Biomedical subjects

T A Springer

Publications and source records attributed to T A Springer.

At least 163 records · Page 9Linked to original sources

PubMed1993

Neutrophils roll on E-selectin.

Using flow conditions that simulate those in post capillary venules, we have found that neutrophils attach and roll on a substrate bearing purified E-selectin. E-selectin resembles P-selectin (CD62) with regard to the dependence of attachment efficiency on wall shear stress and selectin density. In contrast, once attached, neutrophils form rolling adhesions on E-selectin that are much stronger than those on P-selectin. Rolling velocities on E-selectin are slower and have less variance than on P-selectin. With increasing shear stress, rolling velocities reach a plateau level that is dependent on E-selectin density, suggesting that the number of receptor-ligand bonds and the bond dissociation rate limit rolling velocity, and that the bonds are not broken by the applied force.

Adenosine Triphosphate↗

PubMed1993

[Successful prevention of rhinovirus infections with chimeric ICAM-1 immunoglobulin in vitro].

The intercellular adhesion molecule 1 (ICAM-1) is used as cellular receptor by 90% of human rhinoviruses (HRV). Recently, a recombinant, soluble ICAM-1 (sICAM-1) has been shown to be effective in prevention of the cytopathic effect of rhinovirus infection in vitro. Aim of this study was the production of molecules with multivalent binding sites. Different chimeric proteins were constructed by fusion of two or five extracellular domains of ICAM-1 with the constant regions of IgA1, IgM and IgG1 by polymerase chain reactions (PCR). IgA- and IgG-immunoadhesion molecules were expressed in eucaryotic cells as dimers, IgM-immunoadhesion molecules as decamers. Immunoadhesion molecules were compared to sICAM-1 for their ability to neutralize HRV: The chimeric protein of five N-terminal domains of ICAM-1 and the constant regions of IgA1 was 150 times more potent than sICAM-1 in neutralizing HRV. The first and the second N-terminal of ICAM-1 fused to IgA1 or IgM were five times more active, however, fused to IgG1 four times less active than sICAM-1. Sedimentation analyses of [H3]-leucin labled HRV after preincubation with the different immunoadhesion molecules showed a dose-dependent induction of a conformational change of the viral capsid proteins. The order of efficiency of the chimeric proteins was consistent to the neutralizing experiments. Our results showed that HRV neutralizing can be dramatically increased by multimerization of ICAM-1. The chimeric molecule between IgA1 and ICAM-1 seems to be a potential candidate for clinical testing to prevent and treat HRV-infections.

Cell Adhesion Molecules↗

PubMed1993

Cloning and expression of intercellular adhesion molecule 3 reveals strong homology to other immunoglobulin family counter-receptors for lymphocyte function-associated antigen 1.

Based on protein sequence, we have isolated a cDNA for intercellular adhesion molecule 3 (ICAM-3), the most recently defined counter-receptor for lymphocyte function-associated antigen 1 (LFA-1). Expression of the cDNA yields a product that reacts with monoclonal antibody to ICAM-3 and functions as a ligand for LFA-1. The deduced 518-amino acid sequence of the predicted mature protein defines a highly glycosylated type I integral membrane protein with five immunoglobulin (Ig)-like domains. The five Ig-like domains of ICAM-3 are highly homologous with those of human ICAM-1 (52% identity) and human ICAM-2 (37% identity).

Amino Acid Sequence↗

PubMed1993

Characterization of two new CD18 alleles causing severe leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by heterogeneous mutations within the gene encoding the common beta subunit (CD18) of the three leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18). Based on the level of expression of CD18 on patient leukocytes, two phenotypes of LAD have been defined (severe and moderate) which correlate with the severity of the disease. We have investigated the molecular basis of the disease in two unrelated severe patients (HS and ZJO). Both patients share a complete absence of CD18 protein precursor and cell surface expression, but they differ in the level of CD18 mRNA, which is normal in HS and undetectable by Northern blot in ZJO. Determination of the primary structure of the patient HS CD18 mRNA revealed a 10-base pair deletion between nucleotides 190-200 (CD18 exon 3), which eliminates residues 41-43 and causes a frameshift into a premature termination codon 17 base pairs downstream from the deleted region. The 10-base pair frameshift deletion maps to a region of the CD18 gene where aberrant mRNA processing has been detected in HS and two other unrelated LAD patients. In the ZJO patient, amplification of lymphoblast CD18 mRNA demonstrated the presence of a non-sense mutation in the third nucleotide of the triplet encoding Cys534 (TGC-->TGA), within exon 12. Both genetic abnormalities were also detected at the genomic level, and affect the restriction pattern of their corresponding genes, thus enabling the detection of the mutant alleles among healthy heterozygous alleles in family studies. The identification of two new LAD CD18 alleles, either carrying a non-sense mutation (ZJO) or a partial gene deletion (HS), further illustrates the heterogeneity of the genetic alterations in LAD.

