Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin–integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the “focal zone.”

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through α-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.

A successful immune response depends on the migration of lymphocytes into lymph nodes or inflamed tissues where they make contact with antigen-presenting cells. We are interested in how one member of the integrin family, leukocyte function-associated antigen-1 (LFA-1), controls the function and, in particular, the migration of immune cells. We find that this integrin operates not only as an adhesion receptor for T lymphoblasts (T cells) but also induces their migration in vitro at approximately 15 microm/min. Migration requires active myosin light chain kinase at the leading edge and Rho kinase at the trailing edge of the cell. Two active conformations of LFA-1 are differently distributed on the T-cell membrane and regulate independent aspects of migration. High-affinity LFA-1 is located in a midcell 'focal zone' and influences the speed of migration, whereas intermediate affinity LFA-1 controls leading edge adhesions. Manipulating LFA-1 conformation in vivo can be performed, for example, by creating the active conformation in a transgenic mouse, and this model gives further insight into the role of LFA-1 in migration. In humans, the beneficial effect of functioning CD18 integrins in combating infections in vivo is illustrated by rare patients displaying two forms of leukocyte adhesion deficiency. In summary, we speculate that T cells have evolved a mode of rapid migration that is of paramount importance in achieving the high-speed immune surveillance upon which depends the body's protection against diverse invaders from pathogens to cancer cells.

Understanding how the integrins on leukocytes operate is important because these receptors control the activity of leukocytes in all phases of their lives. Thus integrins control leukocyte development and maturation in bone marrow, the circulation of naive cells in secondary lymphoid tissue, e.g. the lymph nodes, and leukocyte responses to inflammatory signals emanating from injured tissues. Using as an example LFA-1, which is expressed by all leukocytes, we outline how the activity of this integrin is modified to meet the challenges posed by these leukocyte activities. Briefly, we discuss three means by which LFA-1 is adapted to bind more efficiently to its chief ligand, ICAM-1. LFA-1 can undergo changes in conformation leading to increased affinity, can be clustered on the membrane and, finally, when activated can move into the lipid raft compartment of the membrane. The study of humans with the beta2 deficiency syndrome termed leukocyte adhesion deficiency (LAD)-1 and analysis of LFA-1 null mice has given further insight into integrin activation mechanisms and the in vivo roles of LFA-1 and other leukocyte integrins.

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca2+-dependent enzyme, calpain 2. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.

On T cells the leukocyte integrin leukocyte functionassociated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg 2؉ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 ␣ subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg 2؉ -induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg 2؉ -activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg 2؉ -activated LFA-1 to ICAM-1 is blocked by peptides covering the ␣4-␤3 loop, the ␤3-␣5 loop, and the ␣5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the ␤-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.Adhesion mediated by the interaction of the integrin LFA-1 1 (CD11a/CD18) with one of its ligands, ICAM-1, ICAM-2, or ICAM-3, is crucial to the inflammatory process (reviewed in Refs. 1-3). LFA-1 is not constitutively able to bind ligand, but requires activation, with the characteristics of the active LFA-1 depending on the method of stimulation. In vitro, triggering of T cells with phorbol ester or by increasing intracellular Ca 2ϩ concentration does not induce a detectable change in the affinity of the individual LFA-1 molecules. These stimuli, however, induce clustering of LFA-1, thereby increasing the overall strength of binding (4, 5), which is described as an increase in avidity. In contrast, activation from the outside of the cell with Mg 2ϩ and EGTA results in the formation of a higher affinity form of LFA-1, as assessed by an increased ability to bind soluble ICAM-1, and in expression of an activation reporter epitope recognized by mAb 24 (1, 6, 7). In this situation there is no evidence for LFA-1 clustering (5). These findings and those of others (8, 9) suggest that physiological stimuli regulate adhesion by two major mechanisms involving alterations either in the affinity of the individual integrin molecules or in the overall avidity of binding. Such different forms of integrin adhesion might dictate the nature of signals transmitted into the cell and have functional consequences. It is not completely understood how these two forms of adhesion relate to the in vivo activation o...

This paper explores the ways in which ward rounds can be conducted to maximize educational opportunities, as part of a project to improve the effectiveness of on-the-job training (OJT) for hospital doctors. Ninety ward rounds taken by 24 trainers in the Anglia region were observed. Each observation produced a note of the ward round's structure and routines and of the contributions made to it by trainers and trainees. Teaching was a feature of all ward rounds and different types of rounds were valued for different reasons. A range of ward round structures was observed and, within each, a range of routines for conducting the round. Ward round structures fell into four categories, with almost three-quarters of trainers making no use of either pre- or post-round meetings. Where such meetings took place, however, opportunities for OJT were created and, in some cases, optimized through routines to encourage trainee contributions. Discussion time away from patients structured into ward rounds enabled trainers and trainees to take advantage of many opportunities to learn from service. Although unplanned and unsystematic opportunities for OJT do arise, far more reliable are those created through systematic planning and preparation. Trainers have choices to make about how they conduct ward rounds and by choosing to make use of pre- and/or post-ward round sessions, valuable opportunities for OJT can be created.