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T A Springer

Publications and source records attributed to T A Springer.

At least 91 records · Page 5Linked to original sources
PubMed1997

Characterization of lymphocyte function-associated antigen 1 (LFA-1)-deficient T cell lines: the alphaL and beta2 subunits are interdependent for cell surface expression.

The leukocyte, or beta2, integrins are heterodimeric cell surface molecules that share a common beta subunit, and have unique alpha subunits. LFA-1 is the predominant beta2 integrin on lymphocytes, and plays an important role in many lymphocyte functions; however, most studies of the cytoplasmic regions of LFA-1 have been performed in transfected epithelial cells, such as COS, in part because no lymphoid cell lines deficient in the LFA-1 alpha subunit have been described. To address structure-function studies of beta2 integrins in relevant cell types, two T lymphoblastoid cell clones that completely lack cell surface LFA-1, J-(beta2).7 and SK-(beta2).7, derived from Jurkat and SKW3, respectively, were prepared by chemical mutagenesis and selection. Biosynthetic labeling and immunoprecipitation showed that the J-(beta2).7 clone did not translate any LFA-1 alpha subunit protein, while the SK-(beta2).7 cells did not synthesize any beta2 subunit protein. Northern blot analysis of poly(A+) RNA from these cells revealed an absence of the corresponding mRNA in each case. By transfection analysis, only the alpha subunit reconstituted LFA-1 expression in the J-(beta2).7 cells, while only the beta subunit restored cell surface LFA-1 expression in the SK-(beta2).7 cells. Functional studies with the parental cell lines, the J-(beta2).7 and SK-(beta2).7 cells, and the transfectants showed that all binding of Jurkat and SKW3 cells to purified ICAM-1 is mediated by LFA-1, and the reconstituted LFA-1 expressed by the J-(beta2).7 and SK-(beta2).7 transfected cells is regulated normally.

CD18 Antigens↗
PubMed1997

The faster kinetics of L-selectin than of E-selectin and P-selectin rolling at comparable binding strength.

Selectins are a family of lectins that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. To test the hypothesis that the kinetics and the strength of rolling interactions can be independently varied for different selectin:ligand pairs, we have directly compared all three selectins with regard to distinct measures of selectin-mediated interactions in shear flow: tethering, rolling velocity, and strength of rolling adhesions. At comparable site densities of E-selectin, P-selectin, and the L-selectin counter-receptor CD34, neutrophils tethered with similar efficiency and developed rolling adhesions of similar strength as measured by resistance to detachment. Under the same conditions, neutrophils rolled 7.5- to 10.5-fold faster on CD34 than on E-selectin and P-selectin. These findings suggest that the kinetics of bond dissociation and bond formation are faster for L-selectin than for E- and P-selectin. We also compared the behavior of neutrophils and lymphocytes on the same selectin. Both cell types showed comparable strength of binding to CD34; however, neutrophils rolled with faster velocities than lymphocytes.

Antigens, CD34↗
PubMed1997

Defining extracellular integrin alpha-chain sites that affect cell adhesion and adhesion strengthening without altering soluble ligand binding.

It was previously shown that mutations of integrin alpha4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha4beta1-dependent cell adhesion. Some reports have suggested that alpha-chain "EF-hand" sites may interact directly with ligands. However, we show here that mutations of three different alpha4 "EF-hand" sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha4beta1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha4 "EF-hand-like" sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin.

Pre-B-cell growth-stimulating factor/ stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability,. B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/ SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Adult↗
PubMed1996

A differential role for cell shape in neutrophil tethering and rolling on endothelial selectins under flow.

We investigated the role of neutrophil microvilli in interactions with E-selectin and P-selectin in hydrodynamic shear flow by disruption with cytochalasin B, hypotonic swelling, and chilling. Cytochalasin B only marginally reduced microvilli numbers (from 30 +/- 6 to 16 +/- 6 per cell perimeter, p < 0.005) as shown by electron microscopy, completely disrupted tethering in shear flow to E-selectin and P-selectin, increased the strength of rolling adhesions on E-selectin and P-selectin, and increased cell deformability in shear flow with a likely increase in the area of cell:substrate contact. Hypoosmotic swelling markedly reduced microvilli number (to 6 +/- 5 per perimeter, p < 0.005), almost completely inhibited tethering on E- and P-selectin, and increased the strength of rolling adhesions on P-selectin but not on E-selectin. Chilling almost completely abolished microvilli (to 3 +/- 3 per perimeter, p < 0.005), but pseudopod-like structures were present, and had little effect on tethering in flow. Immunogold labeling of L-selectin, which is normally clustered on tips of microvilli, showed that in the absence of microvilli it remained in small clusters. Our studies show that alterations in cell morphology and viscoelasticity can have opposing effects on tethering and rolling, showing that they are independently regulatable. Furthermore, our results suggest that the association of molecules that mediate rolling with microvilli tips may be important not just to enhance presentation, but for other functions such as to promote resistance to extraction from the membrane or cooperative interactions among clustered receptors.

