6 mice were incubated with 20 or 200 M PV up to 4 h, and CD21 cell surface expression was measured at the indicated time points on the B220+ gated cells (Fig. 2A). After 15 min of stimulation, the B cells showed a slight peak of cell surface CD21 expression with both PV
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concentrations, which declined rapidly with 200 M PV, and stabilized after 2–3 h to a level around 70% of control. With the lower PV concentration, the cells showed a similar increase within the first 15 min of stimulation, followed by a drop to almost basal levels and finally increased again to approximately 10% above control levels. In parallel experiments, we also followed the kinetics of CD21 cell surface expression of CD21 wt and CD21 KHRtransfected CH27 B cells after treatment with 200 M PV (Fig. 2B). The initial peak of expression seen in ex vivo B cells was not detected with the CD21 expressing cell lines (Fig. 2B). However, a similar rate of surface CD21-reduction was observed in the cells expressing CD21 wt. Noticeably, this reduction was faster than in the CD21 KHR cells. After 4 h, the CD21 cell surface expression was reduced by 50% in both cell types. Although previous studies have failed to detect ligand-induced CD21 internalization, unless cross-linked to a functional BCR (Barrault and Knight, 2004), we were keen to confirm that this down-regulation was indeed a result of shedding rather than internalization. We therefore used an ELISA to assess the presence of soluble CD21 in the supernatant of the treated cells and determined that the relative amounts of sCD21 shed from the CD21 KHR expressing cells were only slightly elevated when compared to the levels obtained from CD21 wt expressing cells (Fig. 2C). Thus, PV stimulation of B cells induced an up-regulation of cell surface CD21 at lower concentrations and induced cytoplasmic domain-dependent CD21 ectodomain shedding at higher concentrations. 3.3. Deletion of the full CD21 cytoplasmic domain has an enhancing effect on CD21 basal and induced shedding rates To identify the regions of the CD21 cytoplasmic domain that may be responsible for the opposed effects of high and low PV concentrations, the CD21 levels in several additional transfected CH27 cell lines expressing various other cytoplasmic domain truncations or point mutations within this domain (see Table 1) were studied after treatment for 4 h with 20 or 200 M PV (Fig. 3A). To control for the differences seen in CD21 expresTable 2 Median fluorescence intensities of the different CD21-transfected CH27 B cell lines Cell line
CD21 median fluorescence intensities (mean ± S.E.M. (n))
CH27 CD21 wt CD21 YSI CD21 LET CD21 YYTK CD21 KHR (IB2) CD21 KHR (IB4) CD21 YYFF CD21 YYAA CD21 YF CD21 YA
24 205 185 169 160 114 141 172 216 123 195
± ± ± ± ± ± ± ± ± ± ±
3 (9) 19 (18) 18 (8) 22 (8) 34 (7) 12 (16) 22 (7) 22 (7) 39 (7) 12 (7) 24 (7)
sion levels in the different untreated transfectants (see Table 2), the respective untreated CD21 levels were converted to 100% CD21 cell surface expression. In addition, sCD21 in the cell culture supernatants was also measured by ELISA (Fig. 3B) and the values calculated in percentages, with sCD21 levels released from untreated cells set to 100%. All transfectants up-regulated CD21 upon 20 M PV stimulation, except for the CD21 KHR expressing cells. With 200 M PV treatment, CD21 cell surface staining was lowered in all lines as well, although to a lesser degree from the CD21 KHR cells and the site-directed mutants. The sCD21 levels were in a comparable range for all cell lines. In order to compare the actual shedding rates of all cell lines, ratios of the cell surface expression and the respective ssCD21 data measured in ELISA were calculated from the raw data of this experiment (Fig. 3C). From this calculation it was evident that the CD21 KHR transfectant was unique in its shedding properties, showing higher basal shedding as well as induced shedding rates as compared to all other cell lines. 3.4. Shedding of murine CD21 can be induced by thiol antioxidants To determine whether murine B cells also shed CD21 upon stimulation with the thiol antioxidants GSH and NAC, we compared CD21 levels on primary B cells obtained from spleens of C57BL/6 mice with CH27-CD21 wt cells. After challenging the cells for 4 h with the respective stimuli the cell surface expression of CD21 was measured by flow cytometry. Upon GSH and NAC treatment, CD21 cell surface expression was down-modulated in both B220+ gated splenocytes (Fig. 4A) and CH27-CD21 wt cells (Fig. 4B) in a concentration-dependent manner. These findings confirm our results from human CD21-expressing cells (Aichem et al., 2006). 