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Biomedical subjects

C G Gahmberg

Publications and source records attributed to C G Gahmberg.

At least 37 records · Page 2Linked to original sources

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.

Amino Acid Sequence↗

The neuronal glycoprotein telencephalin is a cellular ligand for the CD11a/CD18 leukocyte integrin.

Many leukocyte functions depend on interactions between the leukocyte-specific beta2 integrins CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs). Telencephalin (TLN) is a novel member of the Ig superfamily expressed in the central nervous system. The NH2-terminal five Ig-like domains of TLN show the highest homology with the Ig domains of ICAM-1, ICAM-2, ICAM-3, and LW (ICAM-4), the known cellular ligands for CD11a/CD18. Here, we demonstrate that TLN interacts with CD11a/CD18. Peripheral blood T cells, Jurkat T cells, and B lymphoblastoid cells bound to immunopurified recombinant human TLN proteins. This adhesion was through CD11a/CD18 and was significantly inhibited by an Ab to CD11a/CD18. Reciprocally, TLN-transfected L cells also bound to purified CD11a/CD18. Recombinant TLN proteins comprising either the first five Ig domains (TLN(1-5)) or the entire extracellular portion (TLN(1-9)) showed binding to CD11a/CD18. We conclude that TLN is a novel neuronal cell adhesion molecule that may be important in integrin-mediated cell-cell interactions in the central nervous system, and that the CD11a/CD18-dependent recognition site of human TLN is located within the NH2-terminal five domains of this molecule.

Amino Acid Sequence↗

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.

Animals↗

Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alpha-actinin.

Intercellular adhesion molecule-2 (ICAM-2) functions as a ligand for lymphocyte function-associated antigen-1 (LFA-1) and is involved in leukocyte adhesion. We studied intracellular associations of ICAM-2 using a peptide encompassing the cytoplasmic amino acids 231-254 as an affinity matrix. Among the proteins from placental lysates that bound to the peptide was alpha-actinin as demonstrated by immunoblotting. Purified, 125I-labeled alpha-actinin also bound to the peptide. Confocal microscopic analysis of Eahy926 cells demonstrated a colocalization of ICAM-2 and alpha-actinin. Of overlapping octapeptides covering the entire ICAM-2 cytoplasmic amino acids, ICAM-2241-248 bound alpha-actinin most avidly and effectively competed with the longer cytoplasmic peptide for binding. The site of interaction in alpha-actinin was studied using bacterially expressed alpha-actinin fusion proteins. Several constructs covering nonoverlapping regions of alpha-actinin bound to the ICAM-2 cytoplasmic peptide suggesting that multiple regions in alpha-actinin can mediate the interaction. These results, together with previously demonstrated interactions between alpha-actinin and the adhesion proteins ICAM-1, L-selectin, beta1- and beta2-integrins emphasize the role of alpha-actinin as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton.

Actinin↗

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.

Animals↗

Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD11b/CD18 through the A domain.

The interactions between the leukocyte-specific beta 2-integrins cluster of differentiation (CD) Ag CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs), play important roles in many adhesion-dependent leukocyte functions. ICAM-1 is known to be a ligand for both CD11a/CD18 and CD11b/CD18. ICAM-2, whose two extracellular Ig domains show the highest homology to the two NH2-terminal domains of ICAM-1, has been previously shown to be a ligand for CD11a/CD18. We recently found that a 22-amino acid CD11a/CD18-binding peptide, P1, derived from the first domain of ICAM-2, also binds to purified CD11b/CD18. In the present study, we demonstrate that the ICAM-2 protein interacts with CD11b/CD18, and the binding is through the CD11b A domain.

Animals↗

Treatment with okadaic acid reveals strong threonine phosphorylation of CD18 after activation of CD11/CD18 leukocyte integrins with phorbol esters or CD3 antibodies.

The CD11/CD18 leukocyte integrins comprise three heterodimers involved in leukocyte adhesion. CD11/CD18 avidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated in leukocytes after phorbol ester stimulation. The importance of phosphorylation in the regulation of CD11/CD18 avidity has, however, remained unclear. We have now activated T cells using phorbol esters, CD3, and CD44 Abs. Both phorbol ester and CD3 treatment activated protein kinase C. CD18 was shown to become more stably phosphorylated after phorbol ester treatment and more transiently so after CD3 stimulation. The phosphorylation was strongly augmented by okadaic acid, a serine/threonine phosphatase inhibitor. While phorbol ester treatment caused phosphorylation mainly on serine, in okadaic acid-pretreated cells, both phorbol ester treatment as well as CD3 stimulation revealed strong threonine phosphorylation. Since earlier mutational studies have demonstrated the functional importance of cytoplasmic threonine residues in CD18, the threonine phosphorylation reported here indicates the role of threonine phosphorylation in the regulation of CD11/CD18 avidity.

Alkaloids↗

Mutation of the cytoplasmic domain of the integrin beta 3 subunit. Differential effects on cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis.

