Presentation of integrins on leukocyte microvilli: a role for the extracellular domain in determining membrane localization.

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha4beta7, alphaLbeta2, and alphaMbeta2 were heterologously expressed in K562 cells. alpha4beta7 initiated adhesion under flow and localized to microvilli, whereas beta2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha4beta7 cytoplasmic and/or transmembrane domains with the homologous domains of alphaLbeta2 or alphaMbeta2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha4beta7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

Expression and function of the MAdCAM-1 receptor, integrin alpha 4 beta 7, on human leukocytes.

Recirculation of mouse lymphocytes to the gut involves binding of the lymphocyte integrin alpha 4 beta 7 to the mucosal vascular addressin, MAdCAM-1. In humans, indirect evidence suggests that CD4+ T cells that express high levels of alpha 4 beta 7 migrate selectively to the gut. We now report that human adult blood CD8+ T cells and B cells, like CD4+ T cells, have heterogeneous expression of alpha 4 beta 7. In contrast, NK cells, eosinophils, and newborn blood T and B cells have relatively homogeneous expression of alpha 4 beta 7. CD4+ and CD8+ T cell expression of alpha 4 beta 7 was related to age, CD45RA expression, and integrin beta 1 (CD29) expression, suggesting that alpha 4 beta 7 expression changes after primary activation of CD4+ and CD8+ T cells in vivo. To directly determine whether human alpha 4 beta 7 mediates adhesion to MAdCAM-1, we performed in vitro adhesion assays with two alpha 4 beta 7+ human lymphoma cell lines. The results indicate that human alpha 4 beta 7 is a receptor for MAdCAM-1, whereas alpha 4 beta 1 is not. Adhesion of HUT 78 cells to MAdCAM-1 required Mn2+, whereas adhesion of RPMI 8866 cells did not, suggesting that alpha 4 beta 7 may have at least two distinct functional states. The ability of lymphocytes to bind to MAdCAM-1 and recirculate to mucosal organs is likely to be influenced both by the level of alpha 4 beta 7 expression and by the functional state of the alpha 4 beta 7 molecule.

Selective expression of integrin alpha 4 beta 7 on a subset of human CD4+ memory T cells with Hallmarks of gut-trophism.

Human memory CD4+ T lymphocytes are heterogenous in expression of integrins; one subset has the unexpected phenotype beta 1 low alpha 4 high. We demonstrate that this subset is unique among CD4+ cells in expression of high levels of alpha 4 beta 7, detected by a distinctive mAb Act-1. alpha 4 beta 7 is involved in binding to both fibronectin and vascular cell adhesion molecule-1; Act-1 blocks cell binding to the former and augments binding to the latter. Act-1 expression marks a subset of memory cells that, unlike the predominant circulating memory cell, has up-regulated beta 7 rather than beta 1. Their phenotype is distinct from that described for skin-homing T cells and is fully consistent with that described for gut-homing T cells. Differential adhesion capacity of this subset is verified by selective binding to FN and vascular cell adhesion molecule-1 in a beta 1-independent fashion. Thus, alpha 4 beta 7 detected on this subset of circulating normal T cells fits the expectations for a gut-homing receptor.

Crossbridge order and orientation in resting single glycerinated muscle fibres studied by linear dichroism of bound rhodamine labels.

Linear dichroism of iodoacetyl-rhodamine labels attached to the highly reactive thiol of the myosin heads was measured in order to infer the spatial orientation and the degree of order in myosin crossbridges in single glycerinated rabbit psoas fibres at rest. We have previously shown that in rigor the chromophoric labels are well ordered and that in the presence of MgADP and during isometric contraction a large fraction of probes is also ordered but at an attitude different from that of rigor. Here we show that in relaxed muscle the probe order is dependent on total ionic strength: at and above 0.180 M there is little evidence for any preferred probe orientation, implying a high degree of crossbridge disorder. Below 0.160 M there is progressively more order with decreasing ionic strength down to 0.100 M, below which no measurements could be taken at room temperature (because the fibres would not relax). The dichroism observed under these conditions resembles that of the rigor state in that the dichroism peaks at the same polarization of excitation light, implying that the average probe attitude relative to the fibre axis is larger than 54.7 degrees. Stretching the muscle beyond the point of overlap between actin- and myosin-containing filaments does not affect the ionic strength dependence of the amount of order present in relaxed muscle, suggesting that the observed order is due to ionic interactions of crossbridges with the thick filament surface.