A tripeptide mimetic of von Willebrand factor residues 981-983 enhances platelet adhesion to fibrinogen by signaling through integrin alpha(IIb)beta3.

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.

Differential expression of cytokines (IL-2, IFN-gamma, IL-10) and adhesion molecules (VCAM-1, LFA-1, CD44) between spleen and lymph nodes associates with remission in chronic relapsing experimental autoimmune encephalomyelitis.

We have recently established chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) in SJL mice with a modified protocol. In this model, splenectomy aborts the relapsing-remitting course of the disease, and adoptive transfer of lymphocytes of the local draining lymph nodes (LNs) to naive recipients exacerbates the disease. Adoptive transfer of splenic cells converted acute EAE into CR-EAE in the naive recipients. In light of the different roles of the spleen and LNs in the evolution of CR-EAE, we examined by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) whether a differential mRNA expression profile of cytokines and cellular adhesion molecules (CAMs) in spleen versus LN was associated with relapse or remission in CR-EAE. All the cytokines tested (interleukin-1beta (IL-1beta), IL-2, IL-4, IL-7, IL-10, interferon-gamma (IFN-gamma)) as well as CAMs (ICAM-1, ICAM-2, VCAM-1, LFA-1 and CD44) were expressed at substantial levels in both spleen and LNs. Interestingly, disease remission was found to be associated with an increased mRNA expression of IL-2 and IFN-gamma in LNs and a decreased IL-10 mRNA level in the spleen. On the other hand, an increased mRNA expression of VCAM-1, LFA-1 and CD44 was observed in the spleens in comparison with that in LNs of mice, with remission. During relapses, mRNA expression of the tested molecules did not significantly differ between spleens and LNs. Our results suggest that a differential and polarized expression profile of certain cytokines and CAMs in spleen versus LN could provide molecular correlates of the cyclic pathogenesis of CR-EAE.

Release of soluble ICAM-5, a neuronal adhesion molecule, in acute encephalitis.

BACKGROUND: Intercellular adhesion molecule (ICAM)-5 (telencephalin) is an adhesion molecule in telencephalic neurons of the mammalian brain that binds to the leukocyte integrin CD11a/CD18. The authors observed that human cerebral neurons also expressed ICAM-5 and that ICAM-5--mediated neuron--leukocyte binding in cultured hippocampal neurons. This led the authors to examine ICAM-5 expression during clinical CNS inflammation. METHODS: The authors found, by immunoblotting, a 115-kDa soluble form of ICAM-5 (sICAM-5) cleaved from the membrane-bound (130 kDa) ICAM-5, and established an ELISA assay to measure it. CSF samples of patients with acute encephalitis and MS were studied. RESULTS: sICAM-5 was increased in encephalitis (320 plus minus 107 ng/mL; n = 25), as compared with patients with MS (128 plus minus 10 ng/mL; n = 16) and control subjects without CNS disease (137 plus minus 6 ng/mL; n = 42) (p < 0.001). The concentration of sICAM-5 correlated with the performance in the immediate recall task (p = 0.013) and with the leukocyte count in the CSF (p = 0.02), especially in cases caused by herpes simplex virus (HSV) (r = 0.94; p = 0.002). CONCLUSIONS: sICAM-5 is cleaved from CNS into CSF during acute encephalitis, and it may mediate leukocyte--neuron interactions. sICAM-5 release from cerebral neurons may actively regulate immune responses and leukocyte adhesion during microbial neuroinvasion in humans during encephalitis.

Intercellular adhesion molecule-1 in extravasation of normal mononuclear and leukaemia cells.

Interaction of intercellular adhesion molecules (ICAMs) with their receptors has a key role in normal leucocyte adhesion and migration, whereas in leukaemia this has not been well established. In this study, we have evaluated the roles of different adhesion molecules in normal and leukaemia cell extravasation in a novel organotypic model for vessel wall and measured plasma ICAM-1 and -2 levels in acute leukaemia patients at diagnosis and during chemotherapy. We found that both normal mononuclear cells and blast cells from acute leukaemia patients, as well as retinoic acid-treated promyelocytic leukaemia cells, rapidly extravasated through endothelial cell layers into the underlying collagen matrix. ICAM-1 antibody prevented the extravasation, while antibodies to other adhesion molecules showed little (CD18, ICAM-2) or no inhibition (CD11a and ICAM-3). Soluble ICAM-1 (sICAM-1) protein had no effect. We also observed increased plasma sICAM-1 and -2 levels in leukaemia patients and found that they correlated only weakly with the white blood cell count. No correlation was found between sICAM-1 or -2 levels and the response to therapy. Although elevated sICAM-2 levels decreased rapidly during chemotherapy, sICAM-1 levels did not. Because sICAM-1 protein had no effect on leukaemia cell extravasation in vitro, it is probable that the increased plasma sICAM-1 levels in leukaemia patients may not play a role in leukaemia cell infiltration. However, as we showed that ICAM-1 mediated leukaemia cell extravasation on the cell surface, it is possible that cellular ICAM-1 has an important role in disease progression.

