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International Immunology Advance Access originally published online on June 1, 2006
International Immunology 2006 18(7):1171-1178; doi:10.1093/intimm/dxl051
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CD21 and CD62L shedding are both inducible via P2X7Rs

Sarah Sengstake1, Eva-Maria Boneberg2 and Harald Illges1,2

1 Department of Biology, Immunology, Faculty of Sciences, University of Konstanz, D-78457 Konstanz, Germany
2 Biotechnology Institute Thurgau, Konstanzer Strasse 19, CH-8274 Tägerwilen, Switzerland

Correspondence to: H. Illges; E-mail: harald.illges{at}bitg.ch


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Neutrophils and lymphocytes are recruited to sites of inflammation and require the adhesion molecule L-selectin (CD62L) for adherence to endothelial cells. Nucleotides released from activated or dying cells at sites of inflammation can mediate signaling through purinergic receptor family II, resulting in CD62L shedding. Activation of B lymphocytes requires the complement receptor type II (CD21) and at the same time leads to shedding of CD21. Both CD62L and CD21 shedding possibly depends on the same families of proteases. In the present study, we characterized peripheral blood naive and memory cells and neutrophils for CD62L surface expression and analyzed benzoyl-benzoyl triphosphate (BzATP)-induced shedding. BzATP is able to induce CD62L shedding in naive and memory lymphocytes, but not in neutrophils. CD21 shedding can be induced through activation of the B cell receptor (BCR) or with mitogens. Here we show that CD21 is also susceptible to BzATP-induced shedding on peripheral B cells. In addition, using receptor inhibitiors, we show that shedding of CD21 and CD62L is mediated via the P2X7R. P2X7R-mediated CD62L and CD21 shedding could occur as a result of extracellular accumulated ATP and may have an influence on leukocyte migrational behavior and BCR-mediated signaling.

Keywords: CD62L, CD21, ectodomain shedding, purinergic receptors, KN62, oxATP


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Leukocytes require the expression of cell-surface receptors such as L-selectin (CD62L) and complement receptor type II (CD21) for cell migration and cell activation, respectively. Shedding is a mechanism regulating the presence of these and other cell-surface molecules. The shed portion, termed ectodomain, retains all physiological binding capabilities (1).

The adhesion molecule CD62L is constitutively expressed on leukocytes, and interacts with carbohydrate ligands mediating the initial rolling of leukocytes on the endothelium (2). CD62L is also known as lymph node homing receptor and shedding of this receptor is thought to regulate leukocyte extravasation in inflamed tissue. CD62L-deficient mice show impaired transmigration into inflamed tissue upon inflammatory stimuli (3, 4). Replacement of the CD62L membrane-proximal region or deletion of the CD62L cleavage site allowed continued migration of antigen-stimulated lymphocytes to peripheral lymph nodes instead of short-term redirection to the spleen occurring after CD62L shedding (5). Several stimuli are reported to trigger CD62L shedding from the cell surface of leukocytes. Benzoyl-benzoyl triphosphate (BzATP) and ATP induce CD62L shedding from normal B cells and chronic leukemic lymphocytes (6), phorbol-12-myristate-13-acetate (PMA) (7) and CD45-mediated signals also trigger CD62L shedding from B cells (8). Down-regulation of CD62L on lymphocytes also occurs as a consequence of binding to high venular endothelium (9). Neutrophils shed their CD62L upon stimulation with non-steroidal anti-inflammatory drugs via reduction of the intracellular ATP concentration (10), activation with formyl-methionyl-leucyl-phenylanaline (fMLP) (11), hypotonicity (12) or PMA (13). Based on several studies, using protease inhibitors, the protease responsible for CD62L shedding is sought among the metalloproteases (7, 14–16).

P2XRs represent a purinergic receptor subfamily that are ligand-gated ion channels (17). ATP, UTP and ADP and analogs thereof are ligands for the P2XRs and ATP ligand binding results in protein kinase C activation, increased Ca2+ influx and K+ efflux, activation of the p56lck protein tyrosine kinase and MAP kinase, up-regulation of AP-1 and down-regulation of nuclear factor-{kappa}B transcription factors (18). In the immune system, P2X7Rs are expressed on normal B and T cells, monocytes, NK cells, platelets and neutrophils (19). Interestingly, anti-collagen antibody-induced arthritis is attenuated in P2X7R-deficient animals, suggesting that the P2X7R is a regulator of inflammatory cell function (20).

