Results

VII.1.3.1 Pervanadate regulates the cell surface expression of CD21 on murine B-cells

The oxidant PV is an established agent to induce shedding of diverse cell surface molecules (135), and shedding often involves cell signaling and PKC-activation (134,237). Thus, we were interested to determine if PV was also able to induce murine CD21-shedding. For this, the CD21 negative B-cell line CH27 that had been previously transfected with murine wildtype CD21 (CD21 wt) was used (62). In addition, the role of the cytoplasmic domain was investigated using CH27 cells transfected with a truncated version of CD21 (CD21 KHR (62)) where the CD21 cytoplasmic domain consists only of the three transmembrane adjacent residues, lysine (K), histidine (H) and arginine (R) (see Table 1).

Table 1

CD21 constructs used in this study (62).

Residues in the murine sequence are numbered according to the specifications of Molina et al. (247), starting at the first isoleucine in short consensus repeat (SCR) 1.

H.s., Homo sapiens; M.m., Mus musculus.

After challenging the cells for 4 hours with a range of PV concentrations, the cell surface expression of CD21 was measured via flow cytometry (Figure 11A). It can be seen in Figure 11A that whilst lower concentrations of PV initially cause an increase in wt CD21 surface expression, higher concentrations cause a significant decrease.

Figure 11

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 different PV-concentrations and analyzed by flow cytometry for CD21 surface expression. Data represent mean values of at least 3 experiments (+ SEM; untreated control = 100%). Significance of differences between treated and control cells. (B) Immunofluorescence of CD21 wt and CD21 KHR cells stimulated with 20 or 200 µM PV for 4 h followed by staining for CD21 (original magnification

In addition, at the higher concentrations the CD21 cell surface expression in cells transfected with the KHR cytoplasmic domain mutant was not down-regulated to the same degree as compared to wt CD21-expressing cells. This difference seen in PV-induced CD21 regulation between cells expressing either the wt or the KHR CD21 was also observed when the cells were examined by using immunofluorescence (Figure 11B).

VII.1.3.2 Time dependency of the reaction to pervanadate

In order to ascertain if the timing of PV-treatment effected the CD21 levels on B-cells we performed the following kinetic study. Splenocytes isolated from C57/BL6 mice were incubated with 20 and 200 μM PV for up to 4 hours and the cell surface expression of CD21 was measured at the indicated time points on B220⁺ gated cells (Figure 12A). After 15 min of stimulation, the B-cells showed a slight peak of cell surface CD21-expression with both PV-concentrations which declined rapidly at 200 μM PV, and stabilized after 2-3 hours 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 and followed with a drop to a little above control levels and finally again increased to approximately 10% above control levels.

In parallel experiments we also followed the kinetics of CD21 levels of the two CH27 transfectants after treatment with 200 μM PV (Figure 12B). The initial peak of expression seen in ex vivo B-cells was not detectable with the transfected cells (Figure 12B). However, a similar rate of surface CD21-reduction was seen in the cells expressing CD21 wt. Noticeably this rate was faster than that seen in the CD21 KHR cells, while after 4 hours the CD21-reduction from the cell surface was almost identical in the two cell types, with both demonstrating approximately 50% less CD21 surface expression than the untreated control cells. Although previous studies have failed to detect ligand-induced CD21 internalization (unless cross-linked to a functional BCR (62)), we were keen to confirm that this down-regulation was indeed a result of shedding rather than internalization. We therefore used an ELISA to demonstrate the presence of soluble (s)CD21 in the supernatant of the treated cells (Figure 12C). It can be seen that CD21 KHR cells released more soluble CD21 compared to the levels obtained from CD21 wt cells.

0 15 30 60 120 180 240

Time dependency of pervanadate-induced shedding.

(A) Splenocytes of 10 spleens from C57BL/6 mice were pooled and treated with 20 µM or 200 µM 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 µM 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 4 experiments (+ SEM). Significance of differences between CD21 wt and CD21 KHR cells was calculated by unpaired t-test.

Thus, PV-stimulation of B-cells induced an up-regulation of cell surface CD21 at lower concentrations and at higher concentrations induced cytoplasmic domain dependent CD21 ectodomain shedding.

VII.1.3.3 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 as well as those expressing CD21 containing point mutations within this domain (see Table 1) were studied after treatment for 4 hours with 20 and 200 μM PV (Figure 13A). To control for the minor differences seen in CD21-expression levels in the various untreated transfectants (see Table 2), the respective untreated CD21 levels were converted to 100% CD21 cell surface expression.

Table 2

Median fluorescence intensities of the different CD21-transfected CH27 B-cell lines.

