Discussion

Major differences exist between the immune systems of mice and men (248,249) and the organization of CD21-expression is different in the two species (250). We therefore wanted to explore whether variations in shedding of CD21 would also arise. In the current study we have provided evidence that CD21 is shed constitutively and upon stimulation in a concentration- and time-dependent manner in mouse cells.

There are contradictory reports about whether the cytoplasmic tails of the substrates of ADAMs are involved in the shedding process (251). For example, TGF-α and APP (β-amyloid precursor protein) require only a juxtamembrane stalk region and no transmembrane or cytoplasmic tail sequences for efficient shedding (252). In contrast, for L-selectin (253) and L1 (245) susceptibility to shedding seems to be regulated by their own cytoplasmic domain. In our previous work, we could show that the CD21 short consensus repeat (SCR) 16, the extracellular domain adjacent to the transmembrane domain is necessary for induction of shedding (107). In our present work we demonstrated that the lack of the cytoplasmic domain of CD21 enhanced shedding. At least two possibilities could account for this finding. A negative feed-back loop that inhibits CD21-shedding could be accomplished either by signaling with or without downstream gene transcription or by the cytoplasmic domain acting as an anchor, possibly including interactions with the cytoskeleton. Also, one possibility that cannot be excluded is that the CD21 KHR protein is less stable in the membrane without the necessity of interacting with the cytoskeleton. Although the cytoplasmic domain of CD21 is not required for C3dg- or EBV-binding, it is obligatory for internalization of cross-linked C3dg as well as for EBV-infection (80). Also, a 34 amino acid synthetic peptide corresponding to the CD21 cytoplasmic domain could inhibit B-lymphocyte proliferation triggered by EBV or C3d (81). Furthermore, actin rearrangement is crucial for EBV-infection of B-cells (79). It has also been demonstrated that changes in the cytoskeleton occur during PV-stimulation (254), and upon EBV-activation the CD21 cytoplasmic domain interacts with the formin FHOS/FHOD1, an actin-binding protein (77). In a similar fashion to CD21, truncation of the cytoplasmic

domain of the cell adhesion molecule L1 led to strongly increased basal shedding rates. Cytochalasin D treatment reduced basal L1-shedding, implying a direct involvement of the actin cytoskeleton (245). Comparable results were obtained with the angiotensin-converting enzyme (ACE); basal shedding rates were increased in an ACE mutant without cytoplasmic domain, but phorbol ester treatment produced only a slight increase in shedding in comparison with wildtype ACE (255). The authors also suggested a regulatory function for the interaction of the ACE cytoplasmic domain with the actin cytoskeleton. Therefore, an equivalent mechanism for the regulation of CD21-shedding may be expected.

PV is a phosphotyrosine specific phosphatase inhibitor and thus changes the balance between dephosphorylation and phosphorylation. With lower PV-concentrations CD21 was up-regulated in CD21 wt cells and shed upon 200 μM PV. The CD21 KHR cells shed CD21 to the same extent as CD21 wt cells after 4 hours 200 μM PV-treatment but the up-regulation with 20 μM PV was not evident. Using the truncation and site-directed CD21 cytoplasmic domain mutants, only minor differences could be measured as compared to the CD21 wt cells. In a time-course, however, CD21 KHR cells did not reduce CD21 cell surface expression as rapid as CD21 wt cells, but they released more sCD21. When calculating the ratio of soluble CD21 to its cell surface expression it was evident that the absence of the cytoplasmic tail of CD21 increased the spontaneous/ basal as well as the induced shedding rates of CD21. Splenic B-cells from C57BL/6 mice showed essentially the same reaction, but in these cells a small peak of CD21-expression within the first 15 min of activation with both 20 and 200 μM PV could additionally be demonstrated.

The different developmental B-cell stages in the spleen; immature/

transitional, mature/ follicular and marginal zone (MZ-) B-cells can be characterized by their CD21, CD23, IgM and IgD expression pattern. The smallest population, constituting about 5% of the splenic B-cells, the MZ-B-cells, are defined by a CD21^hi, CD23^-, IgM^hi, IgD^low profile (reviewed in (256,257)). We suggest that low PV-induced expression of CD21 on B-lymphocytes could mimic the MZ-B-cells which represent a preactivated type;

MZ-B-cells are able to proliferate and terminally differentiate into

antibody-secreting plasma cells within hours of activation (258). They do not recirculate as the naïve follicular B-cells and participate in very early immune responses playing a pivotal role in the first-line of defense of the humoral response against blood-borne antigens (259). Other preactivated CD21^hi cells are found in human EBV-activated B- and T-cells where CD21 and CD23 are markedly up-regulated by EBNA2 (EBV nuclear antigen 2) expression, which is also leading to increased sCD21 and sCD23 levels in patients with EBV-associated diseases (124). Moreover, allergens induce CD23-expression in CD4⁺ cells and CD21-expression on B-cells in patients with allergic asthma (236). We therefore hypothesize that the activation of B-cells is an (at least) two-step process fine-regulated by CD21-expression and shedding. With the first (or weak; low PV-concentrations) signal, a rapid up-regulation of CD21 on the cell surface of naïve B-cells occurs to increase the signal, i.e. to preactivate 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 (260).

Redox-regulation is important for multiple cellular functions, signal transduction and gene expression, especially of immune cells (109,110).

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 cells (113), NAC does not change the GSH-levels when used on healthy cells (115), and we obtained the same results with both agents, we assume that in our system, GSH and NAC act only extracellularly on CD21 and/or the unknown sheddase (107).

To conclude, we have demonstrated that murine B-cells shed CD21 spontaneously as well as upon stimulation and activation. A regulatory role of the cytoplasmic domain in CD21-shedding was evident, and we thus suggest that shedding of CD21 might be a mechanism for the fine-tuning of B-cell activation. Since changes in sCD21 levels in sera are associated with several pathologic conditions (95,123-126) it will be of considerable interest to study those in different knockout or transgenic mouse models. Moreover, mouse

models could facilitate the search for the so far unidentified CD21 sheddase(s).