Emerging evidence indicates that ectodomain shedding by leukocytes is a key post-translational mechanism for regulating inflammation. In part, this process modulates the activity of various cytokines, cytokine receptors, and adhesion molecules (5
). ADAM17 induction occurs upon cell activation, and its biological functions have been mainly examined in this context. The fate of neutrophils following their infiltration into sites of inflammation is programmed cell death, which is an effector activity that plays a vital role in resolving acute inflammation (20
). In this study, we provide the first direct evidence that ADAM17 cleaves L-selectin upon the induction of neutrophil apoptosis, which appears to be an early apoptotic event. We also report that as the apoptotic process progresses, L-selectin can be released from the surface of leukocytes by a mechanism distinct from ADAM17-mediated shedding and the membrane events of blebbing and microparticle production.
We have previously reported that L-selectin undergoes efficient shedding upon the induction of human neutrophil apoptosis by a mechanism that involves metalloproteases (23
). L-selectin shedding has been reported to occur in an ADAM17-dependent and independent manner (8
), and thus we directly examined its role in L-selectin shedding upon death receptor-induced leukocyte apoptosis using two distinct genetic approaches. Fas signaling of apoptosis in Jurkat cells has been extensively studied (37
), and these cells, which express high levels of surface L-selectin, were employed to knock-down ADAM17 expression using shRNA. This approach decreased the expression of cell surface ADAM17, but not the expression of its most similar family member ADAM10, and also reduced L-selectin shedding upon Fas engagement when compared with control cells. We also examined the shedding of L-selectin by neutrophils lacking functional ADAM17 following the induction of apoptosis. In contrast to wild-type neutrophils, ADAM17-deficient neutrophils demonstrated a near complete abrogation of L-selectin shedding at early time points of induced apoptosis. ADAM8 has been implicated in L-selectin shedding by neutrophils as well (29
), and we also directly examined its role by using neutrophils from ADAM8 knock-out mice. We observed that these cells efficiently down-regulated their surface L-selectin expression soon after the induction of apoptosis. Hence, our findings indicate that in addition to neutrophil activation, ADAM17 is a primary sheddase of L-selectin upon death receptor-induced apoptosis.
Activation-induced L-selectin shedding regulates the receptor’s cell surface density and neutrophil adhesiveness (18
). The purpose of L-selectin shedding during neutrophil apoptosis is less clear at this time, though receptor down-regulation by apoptotic neutrophils likely contributes to a general diminution in their responsiveness to inflammatory cues (22
). Neutrophils that have recently infiltrated sites of inflammation already express low levels of L-selectin due to their activated state (8
), and thus it would seem that nominal levels of cell surface L-selectin would be available for further ADAM17-mediated cleavage upon the induction of neutrophil apoptosis. However, activated neutrophils express L-selectin mRNA, actively synthesize the adhesion molecule, and they can even re-express appreciable levels of surface L-selectin following their activation, but prior to undergoing apoptosis (23
). Consequently, ADAM17-mediated shedding during neutrophil apoptosis at sites of inflammation may produce physiologically relevant levels of localized soluble L-selectin, among additional substrates. Other settings in which ADAM17-dependent shedding may occur upon leukocyte apoptosis is during the clearance of senescent neutrophils from the blood and the turnover of lymphocytes during negative selection, which may be a mechanism for the down-regulation of various receptors and/or the production of assorted soluble agonists and antagonists.