ATP is present in millimolar concentrations in the cytosol of all eukaryotic cell types and extracellular levels are maintained at extremely low levels by ubiquitous ecto-ATPases and ectophosphatases (21). ATP is released from cytosolic stores in platelets, neutrophils and endothelial cells (22) at sites of inflammation. The effusion of ATP and possible down-regulation of ATP diphosphohydrolases, such as CD39, lead to a local accumulation of extracellular ATP as a short-term consequence in inflamed tissues (23). In addition, extracellular ATP seems to play a role in cell differentiation, apoptosis and prevention of necrosis (24).

CD21 is part of the CD19/CD81/Leu-13 co-receptor complex residing on B cells but can be detected on many additional cell types. B cell receptor (BCR) signaling strength, rather than BCR specificity, controls the fate of developing B cells (25, 26). The B cell co-receptor complex has the potential to engage synergistically with the BCR thereby lowering the threshold required for B cell activation (27). Further, alterations in the signaling threshold as a consequence of BCR and co-receptor engagement contribute to the loss of self-tolerance and development of autoimmune disease (28). We have previously reported significantly lower soluble CD21 (sCD21) serum levels in patients with rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome compared with healthy controls (29). Activation of lymphocytes via PMA and Ca2+ ionophores or via cross-linking using anti-IgM/anti-CD40 antibodies induced the proteolytic cleavage of CD21 (30). We show that the CD21 is susceptible to BzATP-induced shedding via purinergic receptors. Shedding could be inhibited with P2X7R inhibitors ATP 2',3'-dialdehyde (oxATP) and 1-(N,O-bis-5-isoquinolinesulfonyl-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62). In addition, we show that significant differences characterize the functional expression of CD62L in various leukocyte cell types and stimulation with BzATP leads to CD62L shedding on peripheral naive and also memory lymphocytes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
P2X7R agonists and antagonists
BzATP, KN62, oxATP were purchased from Sigma (Buchs, Switzerland).

Cell separation and flow cytometry
Human PBMCs were isolated from healthy donors using cell preparation vacutainer from BD Biosciences (Basel, Switzerland) and stained with combinations of mAbs: FITC-labeled anti-CD19 (clone LT19), PE-labeled anti-CD21 (LB21), FITC-labeled anti-CD8 (LT8), PE-labeled anti-CD62L (FMC46), FITC-labeled anti-CD45RA (F8-11-13) were purchased from Serotec (Oxford, UK); FITC-labeled anti-CD27 (CLB-27/1) from Caltag Laboratories (Burlingame, CA, USA) and PerCP-labeled anti-CD20 (L27), PerCP-labeled anti-CD19 (4G7), APC-labeled anti-CD3 (UCHT1), APC-labeled anti-CD45RO (UHCL1), PerCP-labeled anti-CD20 (L27), PerCP-labeled anti-CD4 (SK3), PerCP-labeled anti-CD8 (SK1) were from BD Biosciences. For isolation of human peripheral neutrophils, blood was obtained from healthy donors and collected in Heparin-monovettes (Sarstedt, Nümbrecht, Germany). Neutrophils were isolated using four-layer Percoll/HEPES density gradient centrifugation. Neutrophils were stained with PerCP-labeled anti-CD45 (2D1) and PE-labeled anti-CD62L (FMC46) antibodies from BD Biosciences.

BzATP induced loss of CD62L and CD21
For BzATP stimulation, 106 cells ml–1 purified PBMCs or neutrophils were re-suspended in KCl buffer (150 mM KCl, 5 mM D-glucose, 0.1% BSA, 10 mM HEPES, pH 7.5 with or without BzATP at a final concentration of 0.1 mM and incubated at 37°C for 30 min). In some experiments, PBMCs were pre-stimulated either with 300 µM oxATP for 60 min or with 120 nM KN62 for 15 min at 37°C in KCl buffer. For time-course experiments, an equal volume of MgCl2 buffer (145 mM NaCl, 5 mM KCl, 10 mM MgCl2, 10 mM HEPES, pH 7.5) was added at the indicated time points. Surface CD62L and CD21 were analyzed using a FACS LSR and CellQuest software from BD Biosciences.

sCD62L ELISA
The concentrations of sCD62L released into the supernatants were measured using a human sCD62L sandwich ELISA according to the manufacturer's instructions (Bender MedSystems, Vienna, Austria).

