ADAM17 but not ADAM10 mediates tumor necrosis factor-alpha and L-selectin shedding from leukocyte membranes

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.     

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.     

Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.     

L-选择素在白细胞活化中的作用

白细胞沿着血管内皮滚动、稳定黏附,最终到达炎症部位是一个复杂的、多步骤的过程,该过程需要众多分子协同完成。选择素家族分子对于白细胞沿着血管内皮的滚动起重要作用。L-选择素是选择素家族的一员,组成性的表达在白细胞微绒毛顶端,在白细胞沿血管内皮起始黏附过程中起主要作用。除具有黏附作用外,L-选择素还作为信号分子在黏附事件中发挥作用。该文结合作者的研究工作,综述了L-选择素在白细胞活化过程中的功能。     

Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding

The calmodulin (CaM) hypothesis of ectodomain shedding stipulates that CaM, an intracellular Ca²⁺-dependent regulatory protein, associates with the cytoplasmic domain of l-selectin to regulate ectodomain shedding of l-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of CaM with two peptides derived from human l-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of l-selectin (residues Ala317-Tyr334 in the mature protein), and CLS corresponds to residues Lys280-Tyr334, which contains the entire transmembrane and cytoplasmic domains of l-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that CaM can bind to ARR18 in aqueous solutions or the l-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximately 2 μM. The association is calcium independent and dynamic and involves both lobes of CaM. In a phospholipid bilayer, the positively charged l-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine (PS) lipids at the membrane interface through electrostatic interactions. Under conditions where the PS content mimics that in the inner leaflet of the cell plasma membrane, the interaction between CaM and CLS becomes undetectable. These results suggest that the association of CaM with l-selectin in the cell can be influenced by the membrane bilayer and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.     

differential L-selectin binding activities of human hematopoietic cell L-selectin ligands, HCELL and PSGL-1

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.     

L-selectin shedding is independent of its subsurface structures and topographic distribution

L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells' surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.     

Adhesive dynamics simulations of the mechanical shedding of L-selectin from the neutrophil surface

Here we accurately recreate the mechanical shedding of L-selectin and its effect on the rolling behavior of neutrophils in vitro using the adhesive dynamics simulation by incorporating the shear-dependent shedding of L-selectin. We have previously shown that constitutively expressed L-selectin is cleaved from the neutrophil surface during rolling on a sialyl Lewis x-coated planar surface at physiological shear rates without the addition of exogenous stimuli. Utilizing a Bell-like model to describe a shedding rate which presumably increases exponentially with force, we were able to reconstruct the characteristics of L-selectin-mediated neutrophil rolling observed in the experiments. First, the rolling velocity was found to increase during rolling due to the mechanical shedding of L-selectin. When most of the L-selectin concentrated on the tips of deformable microvilli was cleaved by force exerted on the L-selectin bonds, the cell detached from the reactive plane to join the free stream as observed in the experiments. In summary, we show through detailed computational modeling that the force-dependent shedding of L-selectin can explain the rolling behavior of neutrophils mediated by L-selectin in vitro.     

Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail

BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.     

The tail domain of myosin Va modulates actin binding to one head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.     

Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain

The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin.     

Alternative splicing of the actin binding domain of human cortactin affects cell migration

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.     

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems

This communication is concerned with the binding specficityof the leukocyte-adhesion molecule L-selectin (leukocyte homingreceptor) towards structurally defined sulphated oligosaccharidesof the blood group Le^a and Le^x series, and of the glycolsaminoglycanseries heparin, chondroitin sulphate and keratan sulphate. Therecombinant soluble form of the rat L-selectin (L-selectin-IgGFc chimera) investigated here was shown previously to bind tolipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated atgalactose, such as sulphatides and a mixture of 3-sulphatedLe^a/Le^x type tetrasaccharides isolated from ovarian cystadenoma,as well as to the HNK-1 glycolipid with 3-O sulphated glucuronicacid. In the present study, the L-selectin investigated in bothchromatogram binding and plastic microwell binding experimentsusing neoglycolipids was found to bind to the individual 3-sulphatedLe^a and Le^x sequences (penta-, tetra- and trisaccharides), andwith somewhat lower intensities to their non-fucosylated analogues.Glycosaminoglycan disaccharides of keratan sulphate, heparinand chondroitin sulphate types were also bound by L-selectinin one or both assay systems, leading to the conclusion thatclustered glycosaminoglycan oligosaccharides with 6-O sulphationof N-acetylgalactctosamine, N-acetylglucosamine or glucosamine,4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronicacid, N-sulphation of glucosamine and, to a lesser extent, thenon-sulphated uronic acid-contahing disaccharides, can supportL-selectin adhesion. As inflammatory chemokines (short-rangestimulators of lymphocyte migration which trigger integrin activation)are known to bind to endothelial glycosaminoglycans, we proposethat the binding of the lymphocyte membrane L-selectin to endothelialglycosaminoglycans may provide a link between the selectin-mediatedand integrin-mediated adhesion systems in leukocyte extravasationcascades. The posibility is also raised that lymphocyte L-selectininteractions with glycosaminoglycans may contribute to pathologiesof glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoidarthritis and inflammatory lesions of the cornea. glycosaminoglycans leukocyte adhesion cascades neoglycolipids oligosaccharide presentation sulphated oligosaccharides     

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.     

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.     

Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44

Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies.     

DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro-methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.     

Loss of a glycine in the alpha2 domain affects MHC peptide binding but not chaperone binding.

Prior to the binding of peptide in the endoplasmic reticulum (ER), the major histocompatibility complex (MHC) class I heavy chain associates with an assembly complex that includes the transporter associated with antigen processing (TAP). The proximity of a part of the MHC class I alpha2 domain alpha-helix to areas previously shown to influence assembly complex binding suggests that this region might also be involved in chaperone association. Position 151, found in this part of the alpha2 domain alpha-helix, has a side chain that points up, away from direct contact with peptide, and is occupied by a glycine in all murine MHC class I heavy chains. We found that substitution of this glycine in H-2L(d) with a histidine substantially increased the proportion of peptide-free forms, although TAP binding was not abrogated. Thus, interaction of the heavy chain with peptides, but not with the assembly complex, is influenced by this glycine.     

Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint

SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.     

Selectin-independent leukocyte rolling and adhesion in mice deficient in E-, P-, and L-selectin and ICAM-1

To study selectin-independent leukocyte recruitment and the role of intercellular adhesion molecule-1 (ICAM-1), we generated mice lacking all three selectins and ICAM-1 (E/P/L/I-/-) by bone marrow transplantation. These mice were viable and appeared healthy under vivarium conditions, although they showed a 97% reduction in leukocyte rolling, a 63% reduction in leukocyte firm adhesion, and a 99% reduction of neutrophil recruitment in a thioglycollate-induced model of peritonitis at 4 and 24 h. Mononuclear cell recruitment was almost unaffected. All residual leukocyte rolling and most leukocyte adhesion in these mice depended on alpha(4)-integrins, but a small number of leukocytes (6% of wild-type control) still became adherent in the absence of all known rolling mechanisms (E-, P-, L-selectin and alpha(4)-integrins). A striking similarity of leukocyte adhesion efficiency in E/P/L-/- and E/P/I-/- mice suggests a pathway in which leukocyte rolling through L-selectin requires ICAM-1 for adhesion and recruitment. Comparison of our data with mice lacking individual or other combinations of adhesion molecules reveal that elimination of more adhesion molecules further reduces leukocyte recruitment but the effect is less than additive.