Inhibition of beta(2) integrin-mediated leukocyte cell adhesion by leucine-leucine-glycine motif-containing peptides.

Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific beta(2) integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major beta(2) integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel beta(2) integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the beta(2) integrin-mediated leukocyte adhesion, but not beta(1) or beta(3) integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3 (CD50).

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.

An essential role for calmodulin in regulating human T cell aggregation.

After activation of T cells with either CD3 antibodies or phorbol esters, we have found that T cell-cell aggregation, integrin-dependent actin reorganisation and cell spreading are strongly suppressed by any of three structurally different calmodulin antagonists, without any effect on the amount of CD11/CD18 integrin binding to the actin cytoskeleton. However, only T cell receptor-induced, and not phorbol ester-induced, aggregation and cell spreading are prevented by inhibitors of phosphatidylinositide (PI) 3-kinase. These results suggest that PI 3-kinase lies upstream of calmodulin in the signalling pathway leading to T cell aggregation, cell spreading and actin reorganisation and that cell spreading and actin reorganisation are essential for T cell adhesion.

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion.

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.

Binding sites of leukocyte beta 2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein.

The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LW(ab) block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca(2+) and Mg(2+). Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-4 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-2, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) on the CFG face and Trp(77) on the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.

Binding of T lymphocytes to hippocampal neurons through ICAM-5 (telencephalin) and characterization of its interaction with the leukocyte integrin CD11a/CD18.

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a member of the immunoglobulin superfamily expressed on telencephalic neurons, and serves as a ligand for the leukocyte integrin CD11 a/CD18. We studied here the binding site in ICAM-5 for CD11a/CD18. Protein constructs containing the first immunoglobulin domain of ICAM-5 were able to support CD11a/CD18 interaction, while deletion of the first domain abolished binding. Monoclonal antibodies reacting with the first domain of ICAM-5 also completely blocked the interaction. The soluble first domain of ICAM-5 inhibited the binding of T cells to immobilized ICAM-5 at concentrations of 50 nM and higher. Interestingly, the sixth domain of ICAM-5 was also able to support leukocyte binding, but this binding activity may not involve leukocyte integrins. To test the involvement of ICAM-5 in leukocyte-neuron interactions, an assay using human T cells binding to rat hippocampal neurons was established. This binding was blocked by monoclonal antibodies against CD11a/CD18 and ICAM-5. Thus ICAM-5 may act as a major adhesion molecule for leukocyte binding to neurons in the central nervous system.

Leukocyte adhesion--an integrated molecular process at the leukocyte plasma membrane.

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell-cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific beta2-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Leukocyte adhesion--a fundamental process in leukocyte physiology.

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte beta 2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.

The cytoskeletal association of CD11/CD18 leukocyte integrins in phorbol ester-activated cells correlates with CD18 phosphorylation.

Leukocyte adhesion is a regulated process, which involves CD11/CD18 leukocyte integrins. CD11/CD18 acidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated on serine and threonine after phorbol ester activation of T cells. Increased adhesiveness is believed to be mediated by regulating the overall avidity of cellular contact. CD11/CD18 integrins have earlier been reported to interact with several cytoskeletal proteins. We have now studied the involvement of the CD18 phosphorylation in cytoskeletal associations. We have investigated the distribution of phosphorylated CD18 between soluble, cytoskeletal and nuclear fractions of T cell detergent lysates. A significant amount of phosphorylated CD18 polypeptides was observed to fraction along with the cytoskeleton, while the majority of the cell surface CD18 molecules remained in the soluble fraction. Putative candidates for this altered cytoskeletal binding of CD11/CD18 were shown to be talin and filamin, which were observed to bind to CD18 cytoplasmic peptides and co-precipitate with CD18. The importance of the CD18 cytoplasmic domain in the regulation of the leukocyte adhesion was further strengthened by inhibition of phorbol ester-induced T cell adhesion with a phosphorylated lipopeptide corresponding to the cytoplasmic portion of the CD18. These results indicate that the induced CD18 phosphorylation and the altered cytoskeletal binding of the phosphorylated integrin complex may contribute to the increased avidity of CD11/CD18-mediated leukocyte adhesion.

ICAM-2 and a peptide from its binding domain are efficient activators of leukocyte adhesion and integrin affinity.