Real-time PCR analysis for P2X7 mRNA
For quantitative information about P2X7 mRNA levels in human leukocyte subsets, freshly isolated human PBMCs were sorted using a FACSVantage SE (BD Biosciences). Neutrophils were purified using a Percoll/HEPES four-layer density gradient centrifugation as described before. RNA was isolated using a RNAeasy isolation kit (Qiagen, Basel, Switzerland) according to the manufacturer's instructions. Total RNA (200 µg) of CD19+, CD4+, CD8+ and neutrophils was treated with DNaseI (Qiagen) and reverse transcribed to cDNA using Multiscribe reverse transcriptase (Applied Biosystems, CA, USA) and a T3 Thermocycler, Biometra, Germany, with the following settings: 25°C for 10 min, 48°C for 45 min and 95°C for 5 min. For P2X7 quantification, 2 µl of each cDNA was transcribed using a QuantiTect SYBR Green PCR kit (Qiagen) containing SYBR Green-labeled dNTPs and HotStar Taq polymerase, according to the manufacturer's instructions. Real-time PCR was carried out with a TaqMan AbiPrism7700 Sequence Detector (Applied Biosystems). As forward primer 5'-TCT GCA AGA TGT CAA GGG C-3' (nucleotide position 1356–1374) and as reverse primer 5'-TCA CTC TTC GGA AAC TCT TTC C-3' (nucleotide position 1829–1850; GenBank accession no. NM002562) were used (19). A three-step real-time PCR system with the following settings was employed: HotStar Taq polymerase activation at 95°C for 15 min and 45 cycles of 94°C for 15 s, annealing of primers at 52°C for 30 s and extension at 72°C for 30 s. In parallel, primers for two housekeeping genes ubiquitin c (UBC) and beta-2-microblobulin (B2M) were used for normalization, employing the delta-delta cycle treshold-method (31). CD19+ cells were used as a calibrator to calculate relative P2X7 mRNA levels. All primers were purchased from Microsynth (Balgach, Switzerland) and amplicons were loaded on a 2% agarose gel as a control.

Statistics
Data are shown as mean ± SD or mean ± SEM. For statistical analysis of two groups of parametric data, a paired t-test was used. The non-parametric Mann–Whitney test was used to compare unpaired groups. All calculations were performed with InSTAT 3.0 (GraphPad, San Diego, USA) and are based on raw data. P-values of <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Different expression of CD62L on normal human PBMCs and neutrophils
In order to investigate BzATP-induced shedding of CD62L on different hematopoietic subsets, we first characterized CD62L surface expression on these cells. CD62L expression was analyzed on peripheral blood B cell or T cell sub-populations or neutrophils by flow cytometry. B cells expressed significantly more CD62L on their surface [median fluorescence intensity (MFI) = 563.3 ± 85.6, n = 6] compared with CD4+ T cells (MFI = 237.2 ± 48.3, n = 5) with P = 0.0043, or CD8+ T cells (MFI = 154.7 ± 44.5, P = 0.0043, n = 5) with P = 0.0043. The CD4+ T cells expressed significantly more CD62L (MFI = 237.2 ± 48.3, n = 5) compared with CD8+ T cells (MFI = 154.7 ± 44.5, n = 5) with P = 0.0396 (Fig. 1A). Neutrophils expressed the lowest amount of surface CD62L (MFI = 114.1 ± 82.6, n = 4). Among the B cell populations, CD27+ memory B cells showed higher CD62L expression (MFI = 457.3 ± 110.5, n = 5) compared with naive CD27– B cells (MFI = 418.8 ± 100.1, n = 5) (Fig. 1B). CD4+CD45RO T cells expressed significantly less CD62L than the naive CD4+CD45RA T cells (MFI = 168.2 ± 83.8 versus MFI = 221.1 ± 66.2, n = 5) with P = 0.0158, and memory CD8+CD45RO T cells expressed significantly less CD62L than the naive CD8+CD45RA T cells (MFI = 37.9 ± 35.15 compared with MFI = 244.3 ± 103.3, n = 5, P = 0.0210) (Fig. 1C).


