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T A Springer

Publications and source records attributed to T A Springer.

At least 19 recordsLinked to original sources
PubMed2000

The top of the inserted-like domain of the integrin lymphocyte function-associated antigen-1 beta subunit contacts the alpha subunit beta -propeller domain near beta-sheet 3.

We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.

Amino Acid Sequence↗
PubMed2000

Folding and function of I domain-deleted Mac-1 and lymphocyte function-associated antigen-1.

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.

Animals↗
PubMed2000

Structural and functional studies with antibodies to the integrin beta 2 subunit. A model for the I-like domain.

To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.

Amino Acid Sequence↗
PubMed2000

Computational design of an integrin I domain stabilized in the open high affinity conformation.

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.

Amino Acid Substitution↗
PubMed1999

Lymphocyte function-associated antigen-1 binding residues in intercellular adhesion molecule-2 (ICAM-2) and the integrin binding surface in the ICAM subfamily.

The crystal structure of intercellular adhesion molecule-2 (ICAM-2) revealed significant differences in the presentation of the critical acidic residue important for integrin binding between I and non-I-domain integrin ligands. Based on this crystal structure, we mutagenized ICAM-2 to localize the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1). The integrin binding site runs diagonally across the GFC beta-sheet and includes residues on the CD edge of the beta-sandwich. The site is oblong and runs along a flat ridge on the upper half of domain 1, which is proposed to dock to a groove in the I domain of LFA-1, with the critical Glu-37 residue ligating the Mg2+ in the I domain. Previous mutagenesis of ICAM-1 and ICAM-3, interpreted in light of the recently determined ICAM-1 and ICAM-2 structures, suggests similar binding sites. By contrast, major differences are seen with vascular cell adhesion molecule-1 (VCAM-1), which binds alpha4 integrins that lack an I domain. The binding site on VCAM-1 includes the lower portion of domain 1 and the upper part of domain 2, whereas the LFA-1 binding site on ICAM is confined to the upper part of domain 1.

Amino Acid Sequence↗
PubMed1999

Conformational changes in tertiary structure near the ligand binding site of an integrin I domain.

For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937-938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931-942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C. , Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923-935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (alphaMbeta2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.

Amino Acid Sequence↗
PubMed1999

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear.

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.

Antibodies, Monoclonal↗
PubMed1999

Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration.

We evaluated the relative contribution of ICAM-1 and ICAM-2, known ligands on endothelium for LFA-1 and Mac-1, in spontaneous neutrophil (PMN) transendothelial migration (TEM) across IL-1-activated HUVEC monolayers or TEM induced by C5a or IL-8 across unstimulated HUVEC grown on polycarbonate filters. Adhesion blocking mAb to ICAM-1 [R6.5 F(ab)2] or ICAM-2 [CBR IC2/2 F(ab)2] tended to inhibit TEM under each condition but, in general, inhibition was significant only with both ICAM-1 and ICAM-2 blockade. mAb to LFA-1 partially inhibited migration to C5a or IL-8 across unstimulated HUVEC and inhibition was not altered by additional treatment of HUVEC with mAbs to ICAM-1 and -2. In contrast, with IL-1 HUVEC, mAb to ICAM-1 significantly inhibited this LFA-1-independent TEM. mAb to Mac-1 alone partially inhibited TEM and, when combined with mAb to LFA-1, migration was almost completely blocked with all TEM conditions tested. The contribution of alternate ligands for Mac-1 in mediating Mac-1-dependent but ICAM-1/-2-independent C5a-induced TEM was examined using anti-LFA-1-treated PMN and anti-ICAM-treated resting HUVEC. Addition of RGD peptides, fibronectin, fibrinogen, heparins, collagens alone or in combination, even to heparinase-treated HUVEC, did not inhibit this Mac-1-mediated PMN TEM. The results indicate that: (1) LFA-1 mediates PMN TEM primarily by interaction with ICAM-1 and ICAM-2; (2) ICAM-2 may function in concert with ICAM-1 in this role, especially on unstimulated endothelium, and (3) Mac-1 on PMN also plays a major role in TEM and can utilize yet to be identified ligands distinct from ICAM-1 or -2, especially on unstimulated endothelium.

Antibodies, Monoclonal↗
PubMed1999

Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1).

We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor-1 (SDF-1). High percentages (30-40% of input) of purified bone marrow progenitors including non-B lineage progenitors, pro-B cells, and pre-B cells migrated to SDF-1alpha, demonstrating that SDF-1 is an efficacious chemoattractant of these cells. Pro-B cells responded optimally to a lower concentration of SDF-1 than other subsets, demonstrating that SDF-1 is a more potent chemoattractant of this subset. A lower percentage (10-15% of input) of mature B lymphocytes migrated to SDF-1alpha than pro-B cells, demonstrating that responsiveness of B lineage cells to SDF-1 decreases during differentiation. Inhibition by anti-CXCR4 mAb demonstrated that migration of B lineage cells to SDF-1 was completely dependent on CXC chemokine receptor-4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro-B cells, despite differences in responsiveness to SDF-1. CXCR4 receptors expressed by unresponsive and SDF-1-responsive B cells bound SDF-1alpha with similar affinities (K(D) = 1.7-3.3 x 10(-9) M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF-1. We speculate that SDF-1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis.