3.5. Influence of the cytoplasmic domain of CD21 on thiol antioxidant induced CD21-shedding We were interested if the CD21 KHR transfectant would also show enhanced shedding rates after thiol antioxidant treatment. Therefore, CD21 wt and CD21 KHR expressing CH27 cells were challenged for 4 h with a range of GSH and NAC concentrations and subjected to analysis by flow cytometry. Shedding of cell surface CD21 from CD21 KHR expressing cells occurred at lower GSH or NAC concentrations in comparison to CD21 wt expressing cells (Fig. 5A and B). Even at GSH or NAC concentrations as low as 0.1 mM, CD21 KHR expressing cells showed a marked reduction of surface CD21 levels, whereas CD21 wt expressing cells required a concentration of at least 5 mM GSH for significant CD21 cell surface reduction (p < 0.001). Moreover, CD21 levels decreased to a higher extent from the cell surface of CD21 KHR cells, leaving less than 25% of CD21 molecules on the cell surface when treated with 10 mM GSH. In parallel to these experiments, immunofluorescent stainings followed by microscopic analysis of the two transfectants were also performed. Fig. 5C shows that after treatment of the cells with 5 mM GSH for 4 h, both CD21 wt and CD21 KHR cell lines showed a noticeable decrease of CD21 cell surface staining.
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sion was reduced to 40%, and after 4 h, only 20% of the untreated control levels remained. In contrast, all other cell lines reduced CD21 cell surface expression to approximately 75% compared to control levels after 4 h incubation with GSH. Thus, the absence of the cytoplasmic domain resulted in both faster and increased levels of CD21-shedding, indicating a pivotal regulatory role for the cytoplasmic domain in this process. 4. Discussion
Fig. 4. CD21 on mouse B cells is shed upon stimulation with thiol antioxidants. (A) C57BL/6 mouse splenocytes were treated with different concentrations of GSH or NAC for 4 h and subjected to analysis of CD21 cell surface expression by flow cytometry; the B lymphocyte population was analyzed by gating of B220+ cells. The mean of three independent experiments is shown (±S.E.M.; untreated control = 100%). (B) The CD21-transfected CH27 B cell line (CH27CD21 wt) was treated as described in A. Data represent the mean of at least four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells.
As deficiency of the cytoplasmic domain of CD21 had an enhancing effect on CD21-shedding induced by thiol antioxidants as well, we studied the CD21 levels in the other transfected CH27 cell lines (see Table 1) after treatment with 0.5 or 5 mM GSH (Fig. 6A). Similarly, the CD21 KHR cells showed a significant (p < 0.001) decrease of CD21 cell surface staining with 0.5 mM GSH treatment, which was even more pronounced with 5 mM GSH. However, the CD21-shedding observed in the additional transfectants was similar to those in CD21 wt cells. Hence, it appears that the three membrane proximal residues KHR present in the CD21 cytoplasmic domain are sufficient for this effect. Following, the CD21-shedding kinetics were compared from the transfectants expressing the various CD21 cytoplasmic domain truncation mutants by challenging the cells for up to 4 h and measuring CD21 surface staining at different time points. As discerned in Fig. 6B, all cell lines shed CD21 in a time-dependent manner. Remarkably, the CD21 KHR expressing cells lost CD21 expression much faster. After 1 h the CD21 cell surface expres-
The study of animal models, particularly of mice, has also permitted a better understanding of the human immune system. However, the immune systems of mice and men differ in several aspects (Mestas and Hughes, 2004), including the organization of the CD21 locus (Kurtz et al., 1989). We have previously demonstrated that human CD21 is shed under various conditions. In this current study we provide evidence that mouse CD21 is also shed constitutively as well as upon B cell activation through PV and upon thiol antioxidant stimulation in a concentration- and time-dependent manner. We also define, that the cytoplasmic domain is potent modulator of CD21-shedding. There are contradictory reports about whether the cytoplasmic tails of the substrates of metalloproteases are involved in the shedding process (Schlondorff and Blobel, 1999). For example, transforming growth factor (TGF)-␣ and -amyloid precursor protein (APP) require only a juxtamembrane stalk region and no transmembrane or cytoplasmic tail sequences for efficient shedding (Arribas et al., 1997). In contrast, for L1 (Heiz et al., 2004), l-selectin (Smalley and Ley, 2005), and CD44 (Nagano and Saya, 2004) susceptibility to shedding seems to be regulated by their own cytoplasmic domain. We demonstrated earlier that the CD21 SCR16, the extracellular domain adjacent to the transmembrane domain is necessary for induction of shedding (Aichem et