Alleles↗

PubMed1993

Location of the domains of ICAM-1 by immunolabeling and single-molecule electron microscopy.

Intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin gene superfamily, is a cell surface glycoprotein with an extracellular domain comprising five immunoglobulin-like domains. Soluble ICAM-1, a recombinant protein truncated at the transmembrane domain, has a rod-like shape, 19 nm long overall, with a characteristic bend 7.6 nm from one end of the molecule. Because the link between domain D2 and domain D3 is proline rich, it has been proposed that the short arm contains domains D1 and D2 and the long arm contains domains D3-D5. We used single-molecule electron microscopy of soluble ICAM-1 decorated with monoclonal antibodies specific for domains D1 and D4 to show that the bend instead lies between domains D3 and D4. Therefore, the short arm lies closer to the plasma membrane, whereas the long arm, containing all the known ligand binding sites on ICAM-1, is positioned toward the target cell surface.

Antigen-Antibody Complex↗

PubMed1993

A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen.

We report that a subpopulation (10%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac-1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.

Animals↗

PubMed1993

The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands.

Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-terminal and perhaps divalent cation binding regions but not the COOH-terminal segment may contribute. The recognition sites in the I domain appear overlapping but not identical as individual Mac-1-ligand interactions are distinguished by the discrete patterns of inhibitory mAbs. Additionally, we find that the alpha subunit NH2-terminal region and divalent cation binding region, despite being separated by over 200 amino acids of the I domain, appear structurally apposed because three mAbs require the presence of both of these regions for antigenic reactivity, and chimeras that contain the NH2 terminus of p150,95 require the divalent cation binding region of p150,95 to associate firmly with the beta subunit.

Antibodies, Monoclonal↗

PubMed1993

Functional studies of truncated soluble intercellular adhesion molecule 1 expressed in Escherichia coli.

We have expressed in Escherichia coli the two N-terminal immunoglobulin (Ig)-like domains of the intercellular adhesion molecule 1 (ICAM-1). The first 188 residues of ICAM-1 were expressed with an N-terminal methionine (MP188) or as a maltose-binding fusion protein which was cleaved with factor Xa (XP188). After refolding, both MP188 and XP188 were active in binding to the leukocyte integrin lymphocyte function-associated antigen 1, which has previously been shown to bind to the N-terminal Ig domain of ICAM-1. The major group of rhinoviruses and malaria-infected erythrocytes bind to distinct sites within the first Ig-like domain of ICAM-1. Both MP188 and XP188 bound to malaria-infected erythrocytes; however, only XP188 inhibited human rhinovirus plaque formation. A product (MdQ1P188) with the initiation methionine fused to residue 2, i.e., with glutamine 1 deleted, inhibited plaque formation. MdQ1P188 was able to induce a conformational change of the virus capsid as shown by conversion of 149S particles to 85S particles, whereas MP188 had no effect. These results show that functionally active fragments of ICAM-1 can be produced in E. coli, that glycosylation is not required for ligand binding, and that the N-terminal residue of ICAM-1 is proximal to or part of the human rhinovirus-binding site.

Animals↗

PubMed1993

The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells.

