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 surface origin of human alpha1-acid glycoprotein (orosomucoid).

Specific antibodies against human alpha1-acid glycoprotein reacted with human lymphocytes, granulocytes, and monocytes. The antigen on the leukocytes is an externally located integral membrane glycoprotein which is made by the cells and has an apparent mol wt of 52,000. It is released from cells in vitro to the culture medium. The mol wt of the soluble fragment is 41,000, which corresponds to that of alpha1-acid glycoprotein in serum and urine. Peptide mapping confirmed that the main part of the cellular membrane antigen consists of alpha1-acid glycoprotein with an additional, probably hydrophobic fragment. This finding may partially explain the increase in the serum levels of alpha1-acid glycoprotein observed in many disorders involving leukocyte proliferation. In addition, the known sequence homology of alpha1-acid glycoprotein with immunoglobulins can now be more easily understood by their origin in similar cell types.

Characterization of surface glycoproteins of mouse lymphoid cells.

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.

A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18.

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP-1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.

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.

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.

Why mammalian cell surface proteins are glycoproteins.

Most proteins presented at the external surface of mammalian cells contain carbohydrate. The reason for this is not fully understood, but recent work has shown that such carbohydrate has two major functions. Inside the cell, it helps proteins fold and assembly correctly in the endoplasmic reticulum, and it might also act as a signal for the correct migration of glycoproteins. Outside the cell, it provides specific recognition structures for interaction with a variety of external ligands.

Studies on the defect underlying the lysosomal storage of sialic acid in Salla disease. Lysosomal accumulation of sialic acid formed from N-acetyl-mannosamine or derived from low density lipoprotein in cultured mutant fibroblasts.

Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane.

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

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.

Pericellular matrix and cell surface glycoproteins of virus-transformed mouse epithelial cells.

Mouse embryo Mus musculus castaneous epithelial cells, transformed with Moloney murine sarcoma virus (MSV) or with ecotropic murine leukemia virus (MuLV), were analyzed for production of pericellular matrix glycoproteins. The nontransformed, MSV-transformed, and MuLV-transformed cells produced fibronectin, laminin, type I collagen, and small amounts of type III collagen when studied by immunofluorescence using specific antibodies. The virus-transformed epithelial cells produced enhanced amounts of fibronectin into their growth media. Nontransformed M. musculus castaneous epithelial cells mainly produced type I collagen, as shown by metabolic labeling and polypeptide analysis. A significant increase in the glycoprotein production was seen by the MuLV-transformed cells, whereas small changes in the collagen production were apparent after MSV transformation. MuLV-transformed cells produced increased amounts of type I collagen and also some collagenous polypeptides that comigrated with procollagen type IV chains. The ratio of the procollagen type I chains deposited in the matrix was altered in transformed cells. Radioactive surface labeling of the cells revealed changes of the high-molecular-weight glycoproteins in both the MSV- and the MuLV-transformed cells. Unlike virus-transformed fibroblastic cells, these transformed epithelial cells deposited and retained connective tissue glycoproteins in their pericellular matrices. The results indicate that viral transformation modulates the pericellular matrix and surface glycoproteins of cultured mouse epithelial cels. The ability of virus-transformed epithelial cells to deposit pericellular matrices is a major difference between them and virus-transformed fibroblastic cells.

Changes in cell surface glycoproteins and antigens during differentiation of the human myeloid leukemia cell lines ML-1, ML-2, and HL-60.

Two recently derived human myeloid leukemia cell lines, ML-1 and ML-2, were induced to differentiate by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or with retinoic acid for 5 to 12 days. They were then compared with similarly treated promyelocytic HL-60 cells. TPA-treated ML-1 and ML-2 cells became firmly surface adhesive with a fibroblastoid morphology, while TPA-treated HL-60 cells adhered as rounded macrophages. In contrast, retinoic acid induced only slight morphological changes in all three cell lines. The differentiation-related alterations of the surface membrane glycoproteins were followed by polyacrylamide gel electrophoresis after surface labeling by the periodate-NaB3H4 or galactose oxidase-NaB3H4 methods. The expression of surface membrane differentiation antigens was analyzed with a panel of monoclonal antibodies against myeloid, myelomonocytic, monocytic, and granulocytic determinants using FACS IV flow cytometry. The acquisition of surface adhesiveness by TPA-treated ML-1 and ML-2 cells coincided with the appearance of membrane surface proteins of varying molecular weights, ranging between 90,000 and 155,000, which were not labeled in untreated ML-1 and ML-2 cells. These findings and the results obtained by monoclonal antibody staining and FACS analysis indicate that treatment of the myeloid lines ML-1, ML-2, and HL-60 by TPA induced the expression of antigenic and membrane molecular features compatible with a monocytic-macrophage phenotype, while treatment by retinoic acid induced granulocytic differentiation. The ML-1 and ML-2 cells offer interesting models for studies on the membrane molecular events occurring when nonadherent, monocytic cells become surface adherent.