al., 2006). Here, we have examined the role of the CD21 cytoplasmic domain and found that removal of this region significantly enhanced shedding. Several possibilities could account for this finding. A negative feed-back loop could be accomplished, e.g. through interaction with the cytoplasmic domain of a membrane-type metalloprotease (Dello Sbarba and Rovida, 2002) or by interaction with another protein such as the formin FHOS/FHOD1 (Gill et al., 2004), thus linking to the cytoskeleton. The CD21 cytoplasmic domain could, as a result, act as an anchor. It was already shown that changes occur in the cytoskeleton upon PV stimulation (Faruki et al., 2000), and upon EBV-activation the CD21 cytoplasmic domain interacts with FHOS/FHOD1 (Gill et al., 2004). In a similar fashion to CD21, truncation of the cytoplasmic domain of L1 led to strongly increased basal shedding rates, and cytochalasin D treatment reduced basal wildtype L1-shedding, implying a direct involvement of the actin–cytoskeleton (Heiz et al., 2004). L1 is also released upon stimulation with PV and it was suggested that ectodomain shedding would occur (i) at the cell surface (mostly basal shedding and PMA-induced shedding, through ADAM17/TACE), and (ii) through so-called exosomes (methyl--cyclodextrin (MCD)-induced). Exosomes are small microvesicles (40–80 nm) that are released from the plasma membrane but originate from late endosomal compartments
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Fig. 5. Cells lacking the CD21 cytoplasmic domain shed CD21 at lower thiol antioxidant concentrations. The two transfected CH27 B cell lines, CD21 wt and CD21 KHR, were stimulated with different concentrations of GSH (A) or NAC (B) for 4 h, and analyzed for CD21 cell surface expression by flow cytometry. Data represent mean values of at least three experiments (±S.E.M.; untreated control = 100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. data of control cells. (C) Immunofluorescence stainings of CD21 wt and CD21 KHR cells. Cells were treated with 5 mM GSH for 4 h (original magnification 400× for CD21 wt and 630× for CD21 KHR). Scale bar: 20 m.
and may serve as platform for ADAM10-mediated L1-cleavage (Gutwein et al., 2003; Stoeck et al., 2006). Of note, B cell derived exosomes appear to be enriched in MHC class I and II molecules indicating that they are produced in a later phase of B cell activation (Mignot et al., 2006), and complement receptor 1/CD35 is mainly released via ectosomes, another type of membranevesicles, which in contrast to exosomes do not require prior internalization (Pilzer et al., 2005). While our data indicate that CD21 may be shed in the initial phase of B cell activation, it will be interesting to test the hypothesis whether CD21 is also involved in later stages of B cell activation and perhaps released via exosomes or ectosomes as well. Differential regulation of the putatively involved matrix metalloproteinases (MMPs) and serine proteases, or by-passing the serine-dependent proteolysis step (Aichem et al., 2006), may additionally account for the observed differences in the CD21 wt and CD21 KHR shed-
ding and the differences observed using the different types of shedding induction. PV is a phosphotyrosine-specific phosphatase inhibitor and thus changes the intracellular balance between dephosphorylation and phosphorylation. It is also known to mimic BCR signaling and to activate NF-B through tyrosine phosphorylation and degradation of IB␣ (Brummer et al., 2004; Mukhopadhyay et al., 2000). Furthermore, PV acts as oxidant and may regulate MMP function through oxidation of the MMP pro-domain thiol (Ra and Parks, 2007). In order to compare the different cell lines with each other upon PV-stimulation, we calculated the ratio of soluble CD21 to its cell surface expression and thus revealed that removal of the CD21 cytoplasmic domain increased the spontaneous/basal as well as the PV-induced shedding rates of CD21. We found that lower PV concentrations (20 M) up-regulated wildtype CD21 expression, while shed-
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Fig. 6. Influence of truncation and site-directed mutagenesis of the cytoplasmic domain of CD21 on its shedding properties upon thiol antioxidant stimulation. (A) The transfected CH27 cells (see Table 1) were challenged with 0.5 or 5 mM GSH for 4 h and subjected to CD21 cell surface expression analysis by flow cytometry. Data represent mean values of at least four experiments (±S.E.M.; untreated control = 100%). *p < 0.05, ***p < 0.001 vs. data of control cells. (B) The CD21 truncation mutants were treated with 5 mM GSH over a period of 4 h. At the indicated time points cells were analyzed by flow cytometry for CD21 cell surface expression. One representative experiment out of three is shown.
