Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca2+-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.

L-selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L-selectin undergoes ectodomain shedding. Using approaches that monitor full-length L-selectin in short-term assays, it has been determined that L-selectin shedding is defective in tumor necrosis factor alpha-converting enzyme (ADAM-17)-deficient cells. In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells using a monoclonal antibody to the cytoplasmic region of L-selectin, which allows for the detection of total L-selectin (full-length and the membrane-associated cleavage fragment). We demonstrate that ADAM-17-deficient cells generate a 6-kDa transmembrane fragment of L-selectin. Although inducible L-selectin shedding by phorbol 12-myristate 13-acetate stimulation was not observed by these cells in short-term assays, basal turnover did occur, resulting in the production of soluble L-selectin, as determined by enzyme-linked immunosorbent assay. L-selectin turnover was greatly increased upon ADAM-17 reconstitution. Truncating the juxtamembrane region of L-selectin blocked ADAM-17-independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM-17 can use the cleavage domain of L-selectin. We speculate that separate proteolytic mechanisms of L-selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell-cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L-selectin, a potential adhesion buffer.

The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced Lselectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP-and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/ CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce Lselectin shedding from neutrophils. J. Leukoc. Biol. 67: 415-422; 2000.

Leukocytes are captured directly by E- and P-selectin on activated endothelium and by indirect means, which includes attached leukocytes capturing free-flowing leukocytes. However, controversy exists as to whether the latter mechanism occurs in the presence of red blood cells. We analyzed leukocyte capture mechanisms on P-selectin under circulatory hydrodynamics using whole blood. The selective disruption of leukocyte-leukocyte interactions with an L-selectin monoclonal antibody reduced leukocyte accumulation by >50% under various stringencies (substrate concentrations and shear stresses). In addition, a direct analysis of leukocyte capture events revealed that 69% were indirect. Our data indicate that in the presence of red blood cells, P-selectin-attached leukocytes, individually and as a monolayer, augment leukocyte accumulation by indirect capture. This mechanism may contribute to increasing the density of leukocytes on discrete areas of activated endothelial cells at sites of inflammation. These findings are significant since L-selectin accounts for the majority of the leukocyte rolling flux in small venules at diverse inflammatory settings. Yet, the primary mechanism by which L-selectin mediates leukocyte accumulation remains unresolved.