ADAM17’s enzymatic activity is robustly induced upon cell activation with various stimuli, which occurs in part through an intrinsic process (51
). We speculate that ADAM17’s enzymatic activity is enhanced upon the induction of leukocyte apoptosis as well, though at this time we cannot rule out that changes in L-selectin’s conformation and/or cell surface distribution may also contribute to its shedding during this cellular process. We have recently reported that redox modifications of cysteinyl sulfhydryl groups in the ectodomain of mature ADAM17 up-regulates its enzymatic activity (14
). It will be interesting to determine whether this inducer mechanism may regulate ADAM17’s activity upon death receptor-induced neutrophil apoptosis as well. In addition, increased conversion of ADAM17’s pro-form to mature-form has also been reported during leukocyte apoptosis (53
Our data also reveals that during the late stages of neutrophil apoptosis, L-selectin expression can be down-regulated by a mechanism distinct from ADAM17, which resulted in the production of soluble L-selectin. CD16 and CD43 can undergo ectodomain shedding as well upon neutrophil activation, and have been reported to be released from the surface of neutrophils by membrane blebbing and microparticle production during apoptosis (43
). An approach used to discern soluble adhesion molecules that are cleaved from those that are associated with membrane particles is by sedimentation of the latter using ultracentrifugation (44
). We found that a combination of filtration (≥ 0.22 µm particle size) as well as ultracentrifugation (100,000 × g
) did not significantly reduce the levels of soluble L-selectin in the tissue culture media supernatant from cells undergoing prolonged apoptosis in the presence of the broad spectrum zinc metalloprotease inhibitor TAPI, suggesting that it may have been produced by a proteolytic process.
ADAM10 has been reported to cleave L-selectin in the absence of its primary sheddase ADAM17 (30
). However, it would seem unlikely that ADAM10 is playing a major role in soluble L-selectin production by apoptotic leukocytes considering that it has been shown to down-regulate in expression during this process (23
), and that soluble L-selectin production occurred in the presence of TAPI, which is known to block ADAM10 activity (55
). Serine proteases have been implicated as sheddases as well and can also cleave L-selectin (39
). However, various broad-spectrum serine protease inhibitors, as well as inhibitors of aspartic and cysteine proteases, also did not block soluble L-selectin production by late apoptotic leukocytes. Thus, the precise proteolytic process by which soluble L-selectin production occurs during later stages of leukocyte apoptosis remains to be determined. In addition, it will be interesting to determine whether this putative protease targets other cell surface determinants. For instance, surface expression of PSGL-1, a ligand of L-selectin (58
), can also be down-regulated during neutrophil apoptosis, which appears to involve a non-ADAM17 proteolytic process as well (54
The current study is the first to establish mechanisms of soluble L-selectin production upon death receptor-induced, neutrophil apoptosis. We have previously reported that ADAM17 deficient neutrophils can generate soluble L-selectin upon spontaneous apoptosis, which appears to involve other metalloproteases (8
). The biological significance of ADAM17-independent processes that mediate the release of surface L-selectin from apoptotic neutrophils is unclear at this time. Soluble L-selectin is maintained at high levels in the blood of healthy individuals and mice (45
). We have reported that the levels of soluble L-selectin in the blood of ADAM17 and wild-type chimeric mice are not significantly different (8
), suggesting that ADAM17 may not be the principle means of maintaining blood levels of soluble L-selectin. However, Venturi et al. have reported that soluble L-selectin in the blood is primarily derived by proteolytic cleavage, as gene-targeted mice expressing non-cleavable L-selectin have considerably lower levels of serum L-selectin (18
). In conjunction with our findings, it is tempting to speculate that soluble L-selectin levels in the blood of normal individuals may be maintained by a proteolytic mechanism other than ADAM17, perhaps occurring during leukocyte apoptosis.
In conclusion, ADAM17 appears to be active during the various phases of a neutrophil’s short life span; such as during their time in circulation [e.g. maintains L-selectin at an appropriate cell surface density (8
)], upon their activation (8
), and during death receptor-mediated apoptosis. The induction of ectodomain shedding during leukocyte apoptosis likely has a much broader purpose than just cleaving L-selectin. Indeed, various leukocyte determinants that undergo ectodomain shedding have an important role in regulating the resolution of inflammation, including TNFα, TNFRI, TNFRII, FasL, TRAIL, and IL-6R (53
), and thus this process may be another component of the anti-inflammatory program initiated by neutrophils (20