ding was induced with higher concentrations (200 M). Results from PV-treated primary B cells from C57BL/6 mice essentially confirmed our findings with the CH27-CD21 wt cells. CD21 is a known NF-B target gene and it was shown that PMA and LPS, both recognized inducers of NF-B similar to PV, lead to a rapid but only slight upregulation of CD21 (Tolnay et al., 2002). As PV is known to activate B cells (Brummer et al., 2004), we assumed that the induced up-regulation of CD21 expression could mimic the phenotype of marginal zone (MZ-) B cells (CD21hi , CD23− , IgMhi , IgDlow ), which represent a pre-activated cell type that is equipped to rapidly proliferate and to terminally differentiate into antibody-secreting plasma cells within hours. These cells reveal a much lower activation threshold compared to conventional, follicular B cells. This is due to their unique high CD21 expression levels that allow them to respond in a C3d-dependent fashion to T-independent type 2 antigens such as blood borne antigens, with relatively low-avid B cell receptors (Zandvoort and Timens, 2002). Other pre-activated CD21hi cells are represented by human EBV-activated B cells, which also leads to increased sCD21 levels in patients with EBV-associated diseases (Larcher et al., 1995). Furthermore, mitogenic stimulation of murine splenic B cells up-regulates CD21 expression (Axcrona
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et al., 1996), and allergens induce CD21 expression on B cells in patients with allergic asthma (Bonnefoy et al., 1995). Upon terminal B cell differentiation following the GC reaction, CD21 expression is declined (Arens et al., 2004). We therefore hypothesize, that B cell activation may be (at least) a two-step process fine-regulated by CD21 expression and shedding via NF-B as downstream regulator. With the first (or weak; low PV concentrations) signal, a rapid up-regulation of CD21 on the cell surface of na¨ıve B cells occurs to augment the signal, i.e. to pre-activate B cells, and after a second (or stronger; high PV concentrations) signal, CD21 is shed to prevent continued generation of ligand-induced signals after the initial stimulus has been transmitted. This theory is supported by the two-phase model of B cell activation proposed by Baumgarth (2000). Redox-regulation is important for multiple cellular functions, signal transduction and gene expression, especially of immune cells (Pastore et al., 2003). Here, we demonstrated that murine CD21 is also shed upon NAC and GSH stimulation and that truncation of the cytoplasmic domain led to a strong increase in CD21-shedding. As extracellularly applied GSH is not able to enter intact cells (De Flora et al., 2001), NAC does not change the GSH-levels when used on healthy cells (Nishinaka et al., 2001), and we obtained the same results with both agents, we assume that in our system, GSH and NAC act only extracellularly on CD21 or the unknown sheddase or both. Moreover, as neither GSH nor NAC activated the cells (Aichem et al., 2006), we did not expect any transcriptional activation of CD21 as suggested for the treatment with PV. By-passing the need for primary cleavage by a regulatory serine protease might explain the observed faster and increased shedding rates with the CD21 KHR mutant upon thiol antioxidant stimulation. To conclude, we have demonstrated that murine B cells shed CD21 spontaneously as well as upon stimulation and activation. Our data provide further evidence that the thiol antioxidants GSH and NAC and the oxidant PV activate different pathways, perhaps even different proteases, leading to CD21-shedding. A regulatory role of the cytoplasmic domain in CD21-shedding in this process was evident, and we thus suggest that CD21shedding might be a mechanism for the fine-tuning of early stages in B cell activation. Since changes in sCD21 levels in sera are associated with several pathologic conditions (Huemer et al., 1993; Larcher et al., 1995; Lowe et al., 1989; Masilamani et al., 2004a,b) it will be of considerable interest to study those in different knockout or transgenic mouse models. Ultimately, it is of primary importance to identify the yet unidentified CD21 sheddase(s) and to further elucidate the mechanistic details of CD21-shedding. Acknowledgments We would like to thank S. Grotegut for critical comments on the manuscript and helpful advice for the work on the confocal microscope at the Institute of Biochemistry and Genetics, Department of Clinical and Biological Sciences (DKBW), University of Basel, Switzerland. This work was supported by the Thurgauische Stiftung fuer Wissenschaft und Forschung and the EU through grant LSHM-CT-2004-005264.
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