B-Lymphocytes↗
PubMed1999

Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended hyperresponsiveness.

ICAM-2-deficient mice exhibit prolonged accumulation of eosinophils in lung interstitium concomitant with a delayed increase in eosinophil numbers in the airway lumen during the development of allergic lung inflammation. The ICAM-2-dependent increased and prolonged accumulation of eosinophils in lung interstitium results in prolonged, heightened airway hyperresponsiveness. These findings reveal an essential role for ICAM-2 in the development of the inflammatory and respiratory components of allergic lung disease. This phenotype is caused by the lack of ICAM-2 expression on non-hematopoietic cells. ICAM-2 deficiency on endothelial cells causes reduced eosinophil transmigration in vitro. ICAM-2 is not essential for lymphocyte homing or the development of leukocytes, with the exception of megakaryocyte progenitors, which are significantly reduced.

Administration, Inhalation↗
PubMed1999

The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment.

We report that the chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within fetal liver and bone marrow microenvironment. In CXCR4-deficient embryos, pro-B cells are present in blood but hardly detectable in liver; myeloid cells are elevated in blood and reduced in liver compared to wild-type embryos. Mice reconstituted with CXCR4-deficient fetal liver cells have reduced donor-derived mature B lymphocytes in blood and lymphoid organs. The numbers of pro-B and pre-B cells are reduced in bone marrow and abnormally high in blood. Granulocytic cells are reduced in bone marrow but elevated and less mature in the blood. B lineage and granulocytic precursors are released into the periphery in absence of CXCR4.

Animals↗
PubMed1999

Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains.

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.

Amino Acid Sequence↗
PubMed1998

Interaction of very late antigen-4 with VCAM-1 supports transendothelial chemotaxis of monocytes by facilitating lateral migration.

The transient regulation of very late antigen (VLA)-4 avidity by CC chemokines may promote chemotaxis of monocytes across VCAM-1-bearing barriers, whereas late and prolonged activation of VLA-5 may mediate subsequent localization in the extracellular matrix. We demonstrate that interactions of VLA-4 with VCAM-1, fibronectin, or a 40-kDa fragment but not a 120-kDa fragment of fibronectin supported the lateral random migration of isolated blood monocytes induced by CC chemokines, termed chemokinesis. This effect was optimal at intermediate substrate concentrations. Moreover, coimmobilization of VCAM-1 with ICAM-1 allowed better migration than ICAM-1 alone. Chemokinesis on VCAM-1 appeared to be associated with transient regulation of VLA-4 avidity by CC chemokines, given that locking VLA-4 in a high avidity state markedly inhibited migration and the locomotion rate was inversely correlated with the adhesive strength of VLA-4 to VCAM-1 following stimulation with monocyte chemoattractant protein-1. Induction of VCAM-1 expression by endothelial activation with IL-4 improved chemokinesis and lateral migration toward a monocyte chemoattractant protein-1 or a monocyte inflammatory protein-1alpha gradient on endothelium and increased transendothelial chemotaxis of monocytes by a VLA-4-dependent mechanism. In contrast, endothelial activation with IL-4 did not affect the time required for diapedesis of monocytes itself. Hence, VCAM-1 may facilitate transendothelial chemotaxis by supporting lateral migration of attached monocytes along endothelium.

Animals↗
PubMed1998

Effects of a monoclonal antibody to P-selectin on recovery of neonatal lamb hearts after cold cardioplegic ischemia.