Rhodococcus equi is a facultative intracellular bacterium of macrophages that causes disease in immunocompromised individuals, particularly those with AIDS. In this report, we demonstrate that R. equi binding to mammalian cells requires complement and is mediated primarily by the leukocyte complement receptor, Mac-1. Bacteria bind to macrophages poorly unless exogenous complement is added to the incubation medium. The addition of fresh nonimmune serum, which contains no detectable antibodies to R. equi, greatly enhances bacterial binding to macrophages, whereas heat inactivation of this serum or immunological depletion of C3 from the serum reduces binding to levels only slightly higher than those of binding under serum-free conditions. Human serum depleted of C2 or C4 is fully opsonic, indicating that complement activation and fixation occur by the alternative pathway. The serum-dependent binding of rhodococci to macrophages is mediated primarily by the macrophage complement receptor type 3, Mac-1 (CD11b/CD18). Bacteria do not bind to fibroblastoid or epithelial cells that lack this receptor. Most of the bacterial binding to macrophages is inhibited by a monoclonal antibody to Mac-1 but is unaffected by a monoclonal antibody to complement receptor type 1. Furthermore, opsonized, but not unopsonized, bacteria bind to purified Mac-1 immobilized on plastic. In addition, in the presence of opsonic complement, rhodococci bind efficiently to fibroblastoid cells transfected with cloned Mac-1 but relatively poorly to cells transfected with the complement receptor type 1. Hence, R. equi fixes complement by activating the alternative complement pathway, and this fixation is a requirement for bacterial adhesion and invasion. Furthermore, complement fixation defines rhodococcal host cell tropism, since R. equi binds specifically and exclusively to cells expressing Mac-1.

Animals↗

PubMed1993

Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules.

The intercellular adhesion molecule 1 (ICAM-1) is used as a cellular receptor by 90% of human rhinoviruses (HRVs). Chimeric immunoadhesin molecules containing extracellular domains of ICAM-1 and constant regions of immunoglobulins (Igs) were designed in order to determine the effect of increased valency, Ig isotype, and number of ICAM-1 domains on neutralization and disruption of rhinovirus structure. These immunoadhesins include ICAM-1 amino-terminal domains 1 and 2 fused to the hinge and constant domains of the heavy chains of IgA1, IgM, and IgG1 (IC1-2D/IgA, -/IgM, and -/IgG). In addition, all five extracellular domains were fused to IgA1 (IC1-5D/IgA). Immunoadhesins were compared with soluble forms of ICAM-1 containing five and two domains (sICAM-1 and ICI-2D, respectively) in assays of HRV binding, infectivity, and conformation. In prevention of HRV plaque formation, IC1-5D/IgA was 200 times and IC1-2D/IgM and IC1-2D/IgA were 25 and 10 times more effective, respectively, than ICAM-1. The same chimeras were highly effective in inhibiting binding of rhinovirus to cells and disrupting the conformation of the virus capsid, as demonstrated by generation of approximately 65S particles. The results show that the number of ICAM-1 domains and a flexible Ig hinge are important factors contributing to the efficacy of neutralization. The higher efficiency of chimeras that bound bivalently in disrupting HRV was attributed to higher binding avidity. The IC1-5D/IgA immunoadhesin was effective at nanomolar concentrations, making it feasible therapy for rhinovirus infection.

B-Lymphocytes↗

PubMed1993

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

Cell Compartmentation↗

PubMed1992

Soluble intercellular adhesion molecule 1-immunoglobulin G1 immunoadhesin mediates phagocytosis of malaria-infected erythrocytes.

We describe an immunoadhesin molecule containing intercellular adhesion molecule 1 (ICAM-1) molecularly fused to hinge and CH2 and CH3 domains of the human immunoglobulin G1 H chain that binds Plasmodium falciparum-infected erythrocytes. This receptor-based immunoadhesin is an effective and specific inhibitor of P. falciparum-infected erythrocyte adhesion to ICAM-1-bearing surfaces, but does not inhibit leukocyte function antigen 1 (LFA-1) interaction with ICAM-1. Furthermore, the immunoadhesin promotes phagocytosis and destruction of parasitized erythrocytes by human monocytes. Each of these modes of action has potential for the therapy of malaria.

Animals↗

PubMed1992

Isolation, characterization, and expression of mouse ICAM-2 complementary and genomic DNA.

Intercellular adhesion molecule-2 (ICAM-2), a cell surface glycoprotein, is a second counter-receptor for lymphocyte function-associated Ag-1 (LFA-1). We report here the isolation and characterization of the cDNA and the gene that encode murine ICAM-2 (Accession numbers X65493 and X65490, respectively). The deduced sequence of the cDNA has 60% amino acid identity with its human counterpart and has the same expression pattern in cells and tissues. Furthermore, COS cells transfected with mouse ICAM-2 complementary and genomic DNA bind to purified human LFA-1, demonstrating the conservation of the function of ICAM-2 as a ligand for LFA-1 and conservation across species of sequences that are critical for binding to human LFA-1. COS cells transfected with the ICAM-2 cDNA do not react with mAb PA3, previously suggested to define ICAM-2 in the mouse. The mouse ICAM-2 gene was isolated and its structural organization determined. The gene is present in a single copy in the mouse genome and contains four exons spanning about 5.0 kb of DNA. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members. The gene for ICAM-2, which is constitutively expressed in endothelial cells, has several conserved sequence motifs in its promoter region, including a direct repeat, and lacks transcription factor-binding sites present in the ICAM-1 gene, which is inducible in endothelial cells.