Cell adhesion mediated by the CD11/CD18 integrins and their ligands, the ICAMs, is required for many leukocyte functions. In resting cells the integrins are nonadhesive, but when activated they become adhesive for their ligands. Previous findings have shown that a peptide derived from the first Ig domain of ICAM-2 (P1) binds to LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) and activates leukocyte aggregation. Because its mechanism of action has remained poorly understood, we have now studied the peptide-induced ligand binding in detail. Here we show that P1 was able to induce CD11/CD18-dependent adhesion of human T lymphocytes to immobilized, purified ICAM-1, -2, and -3. The optimal peptide concentration was 150 micrograms/ml, whereas concentrations higher than 400 micrograms/ml did not have any stimulatory effect. The increase in adhesion was detectable within 10 min of treatment with the peptide; it was dependent on energy, divalent cations, temperature, and an intact cytoskeleton but was unaffected by protein kinase C and protein tyrosine kinase inhibitors. Peptide treatment resulted in strong stimulation of the binding of soluble, recombinant ICAMs to T lymphocytes, showing that the integrin affinity toward its ligands was increased. Importantly, soluble ICAM-2Fc was also able to induce T lymphocyte adhesion to purified ICAM-1, -2, and -3, and it was a more potent stimulatory molecule than ICAM-1Fc or ICAM-3Fc.

Characterization of beta2 (CD18) integrin phosphorylation in phorbol ester-activated T lymphocytes.

Integrins are transmembrane proteins involved in cell-cell and cell-extracellular-matrix interactions. The affinity and avidity of integrins for their ligands change in response to cytoplasmic signals. This 'inside-out' activation has been reported to occur also with beta2 integrins (CD18). The beta2 integrin subunit has previously been shown to become phosphorylated in T lymphocytes on cytoplasmic serine and the functionally important threonine residues after treatment with phorbol esters or on triggering of T-cell receptors. We have now characterized the phosphorylation of beta2 integrins in T-cells in more detail. When T-cells were activated by phorbol esters the phosphorylation was mainly on Ser756. After inhibition of serine/threonine phosphatases, phosphorylation was also found in two of the threonine residues in the threonine triplet 758-760 of the beta2 cytoplasmic domain. Activation of T-cells by phorbol esters resulted in phosphorylation in only approx. 10% of the integrin molecules. Okadaic acid increased this phosphorylation to approx. 30% of the beta2 molecules, assuming three phosphorylation sites. This indicates that a strong dynamic phosphorylation exists in serine and threonine residues of the beta2 integrins.

Leukocyte integrins and inflammation.

Leukocyte adhesion is of pivotal functional importance. Without adequate adhesion, T lymphocytes and natural killer cells are not cytotoxic, B cells cannot develop into antibody secreting plasma cells, leukocytes do not home into inflamed tissues and myeloid cells are not able to phagocytize or exhibit chemotactic responses. During evolution several leukocyte adhesion molecules have developed belonging to a few molecular families. Among these, the leukocyte-specific integrins (beta 2 integrins, CD11/CD18 molecules) are among the most important. Much progress has taken place during the past few years, and at present we have a considerable knowledge of their structure and function. Inflammation is critically dependent on integrin activity, and its regulation forms the topic of this short review.

Structural study of the O-linked sugar chains of human leukocyte tyrosine phosphatase CD45.

The O-linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium-labeled oligosaccharides by beta-elimination in the presence of NaB3H4. Mono Q column chromatography revealed that they comprise neutral (64%) and acidic (36%) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio-Gel P-4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15% of which are modified with poly (N-acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB3H4 treatment. Bio-Gel P-6 column chromatography of a mixture of the tritium-labeled glycopeptide/oligosaccharides obtained by pronase-digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.

Leukocyte adhesion: CD11/CD18 integrins and intercellular adhesion molecules.

Leukocyte integrins and intercellular adhesion molecules play pivotal roles in leukocyte adhesion to target cells and extracellular matrices. Recently, novel intercellular adhesion molecules have been identified, and much information has been obtained on the structures and binding sites of leukocyte integrins and of intercellular adhesion molecules. Furthermore, much progress has been made in the study of integrin activation and the role of leukocyte adhesion molecules in disease.

Leukocyte adhesion--structure and function of human leukocyte beta2-integrins and their cellular ligands.

Leukocyte adhesion is of pivotal functional importance and this has resulted in extensive research and rapid development in the field. Leukocyte adhesion involves members of three molecular families: integrins, members of the immunoglobulin superfamily and carbohydrate binding selectins and sialoadhesins. Recently, considerable structural information on leukocyte integrins and members of the immunoglobulin superfamily of adhesion molecules has been obtained. This fact, combined with the identification of several novel adhesion molecules, has increased our understanding of how they function at the molecular level. Furthermore, the important issue of how integrins are activated to become adhesive is rapidly advancing. It is clearly evident that the knowledge accumulated from basic research will increasingly be applied in clinical medicine. In this review we focus on two important families of adhesion molecules, the leukocyte-specific beta2-integrins and their ligands, the intercellular adhesion molecules. Emphasis is put on their structural/functional relationships, their mode of regulation and on novel adhesion molecules recently discovered.