Phospholipid composition and external labeling of aminophospholipids of human En(a--) erythrocyte membranes which lack the major sialoglycorprotein (glycophorin A).

Erythrocytes of the rare human blood group En(a--) lack the major sialoglycoprotein, glycophorin A, and the cell population heterozygous for the En(a) antigen contain half the normal amount of glycophorin A. With such cells we have studied whether glycophorin A influences the phospholipid composition and the availability of aminophospholipids to external labeling reagents. We here demonstrate that the amounts of all phospholipids are closely similar in normal and variant membranes. However, using the amino-reactive reagent trinitrobenzenesulfonate, we show that phosphatidylethanolamine is more easily labeled in intact En(a--) cells as compared to normal cells, whereas phosphatidylethanolamine shows an intermediate labeling in En(a) heterozygous cells.

Isolation and characterization of the blood group A-specific lectin from Vicia cracca.

We have isolated the blood group A-specific lectin from Vicia cracca by affinity chromatography on immobilized porcine blood group A/H substance. A molecular weight of 100 000 was obtained by gel filtration and analytical ultracentrifugation. The subunit size when determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 29 000. The lectin contains no half-cystine or methionine. It is a glycoprotein apparently with one oligosaccharide per subunit. The oligosaccharide contains mainly D-mannose and D-glucosamine but no D-galactose or sialic acid.

Identification of blood group A-active glycoproteins in the human erythrocyte membrane.

Normal human erythrocytes of blood groups A1, A2, B and O, and En (a-) erythrocytes lacking glycophorin A, but with A1B-activity, were surface-labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. A1 cells were also labeled by lactoperoxidase catalyzed iodination. After solubilization in Triton X-100, the blood group A-active glycoconjugates were isolated using the A-specific lectin from Vicia cracca coupled to Sepharose. No radioactivity was bound from erythrocytes of B and O blood groups. The glycoconjugates from A cell membranes which bound to the lectin and were eluted with 0.01 M N-acetyl-D-galactosamine were analyzed using cylindrical or slab gel electrophoresis in the presence of sodium dodecyl sulfate. The A-active glycoproteins included the major integral glycoprotein, band 3, and many minor, previously poorly defined components. Glycophorins A and B did not contain A-activity.

Rotational diffusion of band 3 proteins in membranes from En(a-) and neuraminidase-treated normal human erythrocytes.

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human eythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887-1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a-) and neuraminidase-treated erythrocytres. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a-) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37 degrees C could be detected between En(a-) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a-) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.

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.

The leukocyte surface antigens CD11b and CD18 mediate the oxidative burst activation of human peritoneal macrophages induced by type 1 fimbriated Escherichia coli.

Analysis by immunofluorescence-activated cell sorting of human peritoneal macrophages from patients undergoing intermittent peritoneal dialysis revealed that they express the CD11/CD18 surface antigens, with CD11b and CD18 as the predominant ones. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from lysates of 125I-labeled macrophages with rabbit polyclonal antibodies against the CD11a-c/CD18 complex or against CD18, revealed four radioactive bands corresponding to CD11a, CD11b, CD11c, and CD18. Monoclonal antibodies against CD11b and CD18 inhibited by 80 and 90%, respectively, the oxidative burst activation of the macrophages by type 1 fimbriated Escherichia coli, whereas monoclonal antibodies against CD11a, CD11c, and CD43 were without effect. Our results suggest that CD11b and CD18 (receptors for C3bi) serve also as receptors for mannose-specific E. coli on human peritoneal macrophages and may be involved in the lectinophagocytosis of the bacteria by these cells.

Pre-replicative changes of the rat sinusoidal plasma membrane glycoproteins during hepatic regeneration.

Cell-surface glycoproteins of rat liver sinusoidal plasma membranes from control and regenerating livers were studied. The glycoproteins were labeled using specific methods for sialic acid (NAIO4/NaB3H4) and galactosyl/N-acetyl galactosaminyl residues (galactose oxidase/NaB3H4 and neuraminidase-galactose oxidase (NaB3H4) and the solubilized proteins were analyzed by gel electrophoresis. The patterns obtained with regenerating livers were quantitatively different from controls. This shows that cell surface glycoproteins change during liver regeneration.

Fibronectin isoforms in plasma membrane domains of normal and regenerating rat liver.

Plasma membrane fractions were obtained from the three surface domains of normal and regenerating adult rat livers. It was shown by immunoblotting that sinusoidal plasma membranes contained the characteristic 220 and 210 kDa fibronectin doublet, whereas bile canalicular plasma membranes contained a 220 kDa component. In lateral plasma membranes, 180, 190 and 220 kDa fibronectin isoforms were present. Fibronectin in the sinusoidal and canalicular plasma membranes was shown, by detergent/aqueous phase partitioning, to be more hydrophilic than isoforms in lateral plasma membranes. Changes in the distribution of fibronectin between plasma membrane domains occurred during liver regeneration and their significance, especially in relation to cell division, is discussed.