Cell Adhesion↗
PubMed1996

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

CD18 Antigens↗
PubMed1996

Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen 1 (LFA-1).

Intercellular adhesion molecule 3 (ICAM-3; CD50) is the predominant counter-receptor on resting T cells and monocytes for the leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), and may play an important role in the initial stages of the T cell-dependent immune response. Deletion of individual immunoglobulin superfamily (IgSF) domains of ICAM-3 and ICAM-3 IgSF domain chimeras with CD21 showed there is a single LFA-1 binding site in ICAM-3 and that IgSF domain 1 is necessary and sufficient for LFA-1 binding. Epitope mapping and functional studies performed with 17 anti-ICAM-3 monoclonal antibodies demonstrated that only some monoclonal antibodies, with epitopes wholly within domain 1 of ICAM-3, were able to block binding of ICAM-3 bearing cells to purified LFA-1, in agreement with the data obtained from the domain deletion mutants and CD21 chimeras. Analysis of a panel of 45 point mutants of domain 1 of ICAM-3 identified five residues that may contact LFA-1 as part of the binding site, Asn23, Ser25, Glu37, Phe54, and Gln75. These five residues are predicted by molecular modeling, based on the structure of vascular cell adhesion molecule 1 (VCAM-1), to cluster in two distinct locations on domain 1 of ICAM-3 on the BED face (Asn23 and Ser25) and on the C strand or CD loop (E37), the E strand (F54), and the FG loop (Q75). The residues, Asn23 and Ser25, comprise a consensus N-linked glycosylation site.

Amino Acid Sequence↗
PubMed1996

A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

Animals↗
PubMed1996

The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.

Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. There are two subfamilies, the CXC and the CC chemokines. We recently found that the CXC-chemokine stromal cell-derived factor-1 (SDF-1) is a highly efficacious lymphocyte chemoattractant. Chemokines act on responsive leukocyte subsets through G-protein-coupled seven-transmembrane receptors, which are also used by distinct strains of HIV-1 as cofactors for viral entry. Laboratory-adapted and some T-cell-line-tropic (T-tropic) primary viruses use the orphan chemokine receptor LESTR/fusin (also known as fusin), whereas macrophage-tropic primary HIV-1 isolates use CCR-5 and CCR-3 (refs 7-11), which are receptors for known CC chemokines. Testing of potential receptors demonstrated that SDF-1 signalled through, and hence 'adopted', the orphan receptor LESTR, which we therefore designate CXC-chemokine receptor-4 (CXCR-4). SDF-1 induced an increase in intracellular free Ca2+ and chemotaxis in CXCR-4-transfected cells. Because SDF-1 is a biological ligand for the HIV-1 entry cofactor LESTR, we tested whether it inhibited HIV-1. SDF-1 inhibited infection by T-tropic HIV-1 of HeLa-CD4 cells, CXCR-4 transfectants, and peripheral blood mononuclear cells (PBMCs), but did not affect CCR-5-mediated infection by macrophage-tropic (M-tropic) and dual-tropic primary HIV-1.

Animals↗
PubMed1996

Platelet-mediated lymphocyte delivery to high endothelial venules.

Circulating lymphocytes gain access to lymph nodes owing to their ability to initiate rolling along specialized high endothelial venules (HEVs). One mechanism of rolling involves L-selectin binding to peripheral node addressin (PNAd) on HEVs. Activated platelets are shown to bind to circulating lymphocytes and to mediate rolling in HEVs, in vivo, through another molecule, P-selectin, which also interacts with PNAd. In vitro, activated platelets enhanced tethering of lymphocytes to PNAd and sustained lymphocyte rolling, even in the absence of functional L-selectin. Thus, a platelet pathway operating through P-selectin provides a second mechanism for lymphocyte delivery to HEVs.

Animals↗
PubMed1996

Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.

Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.

Blood Platelets↗
PubMed1996

Characterization of murine intercellular adhesion molecule-2.

Rat mAbs were raised against murine intercellular adhesion molecule-2 (ICAM-2). Immune precipitation and purification reveal that the murine ICAM-2 glycoprotein is 55 kDa and is similar in size to human ICAM-2. ICAM-2 is expressed on a variety of leukocyte cell lines, including T and B lymphoma, mastocytoma, and macrophage lines. ICAM-2 is well expressed on endothelioma cell lines, and in contrast to ICAM-1, expression is not increased by inflammatory cytokines. One of the mAb to ICAM-2 partially or completely inhibits binding of cells expressing LFA-1 to purified ICAM-2, and binding of cells expressing ICAM-2 to purified LFA-1. The findings in the mouse are congruent with those in the human, suggesting functional conservation of ICAM-2 across species.

