Complete Downmodulation of P-Selectin Glycoprotein Ligand in Monocytes Undergoing Apoptosis
Objectives— Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)–mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells.
Methods and Results— PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding. This was accompanied by a complete loss of PSGL-1–mediated platelet–leukocyte (flow cytometry) and leukocyte–endothelial cell (parallel plate flow chamber) interactions. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an N-ethylmaleimide-inhibitable metalloproteinase of the ADAM family.
Conclusions— Downmodulation of PGSL-1 in apoptotic monocytes may prevent ectopic cell clearance in the peripheral vasculature to reduce local inflammatory and proliferative responses. Depletion of PSGL-1 expression on apoptotic microparticles may also act as a molecular switch to modulate their thrombogenic activity.
The cell adhesion molecules P-selectin glycoprotein ligand (PSGL-1) and its receptor P-selectin (CD62P) are important mediators in the primary interaction between leukocytes and both endothelial cells and activated platelets.1 PSGL-1–P-selectin interaction has been also recognized as a crucial event in the incorporation into growing thrombi of tissue factor-containing microparticles.2 PSGL-1 expression was long considered unregulated. Evidence has emerged, however, to link a reduction of cell surface PSGL-1 expression to stimulation of leukocytes.3,4 An upregulation of PSGL-1 on leukocytes from patients with chronic obstructive pulmonary disease was also observed.5 Recent data demonstrate that PSGL-1 surface expression may be decreased in apoptotic monocytic cells.6 We have, therefore, studied systematically the effects of apoptosis on PSGL-1 expression in function in monocytes.
Materials and Methods
Cell Systems and Transfection
U937 cells, human umbilical vein endothelial cells (HUVECs; kindly provided by Dr U. Rauch, Charité, Berlin, Germany), immortalized ADAM-10−/− and -17−/− MEFs (kindly provided by Drs P. Saftig and S. Rose-John, University of Kiel, Germany), and HEK293 cells were used. Human monocytes and platelets were isolated from citrated blood. A human PSGL-1 cDNA clone was kindly provided by Dr D. Vestweber (University of Münster, Germany). MEFs were transfected using Optifect transfection reagent (Qiagen). For siRNA experiments, HEK293 cells were cotransfected with PSGL-1 cDNA and with ADAM-8 or ADAM-9 siRNA using the DharmaFECT 1 siRNA Transfection Reagent.
PSGL-1 expression was quantified using anti–PSGL-1–PE antibodies. Annexin-V–PE binding was measured using EDTA as a negative control. Monocyte-platelet aggregates were detected using anti–CD61-fluorescein isothiocyanate (FITC) antibodies and gating of monocytes on CD45-PE.
Immunofluorescence Microscopy, Immunoblotting, and Zymography
Methanol-fixed cells were stained with anti–PSGL-1 antibodies and Cy3-conjugated secondary antibodies. Protein expression was also determined by SDS-PAGE/Western blotting. Zymography was performed using SDS/polyacrylamide gels, containing gelatin.
qPCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems).
Parallel flow chamber perfusion experiments were performed at a shear rate of 125 s−1 using confluent HUVECs and calcein-AM-labeled U937 cells.
For details, see the supplemental materials (available online at http://atvb.ahajournals.org).
Staurosporine, dactinomycine, and etoposid led to an almost complete downmodulation of PSGL-1 surface expression in U937 cells and in human blood monocytes, whereas cell activation resulted in a modest reduction of PSGL-1 (Figure 1A and 1B). The kinetics of PSGL-1 reduction was nearly indentical with the occurrence of apoptosis markers, (Figure 2C through 2F) and was prevented by VAD fmk, indicating an apoptosis-related mechanism. Downmodulation of PSGL-1 was confirmed by Western blotting and immunofluorescence microscopy (Figure 1G and 1H). A marked reduction of PSGL-1 mRNA was also observed. However, because PSGL-1 was found to be a long-living protein, transcriptional downregulation is unlikely to account for the complete loss of the protein. Ultracentrifugation pellets and supernatants of conditioned media were examined to discriminate soluble from microparticle-bound PSGL-1 fragments. Staurosporine reduced whereas PMA increased microparticle PSGL-1 expression, the latter possibly reflecting the occurrence of a proteolytic fragment (Figure 2A and 2B). PE-conjugated anti–PSGL-1 antibodies were released from the cell surface on apotosis induction (Figure 2C), indicating a proteolytical shedding. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an NEM-inhibitable metalloproteinase of the ADAM family (Figure 2E through 2G and supplemental materials). Finally, both adhesion of activated platelets to U937 cells and adhesion of U937 cells to HUVECs were almost completely abolished by staurosporine treatment (Figure 2H and 2I).
For further details, see the supplemental materials.
We demonstrate that PSGL-1 is completely downmodulated in apoptotic monocytes, unlike the partial reduction observed in response to cell activation.3 The mechanism underlying the depletion of PSGL-1 on monocytic cells involved primarily proteolytic shedding by an NEM–inhibitable metalloproteinase of the ADAM family. Apoptosis-related PSGL-1 downmodulation resulted in a complete loss of PSGL-1–dependent cell functions (platelet–leukocyte and leukocyte–endothelial cell interactions). These findings have several important physiological and pathophysiological implications. Microparticle-associated, blood-borne TF is incorporated into growing thrombi via PSGL-1 and contributes to thrombus propagation.2 Thus, downmodulation of this molecule during the apoptotic process may serve as a protective molecular switch to alter this function of microparticles. Phagocytosis of circulating apoptotic cells by the reticulo-endothelial system occurs devoid of coincidental inflammation. One might hypothesize that downmodulation of PSGL-1 in apoptotic monocytes serves to prevent ectopic clearance of these cells in the peripheral vasculature, thus preventing local inflammatory responses.7 Similarly, the load of platelet-derived mitogenic and inflammatory mediators8 would be reduced markedly, when activated platelets cannot adhere to PSGL-1–depleted apoptotic monocytes.
The authors thank U. Rauch, S. Rose-John, P. Saftig, and D. Vestweber for valuable reagents.
Sources of Funding
This work was supported by the Studienstiftung des Deutschen Volkes (to J.J.S.) and in part by the SFB612 (to J.W.F.).
Original received September 10, 2007; final version accepted April 17, 2008.
McEver RP, Cummings RD. Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest. 1997; 100: 97–103.
Davenpeck KL, Brummet ME, Hudson SA, Mayer RJ, Bochner BS. Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro. J Immunol. 2000; 165: 2764–2772.