Figure 1
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  		Fig. 1 BzATP induced CD62L shedding from leukocyte subsets. Purified human PBMCs or neutrophils from independent experiments were incubated in KCl buffer with or without 0.1 mM BzATP for 30 min, stained for CD62L expression and analyzed by flow cytometry. (A) CD4+, CD8+ T and CD19+ B cells showed a reduction of CD62L surface expression after BzATP stimulation. (B) Memory CD27+ B cells and (C) memory CD4+CD45RO T cells showed a reduced CD62L surface expression after BzATP stimulation, but not memory CD8+CD45RO T cells. Both naive CD4+CD45RA and CD8+CD45RA T cells expressed more CD62L as compared with their memory counterparts and both reduced CD62L surface expression after BzATP stimulation. Open symbols represent BzATP-stimulated cells and closed symbols unstimulated cells. Horizontal bars indicate mean of each population; *P < 0.05, **P < 0.001.

 
The P2X7 agonist BzATP reduces CD62L surface expression on human PBMCs to background levels
PBMCs or neutrophils were purified and populations of B cells, T cells or neutrophils were analyzed by flow cytometry. Cells were re-suspended in 150 mM KCl buffer and stimulated for 30 min with 0.1 mM BzATP. Stimulated T cells and B cells showed a significant reduction of CD62L to background levels. In contrast, neutrophils did not react significantly to BzATP stimulation (Fig. 1A). BzATP-induced reduction of CD62L surface expression on CD4+, CD8+and CD19+cells was significant for all populations with P-values <0.01. Significant reduction was found for CD27– B cells, CD27+ memory B cells, CD8+CD45RA+ T cells and CD4+CD45RA+ T cells with P-values <0.05. The reduction on memory CD4+CD45RO T cells was not quite significant, P = 0.0952. Memory CD8+CD45RO+ T cells expressed only very low amounts of CD62L on their surface compared with memory CD4+CD45RO+ T cells and BzATP stimulation resulted in a reduction of this expression, but this reduction was not significant.

BzATP-induced CD62L shedding corresponded to elevated levels of sCD62L
To verify that BzATP-induced reduction of cell surface CD62L results from shedding, we measured sCD62L in the supernatants of stimulated cells using an ELISA. Human PBMCs or neutrophils were re-suspended in KCl buffer and stimulated for 30 min with or without BzATP. Supernatants were harvested by centrifugation. Supernatants of unstimulated cells already had mean concentration of 1.8 ± 0.3 ng ml–1 for sCD62L (Fig. 2A). BzATP stimulation led to a significant increase of sCD62L to a mean concentration of 3.6 ± 0.1 ng ml–1 (P = 0.0129). In the supernatants of unstimulated neutrophils, a concentration of 0.8 ± 0.0 ng ml–1 sCD62L was measured, whereas only a slight increase to a mean 0.9 ± 0.3 ng ml–1 could be measured in supernatants of BzATP-stimulated neutrophils, considered not significant (P = 0.5709) (Fig. 2B).


Figure 2
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  		Fig. 2 CD62L is shed into supernatants from human PBMCs after exposure to BzATP. Human PBMCs or neutrophils were re-suspended in KCl buffer and exposed for 30 min to 0.1 mM BzATP stimulation. Cells were centrifuged and supernatants harvested for sCD62L ELISA. (A) BzATP induced CD62L shedding in supernatants from PBMC. (B) Analysis of supernatants from human neutrophil preparations showed no increases in sCD62L upon BzATP stimulation. Data are pooled from three independent experiments and means ± SDs are indicated.