CD21 wt

Ratio sCD21 / surface CD21 expression*103

******

Ratio sCD21 / surface CD21 expression*103

******

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 for 4 h with 20 μM or 200 μM PV and subjected to analysis by flow cytometry for measurement of CD21 surface levels. Data represent mean values of 4 experiments (+ SEM; untreated control = 100%). (B) Quantitation of sCD21 present in the cell culture supernatants as determined by capture ELISA (+ SEM, untreated control = 100%). Significance of differences between treated and control cells. (C) From the raw data of C and B the ratios of soluble CD21 to cell surface expression of CD21 were calculated (+ SEM), showing that the CD21 KHR cells shed more CD21 both spontaneously and induced compared to all other cell lines. Significance of differences between CD21 wt and truncated or site-directed mutant cells with the respective treatment.

In addition, sCD21 in the cell culture supernatants was also measured by ELISA (Figure 13B). All transfectants up-regulated CD21 upon 20 μM PV-stimulation, except for the CD21 KHR cells.

With 200 μM PV treatment, CD21 cell surface expression 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, when expressed in %, in a comparable range in all cell lines.

Again to take the different expression levels and the sCD21 raw data into consideration, ratios of the cell surface expression and the respective soluble CD21 data measured in ELISA were calculated (Figure 13C). 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.

VII.1.3.4 Shedding of cell surface CD21 can be induced by thiol antioxidants in murine B-cells

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 B-cells. After challenging the cells for 4 hours with the respective stimuli the cell surface expression of CD21 was measured by flow cytometry. As can be seen in Figure 14, cell surface CD21 was down-modulated in both B220⁺ gated splenocytes (Figure 14A) and CH27-CD21 wt cells (Figure 14B) upon GSH and NAC treatment in a concentration-dependent manner.

control

CD21 on mouse B-cells is shed upon stimulation with thiol antioxidants.

(A) C57BL/6 mouse splenocytes were treated for 4 h with different concentrations of GSH or NAC and then 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 3 independent experiments is shown (+ SEM; untreated control = 100%).

(B) The CD21-transfected CH27 B-cell line (CH27-CD21 wt) was treated as described in A. Data represent the mean of at least 4 experiments (+ SEM; untreated control = 100%). Significance of differences between treated and control cells.

VII.1.3.5 Influence of the cytoplasmic domain of CD21 on the thiol antioxidant-induced shedding of CD21

We were interested if the CD21 KHR transfectant would show enhanced shedding rates with thiol antioxidant treatment, too. Therefore, CH27-CD21 wt

and CH27-CD21 KHR cells were challenged for 4 hours with a range of GSH and NAC concentrations and subjected to analysis by flow cytometry.

Figure 15

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 for 4 h with different concentrations of GSH (A) or NAC (B), and analyzed for CD21 cell surface expression by flow cytometry. Data represent mean values of at least 3 experiments (+ SEM; untreated control = 100%). Significance of differences between treated and control cells. (C) Immunofluorescence stainings of CD21 wt and CD21 KHR cells. Cells were treated with 5 mM GSH for 4 h (original magnification x400 for CD21 wt and x630 for CD21 KHR). Scale bar: 20 µm.

Shedding of cell surface CD21 from CD21 KHR cells occurred at lower GSH- or NAC-concentrations when compared to that seen in CD21 wt cells (Figure 15A + B).

Even at concentrations as low as 0.1 mM, CD21 KHR-expressing cells showed a marked reduction of surface CD21 compared to that seen in cells expressing CD21 wt that required a concentration of at least 5 mM GSH for significant surface reduction (p<0.001). In addition, CD21 levels decreased to a higher degree 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 confocal microscopic analysis of the two transfectants were also performed.

Figure 22C shows that after treatment of the cells with 5 mM GSH for 4 hours, both CD21 wt and CD21 KHR cell lines showed a noticeable decrease of CD21 cell surface staining.

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 additional transfected CH27 cell lines (see Table 1) after treatment with 0.5 or 5 mM GSH (Figure 16A). As seen before, the CD21 KHR cells showed a significant (p<0.001) decrease of CD21 cell surface staining with 0.5 mM GSH addition, which was even more pronounced with 5 mM GSH. However, the CD21-shedding observed in the additional transfectants was similar to that seen 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.

Then, the kinetics of CD21-shedding were compared from the transfectants expressing the various CD21 cytoplasmic domain truncation mutants by challenging the cells for up to 4 hours and measuring CD21 surface staining at different time points. As discerned in Figure 16B, all cell lines shed CD21 in a time-dependent manner. The CD21 KHR cells, however, were much faster in their rate of CD21 loss. After one hour the CD21 cell surface expression was 40%, and after 4 hours, only 20% of the untreated control levels remained. In contrast, all the other cell lines were seen to reduce CD21 cell surface expression by approximately 75% of control levels after 4 hours

incubation with GSH. Thus, the absence of the cytoplasmic domain resulted in both faster and increased levels of CD21-shedding.

CD21 wt

% CD21 cell surface expression ^*

***

% CD21 cell surface expression ^*

***

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 for 4 h with 0.5 mM or 5 mM GSH and subjected to analysis of CD21 cell surface expression by flow cytometry. Data represent mean values of at least 4 experiments (+ SEM; untreated control = 100%). Significance of differences between treated and 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 3 is shown.