The cytoplasmic domain of the beta subunit of the alpha IIb beta 3 integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the beta 3 (I757TYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ile757 partially preserved these functions. We further found that substitution of Tyr747 with Ala also abolished alpha IIb beta a-mediated cell spreading. The effects of these and other mutations on additional functions of alpha IIb beta 3 were also studied. Progressive truncations of beta 3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of alpha IIb beta 3 to preestablished adhesion plaques and a gradual decrease in the ability of alpha IIb beta 3 to mediate internalization of fibrinogen-coated particles. The Tyr747-->Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr759, and at a conserved area close to the transmembrane domain of beta 3, decreased integrin recruitment to preestablished adhesion plaques but allowed alpha IIb beta 3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of beta 3 did not affect the constitutive endocytosis of alpha IIb beta 3.

Amino Acid Sequence↗

Activation of natural killer cell migration by leukocyte integrin-binding peptide from intracellular adhesion molecule-2 (ICAM-2).

Intracellular adhesion molecule-2 (ICAM-2), one of the ligands of CD11a/CD18 (LFA-1), is mainly expressed on endothelial and hematopoietic cells. The biological significance of ICAM-2 has remained unclear. Previous findings have shown that a peptide from ICAM-2, spanning residues 21-42 from the first immunoglobulin domain, enhances natural killer (NK) cell cytotoxicity and induces T cell aggregation. We have now studied the effect of the same ICAM-2 peptide on NK cell migration in the Boyden chamber assay. The peptide significantly increased NK cell migration up to 215 +/- 21%, as compared to migration of control cells (100%), and the induction was inhibited by anti-CD11a monoclonal antibodies. The ICAM-2 peptide also induced polymerization of F-actin at the leading edge of migratory NK cells. Cross-linking of CD11a/CD18 receptors with anti-CD11a or anti-CD18 monoclonal antibodies and secondary antibodies resulted in receptor recycling, increased migration, and actin polymerization, but led to slight inhibition of cytotoxicity. The ICAM-2 peptide did not induce such a receptor recycling. Phosphotyrosine immunoblotting experiments showed that the ICAM-2 peptide increased the phosphorylation of 150- and 35-kDa proteins. During cross-linking with antibodies, only the 150-kDa protein showed increased phosphorylation. The results show that depending on the type of CD11a/CD18 receptor ligation different kinds of signals are transduced in NK cells. These signals may either trigger only locomotion, or both locomotion and cytotoxicity. Based on these findings, a major function for ICAM-2 on endothelium may be triggering of migration of adhering leukocytes.

Actins↗

Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1).

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S-transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose-dependent manner, and the active site resides within the 104 carboxy-terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.

Amino Acid Sequence↗

A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50.

We have made a monoclonal anti-CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM-1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM-1 and ICAM-3. The binding to ICAM-2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand-specific manner by engagement of CD44.

Antibodies, Monoclonal↗

The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins.

Leukocyte adhesion involves the leukocyte-specific integrins CD11a/CD18, CD11b/CD18 and CD11c/CD18, which bind to intercellular adhesion molecules (ICAM). Three ICAM have been described, and are expressed on leukocytes and various other cells, but are absent from red cells. Here, we show that the red cell Landsteiner-Wiener (LW) blood group glycoprotein is an ICAM which binds to the leukocyte-specific integrins. This finding has important implications in red cell physiology.

Blood Group Antigens↗

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.

Actin Cytoskeleton↗

Sialyl Lewis(x)- and L-selectin-dependent site-specific lymphocyte extravasation into renal transplants during acute rejection.

Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM-1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site-specific restriction. VCAM-1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex-negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti-sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low-dose chymotrypsin treatment of lymphocytes, known to remove L-selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti-sLex- and anti-L-selectin-antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L-selectin-IgG fusion protein, indicating that ligands for L-selectin were induced during rejection.

Animals↗

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.

Amino Acid Sequence↗

Structural study of the sugar chains of human leukocyte common antigen CD45.

The leukocyte cell surface glycoprotein CD45 is a protein tyrosine phosphatase and is involved in signal transduction mediated by the T cell antigen receptor. The asparagine-linked sugar chains were released as oligosaccharides from purified CD45 by hydrazinolysis. Approximately 6 mol of sugar chains was released from 1 mol of CD45. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. Binding of the sialylated oligosaccharides to an SNA-agarose column as well as methylation analysis revealed that the oligosaccharides have only alpha-2,6-linked sialic acid residues. 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 endoglycosidase digestion and by methylation analysis revealed that CD45 contains mainly bi-, tri-, and tetraantennary complex-type sugar chains. About 46% of the tetraantennary complex-type sugar chains had the poly(N-acetyllactosamine) groups and 18% of the 2,4-branched triantennary complex-type sugar chains had the fucosyl N-acetyllactosamine group. A portion of the bi- and 2,4-branched triantennary complex-type sugar chains were bisected. In addition to these sugar chains, a small amount of high mannose-type and hybrid-type sugar chains were detected.

Asparagine↗