 
P2X7 antagonists prevented CD62L shedding on human PBMCs
In order to determine whether the induced shedding in our experimental culture systems utilizes P2X7Rs, we performed time-dependent experiments using BzATP and either the inhibitor KN62 or oxATP. PBMCs were pre-incubated with either 120 nM KN62 for 15 min or 300 µM oxATP for 60 min before BzATP stimulation (Fig. 3A–D, closed symbols). In parallel, PBMCs were stimulated with BzATP in the absence of inhibitor (Fig. 3A–D, open symbols). CD62L cell-surface expression was detected by flow cytometry on B cells and CD4+ T cells over a period of 15 min. In both investigated populations, BzATP induced time-dependent CD62L shedding. Pre-incubation with either KN62 or oxATP attenuated CD62L shedding ~84% for KN62 and 85% for oxATP on peripheral B cells as well as on CD4+ T cells after 15 min BzATP stimulation compared with unstimulated cells. Elevated amounts of the sCD62L were measured in the respective supernatants of BzATP-stimulated PBMC compared with unstimulated cells (14.4 ± 1.9 ng ml–1 versus 9.9 ± 1.9 ng ml–1) (Fig. 3E). In the presence of the inhibitor oxATP, we measured 7.2 ± 1.3 ng ml–1 sCD62L. Pre-incubation with KN62 resulted in 12.6 ± 1.3 ng ml–1 sCD62L. Thus, shedding is inhibited with both oxATP and KN62 and therefore mediated via the P2X7R.


Figure 3
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  		Fig. 3 Kinetics of CD62L shedding in the presence of P2X7 antagonists. Purified human PBMCs were re-suspended in KCl buffer and stimulated for indicated points of time with 0.1 mM BzATP in the presence or absence of KN62 and oxATP. BzATP was inactivated by adding an equal volume of MgCl2 buffer at the indicated time points and CD62L expression was detected by flow cytometry and sCD62L was measured in the supernatants by ELISA. (A) B cells, KN62 (B) CD4+ T cells, KN62, (C) B cells, oxATP and (D) CD4+ T cells, oxATP. Pre-incubation with KN62 and oxATP resulted in inhibition of CD62L shedding. Data are expressed as means ± SEMs. (E) Decreased levels of sCD62L in harvested supernatants after 15 min stimulation showed KN62- and oxATP-induced inhibition of BzATP-induced CD62L shedding. Concentrations of sCD62L are expressed as means ± SDs, n = 4 donors.

 
Human peripheral B cells also shed CD21 upon BzATP stimulation
Since metalloproteases are found to be the target enzymes for CD62L and are discussed to be involved in CD21 shedding (32, 33), we analyzed whether activation of P2X7Rs could induce CD21 shedding on B cells as well. Analysis of unstimulated PBMC revealed CD21 expression of an MFI = 55.8 ± 12.31 (Fig. 4A). Exposure of purified PBMC to 0.1 mM BzATP stimulation for 30 min led to a significant reduction of CD21 expression (MFI = 44.0 ± 9.6, P < 0.0001, n = 7). To measure sCD21 in the supernatants of PBMCs, we employed our in-house developed sCD21 ELISA. Supernatants were harvested and analyzed at time point 0, 15 min and 30 min of stimulation with BzATP. A significant increase of sCD21 could be detected with prolonged incubation time. Thus, CD21 shedding can be induced with BzATP.


Figure 4
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  		Fig. 4 BzATP induced shedding of CD21 on B cells via the P2X7R. (A) Flow cytometry analysis of CD21 surface expression on B cells. Human PBMCs were re-suspended in KCl buffer and stimulated with 0.1 mM BzATP over a 30-min period. CD21 surface expression was decreased after stimulation with BzATP. Data indicate means ± SEMs of seven independent experiments. (B) Purified human PBMCs were re-suspended in KCl buffer and stimulated with 0.1 mM BzATP for 30 min in the presence or absence of the P2X7 inhibitors KN62 and oxATP. CD21 surface expression was detected on human peripheral B cells by flow cytometry. Both P2X7R inhibitors led to reduction of induced CD21 shedding. Percentage expression of CD21 is indicated as means ± SEMs versus unstimulated cells, n = 3. (C) PBMCs from three donors were cultured in the presence (1–3) or absence (4) of BzATP for 30 min. Supernatants were analyzed for sCD21 at timepoint 0 (1), 15 min (2) and 30 min (3) of stimulation with BzATP.

 
Inhibition of P2X7R signaling and ligand binding decreases CD21 shedding
As demonstrated above, CD21 shedding can be induced with BzATP. To prove the involvement of P2X7Rs in CD21 shedding from peripheral blood B cells, we employed the P2X7 inhibitors KN62 and oxATP, respectively.