Animals↗
PubMed1996

A schiff base with mildly oxidized carbohydrate ligands stabilizes L-selectin and not P-selectin or E-selectin rolling adhesions in shear flow.

Selectins are a family of lectins, that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. Mild periodate oxidation of the L-selectin ligand CD34, or L-selectin ligands on leukocytes, enhanced resistance to detachment in shear and decreased rolling velocity equivalent to an 8-fold increase in ligand density, yet had little effect on the rate of tethering. Enhanced interactions were also seen with mildly oxidized sialyl Lewisa and sialyl Lewisx glycolipids. Enhancement was completely reversed by borohydride reduction, yielding a strength of interaction equivalent to that with the native ligands. No effect on the strength of P-selectin and E-selectin interactions was seen after mild oxidation of their ligands. Completeness of modification of sialic acid by mild periodate was verified with monoclonal antibody to sialyl Lewisx-related structures and resistance to neuraminidase. The addition of cyanoborohydride to leukocytes rolling through L-selectin on mildly oxidized but not native CD34 caused arrest of rolling cells and formation of EDTA-resistant bonds to the substrate, suggesting that a Schiff base was reduced. Cyanoborohydride reduction of mildly oxidized cells rolling on P-selectin and E-selectin also caused arrest and formation of EDTA-resistant bonds but with slower kinetics. These data suggest that interactions with a sialic acid aldehyde group on mildly oxidized ligands that include interconversion to a Schiff base can occur with three selectins yet only stabilize binding through the selectin with the fastest koff, L-selectin.

Antibodies, Monoclonal↗
PubMed1996

Interactions of human alpha/beta and gamma/delta T lymphocyte subsets in shear flow with E-selectin and P-selectin.

We have compared the ability of human alpha/beta and gamma/delta T lymphocytes to adhere to selectin-bearing substrates, an interaction thought to be essential for homing and localization at sites of inflammation. Both T cell populations form rolling adhesions on E- and P-selectin substrates under physiologic flow conditions. Although equivalent to alpha/beta T cells in binding to E-selectin, gamma/delta T cells demonstrated greater ability to adhere to P-selectin that was purified or expressed on the surface of activated, adherent platelets. Under static conditions, 80% of gamma/delta T cells and 53% of alpha/beta T cells formed shear-resistant adhesions to P-selectin, whereas only 30% of gamma/delta and alpha/beta T cells adhered to E-selectin. The enhance ability of gamma/delta T cells to adhere to P-selectin cannot be attributed to differences in expression of the P-selectin glycoprotein ligand (PSGL-1), as all alpha/beta T cells versus approximately 75% of gamma/delta T cells expressed PSGL-1. Both cell populations expressed a similar percentage of the carbohydrate antigens sialyl LewisX and cutaneous lymphocyte-associated antigen. Depletion of lymphocyte populations or T cell clones bearing these oligosaccharides with the monoclonal antibody CSLEX-1 and HECA-452, respectively, resulted in a substantial reduction in adhesion to E-selectin and slight reduction in adhesion to P-selectin under flow conditions. Treatment of cells with an endopeptidase that selectively degrades O-sialomucins such as PSGL-1, abolished P-selectin but not E-selectin adhesion. Removal of terminal sialic acids with neuraminidase or protease treatment of cells abrogated cell adhesion to both selectin substrates. These results provide direct evidence for the presence of distinct E- and P-selectin ligands on T lymphocytes and suggest that gamma/delta T cells may be preferentially recruited to inflammatory sites during the early stages of an immune response when P-selectin is upregulated.

Antigens, CD↗
PubMed1996

Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules.

High endothelial venules (HEV) are specialized plump postcapillary venules in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. We have recently identified a novel human transcript, expressed to high levels in HEV, that encodes a secreted, acidic protein closely related to the anti-adhesive extracellular matrix protein known as BM-40, osteonectin, and SPARC (secreted protein acidic and rich in cysteine). Here, we show that this protein, designated hevin, is associated with basal, lateral, and apical surfaces of HEV cells, and unlike MECA-79 antigen, is not expressed on the underlying basement membrane. In contrast to fibronectin or other adhesive extracellular matrix proteins, purified hevin does not support endothelial cell adhesion in vitro. Moreover, addition of soluble exogenous hevin inhibits attachment and spreading of endothelial cells on fibronectin substrates. Hevin-treated cells do not form focal adhesions and exhibit a rounded morphology. Together, these results suggest that hevin is an abundant extracellular protein that modulates high endothelial cell adhesion to the basement membrane.

Amino Acid Sequence↗
PubMed1996

Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury.

Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.

Animals↗