Amino Acid Sequence↗

PubMed1992

Lymphocyte adhesion through very late antigen 4: evidence for a novel binding site in the alternatively spliced domain of vascular cell adhesion molecule 1 and an additional alpha 4 integrin counter-receptor on stimulated endothelium.

Recent studies demonstrate that alternative splicing of mRNA from a single gene can produce two forms of vascular cell adhesion molecule 1 (VCAM-1): a six-immunoglobulin (Ig) domain form (VCAM-6D) and a seven-Ig domain form (VCAM-7D). Using a COS cell transient expression assay, we investigated whether VCAM-6D and VCAM-7D differ functionally in adhesion to the integrin VLA-4 (CD49d/CD29) on lymphoid cells. Binding of lymphoid cell lines and peripheral blood lymphocytes was completely blocked by VLA-4 monoclonal antibody (mAb) and one VCAM-1 mAb (4B9) to both VCAM-6D and VCAM-7D, whereas one VCAM-1 mAb (E1/6) completely blocked binding to VCAM-6D but only partially inhibited binding to VCAM-7D. We conclude that there is one VLA-4 binding site in the six Ig domains shared between VCAM-6D and VCAM-7D, and that the alternatively spliced domain 4 present in VCAM-7D provides a second VLA-4 binding site that is blocked by 4B9 but not the E1/6 mAb. We compared the inhibitory effects of anti-VCAM-1 and anti-VLA-4 mAbs on lymphoid cell adhesion to cultured human umbilical vein endothelial cells (HUVEC). The anti-VCAM-1 mAb 4B9 blocked the binding of PBL and lymphoid tumor cells to stimulated HUVEC better than the anti-VCAM-1 mAb E1/6. Because VCAM-7D is the predominant form of VCAM-1 expressed by stimulated endothelial cells, this difference in VCAM-1 mAb inhibition is attributed to lymphoid cell binding to VCAM-7D on stimulated HUVEC. Although the anti-VLA-4 mAb and anti-VCAM-1 mAb 4B9 equally inhibited PBL binding to stimulated HUVEC, mAb 4B9 inhibited the binding of two lymphoid cell lines significantly less than anti-VLA-4 mAb. Combination of 4B9 mAb with function-blocking antiserum to human fibronectin, a second known ligand for VLA-4, also failed to inhibit as much as anti-VLA-4 mAb. These findings suggest that adhesion of lymphoid cell lines through VLA-4 or other alpha 4 integrins may involve inducible counter-receptor(s) on endothelium distinct from either VCAM-1 or fibronectin. Time course experiments indicate that the fraction of alpha 4 integrin-dependent binding that can be blocked by anti-VCAM-1 mAb E1/6 rises and peaks within 2 h of tumor necrosis factor (TNF) stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Internalization of a major group human rhinovirus does not require cytoplasmic or transmembrane domains of ICAM-1.

Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidyl-inositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.

Cell Adhesion Molecules↗

PubMed1992

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.

B-Lymphocytes↗

PubMed1992

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.

Amino Acid Sequence↗

PubMed1992

Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes.

Recent studies suggest that some T and B lymphocyte cell lines bind to the integrin lymphocyte function-associated molecule 1 (LFA-1) chiefly through a pathway independent of its two known counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. A monoclonal antibody (mAb) was raised that, in combination with blocking mAb to ICAM-1 and ICAM-2, can completely inhibit binding of these cell lines to purified LFA-1. This third ligand, designated ICAM-3 based on its functional relatedness to ICAM-1 and -2, is a highly glycosylated protein of 124,000 Mr. It is well expressed on all leukocytes and absent from endothelial cells. In assays of adhesion of resting lymphocytes to purified LFA-1, ICAM-3 is by far the most functionally important ICAM, implying an important role for ICAM-3 in the generation of immune responses.

Animals↗