Purified PBMCs were pre-incubated with either 120 nM KN62 for 15 min or 300 µM oxATP for 60 min followed by 0.1 mM BzATP stimulation. CD21 surface expression on peripheral B cells was detected by flow cytometry (Fig. 4B). Stimulation with BzATP alone led to a reduction of CD21 surface expression of 65 ± 5.9%. Pre-incubation with oxATP inhibited CD21 shedding (86 ± 11.4% CD21 surface expression compared with unstimulated cells). The inhibitor KN62 prevented CD21 shedding (79 ± 7.3% CD21 surface expression compared with unstimulated cells). Thus, BzATP-induced CD21 shedding is mediated via the P2X7Rs and can be diminished by P2X7 antagonists.

Neutrophils do express P2X7 mRNA, albeit less compared with human peripheral cells
The observation that neutrophils do not shed CD62L upon BzATP stimulation could be explained by a lack of P2X7R expression. Therefore, we used real-time PCR to measure the relative P2X7 gene expression. UBC and B2M were used as control housekeeping genes for normalization. Agarose gel analysis showed P2X7R expression in all analyzed cell types (Fig. 5A). Real-time PCR showed that neutrophils expressed P2X7 mRNA. Among the cell types analyzed, the order of P2X7R mRNA expression was CD19+ > CD4+ > CD8+ > neutrophils (Fig. 5B).


Figure 5
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  		Fig. 5 P2X7 gene expression in human blood cell subsets. Real-time PCR was done with cDNA from FACS-isolated human PBMCs or gradient purified human neutrophils. (A) Agarose gel electrophoresis of real-time PCR products from analyzed cells. (B) Relative P2X7 gene expression, normalized to UBC and B2M houskeeping genes showed that CD19+ B cells expressed most P2X7 mRNA while decreasing amounts were found in other cell types (CD19+ > CD4+ > CD8+ > neutrophils). Data indicate relative gene expression of one donor for CD19+, CD4+, CD8+ cells and two donors for neutrophils.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have shown here that significant differences characterize the CD62L surface expression of peripheral blood naive and memory leukocytes. BzATP stimulation resulted in the proteolytic cleavage of CD62L from both naive and memory B cells as well as from naive and memory T cells. Furthermore, we show that proteolytic cleavage of CD21 from peripheral B cells can be induced with BzATP and both CD62L shedding and CD21 shedding are mediated via the P2X7R.

Human naive and memory T cells can be identified by the expression of the CD45RA or CD45RO isoforms and by the expression of CD62L (34, 35). CD45RA-positive T cells are thought to represent naive T cells and CD45RO to represent memory T cells. Further, there is a discrimination within naive and memory T cells in regard to the expression of CD62L. Naive and (central) memory T cells express high levels of CD62L, whereas (effector) memory T cells express low levels of CD62L (35, 36). Although we did not further discriminate the memory T cell pool in effector and central memory T cells, our data show the presence of CD62Lhigh naive T cells and CD62Llow memory T cells in the peripheral blood. We also show that all investigated types of T cells can be activated with BzATP to shed CD62L. We found high expression of CD62L on memory B cells compared with naive B cells. Most memory B cells are CD62L negative following a primary immune response and express high levels of CD62L after several weeks (37). This suggests that the memory B cell population we detected contained largely of cells which had already undergone an immune response. BzATP induces CD62L shedding from the cell surface of B cell lines (7). Here we analyzed CD62L shedding on normal peripheral naive and memory B and T cells after BzATP stimulation. We show that both naive and memory B cells undergo rapid CD62L shedding after induction with BzATP.

Neutrophils expressed the lowest amount of surface CD62L within the analyzed leukocyte population. Circulating neutrophils have a homogenous CD62L surface expression (13), but the apparent molecular mass of neutrophils CD62L (95 kDa) is distinct from lymphocyte CD62L (74 kDa). All investigated leukocytes expressed CD62L on their surface but only neutrophils were not susceptible to BzATP-induced cleavage of CD62L. Since we knew that CD62L shedding is mediated via the P2X7Rs, we investigated if there is a correlation between P2X7R expression and CD62L shedding. Real-time analysis of the P2X7R mRNA expression revealed relative gene expression levels as follows: CD19+ cells > CD8+ T cells > CD4+ T cells > neutrophils. However, we cannot exclude that the P2X7 mRNA levels from analyzed neutrophils were due to contaminating lymphocytes, which do express high amounts of P2X7 mRNA. This would lead to a false positive signal even at small numbers within the purified neutrophils.