BACKGROUND: The interaction between endothelium and leukocytes plays a crucial role in ischemia-reperfusion injury. P-selectin, which is expressed on activated endothelium, mediates the first step in leukocyte adherence to the endothelium. This study examined the effects of a monoclonal antibody (mAb) against P-selectin on the recovery of cardiac function and myocardial neutrophil infiltration after ischemia. METHODS AND RESULTS: Thirteen blood-perfused, isolated neonatal lamb hearts underwent 2 hours of hypothermic cardioplegic arrest and 2 hours of reperfusion. Immediately before reperfusion, mAb to P-selectin was administered to the perfusate (15 micrograms/mL) in 6 hearts (group P-sel). In control (n = 7), the same volume of saline was added. Isovolumic left ventricular function and coronary blood flow were measured. At 2 hours after reperfusion, myocardial myeloperoxidase activity, an index of neutrophil accumulation, was assayed. At 30 minutes of reperfusion, hearts treated with mAb to P-selectin achieved significantly greater recovery of maximum developed pressure (70 +/- 4% in control versus 77 +/- 2% in group P-sel, P < 0.01), maximum positive first derivative of pressure (dP/dt) (64 +/- 7% in control versus 73 +/- 5% in group P-sel, P < 0.05), and maximum negative dP/dt (61 +/- 6% in control versus 70 +/- 6% in group P-sel, P < 0.05) compared with control. Percent baseline of coronary blood flow was also significantly increased in group P-sel (135 +/- 40% in control versus 205 +/- 43% in group P-sel, P < 0.05). Myocardial myeloperoxidase activity was significantly lower (P < 0.05) in group P-sel (4.7 +/- 3.2) versus control (16.0 +/- 10.1). (Units are change in absorbance/min/g tissue.) CONCLUSIONS: The functional blockade of P-selectin resulted in better recovery of cardiac function and attenuated neutrophil accumulation during early reperfusion. Strategies to block P-selectin mediated neutrophil adherence may have clinical application in improving myocardial function at early reperfusion.

Animals↗
PubMed1998

An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components.

An abundant, widely dispersed, extracellular sequence repeat that contains a consensus YWTD motif is shown here to occur in groups of six contiguous repeats. Thirteen lines of evidence, including experimental and computational data, predict with p<3x10(-9) that the repeats do not form tandem domains, but rather each group of six repeats folds into a compact beta-propeller structure. The six beta-sheets are arranged about a 6-fold pseudosymmetry axis, and each repeat contributes loops to the faces surrounding the pseudosymmetry axis. Seven different endocytic receptors that contain from one to eight YWTD beta-propeller domains act as lipoprotein, vitellogenin, and scavenger receptors. In the low density lipoprotein receptor (LDLR), the many mutations in familial hypercholesterolaemia that map to the YWTD domain can now be interpreted. In the extracellular matrix component nidogen, the YWTD domain functions to bind laminin. Three YWTD domains and interspersed fibronectin type III (FN3) domains constitute almost the entire extracellular domain of the sevenless and c-ros receptor tyrosine kinases. YWTD domains often are bounded by epidermal growth factor (EGF) modules, including in the EGF precursor itself. YWTD beta-propellers have a circular folding pattern that brings neighboring modules into close proximity, and may have important consequences for the architecture of multi-domain proteins.

Amino Acid Sequence↗
PubMed1998

The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes.

The kinetics of rolling and transient adhesions through selectins may depend on the kinetics and mechanical properties of the selectin:ligand bond, as well as on cellular properties including receptor-anchoring to the cell membrane and cytoskeleton. Kinetics are known to depend on the selectin and may also be ligand dependent. Here, we study the kinetics of transient and rolling interactions of leukocytes with L-selectin immobilized on a substrate. Remarkably, all properties examined are similar to those seen when the sidedness is opposite, i.e., when the L-selectin ligand is on the substrate and when the ligand is isolated from HEV rather than present on leukocytes. The similar properties include rolling velocity, a threshold shear stress above 0.4 dyn/cm2 required to support rolling, a k degreesoff of 7.0 to 6.8 s-1 for the L-selectin tether bond, and a mechanical bond length of 0.24 to 0.20 A. Our results argue against a model in which L-selectin shedding mediates rolling. Furthermore, the fast and force-resistant kinetic properties suggest that L-selectin is specialized dynamically for tethering leukocytes to vessel walls and adherent leukocytes.

Antibodies, Monoclonal↗
PubMed1998

Protection from lymphoma cell metastasis in ICAM-1 mutant mice: a posthoming event.

It has been hypothesized that the intercellular adhesion receptors used by normal cells could also be operative in the spreading of circulating malignant cells to target organs. In the present work, we show that genetic ablation of the ICAM-1 gene confers resistance to T cell lymphoma metastasis. Following i.v. inoculation of LFA-1-expressing malignant T lymphoma cells, we found that ICAM-1-deficient mice were almost completely resistant to the development of lymphoid malignancy compared with wild-type control mice that developed lymphoid tumors in the kidneys, spleen, and liver. Histologic examinations confirmed that ICAM-1-deficient mice, in contrast to wild-type mice, had no evidence of lymphoid infiltration in these organs. The effect of ICAM-1 on T cell lymphoma metastasis was observed in two distinct strains of ICAM-1-deficient animals. Nonetheless, lymphoma cells migrated with the same efficiency to target organs in both normal and ICAM-1-deficient mice, indicating not only that ICAM-1 expression by the host is essential in lymphoma metastasis, but also that this is so at stages subsequent to homing and extravasation into target organs. These results point to posthoming events as a focus of future investigation on the control of metastasis mediated by ICAM-1.

Animals↗