The protease that mediates the cleavage of CD62L may be the same enzyme in T cells and B cells. Consistent with our data, for all studies that used BzATP, ATP or PMA to induce CD62L shedding on B cells, and PMA or fMLP to induce CD62L shedding on neutrophils, a stimulation of 15–30 min was sufficient to detect a drastic decrease in CD62L surface expression. Ectodomain shedding of CD62L releases the soluble form of CD62L into the extracellular milieu. Since there is no information about the function of the remaining transmembrane domain to date, the focus is on the biological relevance of sCD62L. Soluble CD62L inhibits lymphocyte attachment to cytokine-activated endothelium via binding to CD62L ligands (38). The domain structures of CD62L and CD21 share some homologies. The cleavage sites of both receptors reside within the membrane-proximal region consisting of complement homology domains (in case of CD62L 2 and for CD21 15 or 16) that are followed by the transmembrane and the cytoplasmic domains (39, 40). Moreover, proteases cleaving CD62L are found to be metalloproteases (7, 15, 16) and protease inhibitor studies revealed that metalloproteases are also involved in CD21 shedding (33). This leads to the assumption that the same family of proteases activates both CD62L and CD21 shedding.

Compared with the BzATP-induced CD62L shedding on peripheral B cells, BzATP-induced CD21 shedding on the same cells had not the same kinetic. Stimulation of B cells with BzATP resulted in a significant 20% reduction of CD21 expression. From previous studies, we know that CD21 shedding can be induced by PMA or anti-IgM/anti-CD40 stimulation for 4.5 h (30), while as shown here, a short time period of 30 min BzATP stimulation was sufficient to activate the proteolytic cleavage of CD21. BzATP was not able to induce shedding of CD21 to background levels, as it was observed for CD62L. Even repeated stimulation with 0.1 mM BzATP stimulation did not increase CD21 shedding (data not shown). The soluble form of CD21 binds to complement fragments and forms complexes with trimeric CD23, inhibiting CD23-induced IgE synthesis (41). The protease responsible for CD21 shedding might be different from the one involved in CD62L shedding, although both are inducible with BzATP and there might be a more complex regulation involved in activating the protease responsible for CD21 shedding to ensure further B cell differentiation and an ongoing immune response.

Applying the P2X7R antagonist's oxATP and KN62, we found that both CD62L and CD21 shedding are stimulated with BzATP via the purinergic receptors.

Acute inflammation is associated with cell damage and locally accumulated ATP is released from platelets, neutrophils and endothelial cells during rupture and shear stress (22). CD62L and CD21 shedding could occur as a result of extracellular accumulated ATP and could have an influence on leukocyte migrational behavior and BCR-mediated signaling.

We present here two examples for ATP-dependent purinergic receptor family II-mediated effects: ATP as an agent that potentially influences the interaction of leukocytes with the endothelium at sites of inflammation, and ATP as a modulator of immuno-coreceptors involved in B cell activation and the development of autoimmune diseases.


   Acknowledgements
 
We thank Elisabeth von Seydlitz for excellent assistance with real-time PCR. This work was supported by the Thurgauische Stiftung für Wissenschaft und Forschung, the Hans-Hench-Stiftung, the EU through grant LSHM-CT-2004-00524 and the Deutsche Stiftung Sklerodermie to H.I.


   Abbreviations
 
BCR, B cell receptor
BzATP, benzoyl-benzoyl triphosphate
B2M, beta-2-microblobulin
CD21, complement receptor II
CD62L, L-selectin
fMLP, formyl-methionyl-leucyl-phenylanaline
KN62, 1-(N,O-bis-5-isoquinolinesulfonyl-N-methyl-L-tyrosyl)- 4-phenylpiperazine
MFI, median fluorescence intensity
oxATP, ATP 2',3'-dialdehyde
PMA, phorbol-12-myristate-13-acetate
sCD21, soluble CD21
UBC, ubiquitin c

   Notes
 
Transmitting editor: A. Radbruch

Received 13 December 2004, accepted 27 April 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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