Lymphocyte migration in lymphocyte function-associated antigen (LFA)-1-deficient mice

Using lymphocyte function-associated antigen (LFA)-1(-/-) mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the alpha4 integrins, alpha4beta7 and alpha4beta1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and alpha4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the alpha4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the alpha4beta7-specific ligand, dominates. Both alpha4beta1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.     

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.     

The use of lymphocyte function-associated antigen (LFA)-1-deficient mice to determine the role of LFA-1, Mac-1, and alpha4 integrin in the inflammatory response of neutrophils.

After injury or infection, neutrophils rapidly migrate from the circulation into tissues by means of an orderly progression of adhesion receptor engagements. Neutrophils have been previously considered to use selectins exclusively to roll on vessels before an adhesion step mediated by the beta2 integrins, lymphocyte function-associated antigen (LFA)-1, and Mac-1. Here we use LFA-1(-/-) mice, function blocking monoclonal antibodies, and intravital microscopy to investigate the roles of LFA-1, Mac-1, and alpha4 integrins in neutrophil recruitment in vivo. For the first time, we show that LFA-1 makes a contribution to neutrophil rolling by stabilizing the transient attachment or tethering phase of rolling. In contrast, Mac-1 does not appear to be important for either rolling or firm adhesion, but instead contributes to emigration from the vessel. Blocking Mac-1 in the presence of LFA-1 significantly reduces emigration, suggesting cooperation between these two integrins. Low levels of alpha4beta1 integrin can be detected on neutrophils from LFA-1(+/+) and (-/-) mice. These cells make use of alpha4beta1 during the rolling phase, particularly in the absence of LFA-1. Thus LFA-1 and alpha4beta1, together with the selectins, are involved in the rolling phase of neutrophil recruitment, and, in turn, affect the later stages of the transmigration event.     

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins.Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.     

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.     

Decreased stroma adhesion capacity of CD34+ progenitor cells from mobilized peripheral blood is not lineage- or stage-specific and is associated with low beta 1 and beta 2 integrin expression

Molecular mechanisms leading to mobilization of hematopoietic cells from bone marrow (BM) to peripheral blood (PB) involve modulation of adhesion molecule expression on these cells that probably result in changes in adhesion capacity to the microenvironment. However, it is not clear whether these changes involve different stages or lineages of progenitor cells. In this study, we compared the capacity of mature and immature clonogenic progenitor cells from granulocyte colony-stimulating factor (G-CSF)-mobilized PB and normal BM CD34+ cells to adhere to complete marrow stroma. This functional capacity was assessed concurrently with molecular expression on CD34+ cells of integrins VLA-4 (alpha 4/beta 1), VLA-5 (alpha 5/beta 1), and LFA-1 (alpha L/beta 2) by interindividual (between mobilized PB and normal BM) and intraindividual (between mobilized PB and steady-state BM and PB in the same patient) analysis. The proportion of adherent clonogenic progenitor cells was significantly lower in PB than in BM, not only for total progenitor cells but also for mature and immature progenitor cells, and the difference was found for granulocytic and particularly for erythroid lineages. The lower adhesion capacity of PB CD34+ cells to stroma was associated with decreased expression (signal/noise MFI ratio) of integrin alpha 4, beta 1, alpha L, and beta 2 chains whereas that of alpha 5 chain did not differ from BM cells with the lowest expression level. Similar differences in integrin expression levels were also found between mobilized PB and steady-state BM CD34+ cells in the same patient except for the alpha L chain. Moreover, we demonstrated for the first time a strong positive correlation between mobilizing capacity and expression levels on mobilized CD34+ cells for the LFA-1 alpha L chain but not for VLA-4 or VLA-5. In conclusion, the decreased adhesion capacity of mobilized PB progenitor cells to stroma involves different maturation stages and different lineages. This is associated with down-regulation of integrins VLA-4 and LFA-1, but mobilizing capacity appears positively correlated with LFA-1 levels.     

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.     

The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester

Lymphocytes become adherent and aggregate after stimulation with phorbol esters such as PMA. Time-lapse video showed that aggregating cells were motile and exhibited vigorous pseudopodial movements. Adhesion sites were initiated between pseudopodia of neighboring cells, and then moved to the uropod. PMA-stimulated aggregation by EBV-transformed B cell lines, SKW-3 (a T cell line), differentiated U937 (a monocytic line), and blood lymphocytes was inhibited by mAbs to LFA-1. A number of different mAb to the LFA-1 alpha and beta subunits and F(ab')2 and Fab' fragments inhibited aggregation. Furthermore, lymphoblasts from normal individuals, but not from LFA-1-deficient patients, aggregated in response to PMA. These findings suggest LFA-1 is critically involved in stimulated lymphocyte adhesion. LFA-1 expression was not increased by PMA stimulation, showing that other mechanisms regulate LFA-1-dependent adherence. LFA-1-deficient patient cells were able to coaggregate with LFA-1+ cells, showing that aggregation is not mediated by like-like interactions between LFA-1 molecules on opposite cells. Aggregation was Mg+2-dependent, inhibited by cytochalasin B, and was reversed when LFA-1 mAb was added to preformed aggregates. Previous findings suggesting that LFA-1 is important in a wide variety of leukocyte functions are elucidated by this work, which shows that LFA-1 is a general leukocyte cell adhesion molecule, the activity of which is regulated by cell activation.     

Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords

The normal migration route of B cells into follicular areas of spleen and lymph nodes is altered in the case of autoreactive cells that have bound self-antigen. To begin characterizing the molecular requirements for B cell migration into follicles, cells were treated with pertussis toxin (PTX), an inhibitor of signaling by many G protein-coupled chemokine receptors. Lymphocyte accumulation in the spleen is not inhibited by PTX and, therefore, the distribution of transferred cells was examined in this tissue. In contrast to untreated cells that localized predominantly in follicular areas within white pulp cords, PTX-treated B cells failed to enter white pulp areas altogether and accumulated in the splenic red pulp. T cells were also excluded from white pulp cords and in the case of both cell types, the adenosine diphosphate-ribosylating subunit of the toxin was required to block white pulp entry. These findings implicate a G protein-coupled receptor in lymphocyte migration into splenic white pulp cords. Exclusion of PTX- treated cells from all organized areas of secondary lymphoid tissues raises the possibility that the association observed between PTX treatment and predisposition to autoimmune disease results from inhibition of tolerance mechanisms that normally operate within secondary lymphoid tissues.     

Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma

Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-Au were isolated and used to analyze the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Th1) clones induced disease, while Th2 clones did not. Using variants of a single cloned Th1 line, the surface expression of alpha 4 integrins (very late antigen 4 [VLA-4]) was identified as a major pathogenic factor. Encephalitogenic clones and nonencephalitogenic variants differ by 10-fold in their level of surface expression of alpha 4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The alpha 4 integrin-high, disease-inducing cloned Th1 T cells enter brain parenchyma in abundance, while alpha 4 integrin-low, nonencephalitogenic Th1 cells do not. Moreover, antibodies to alpha 4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this encephalitogenic clone in vivo. The importance of the expression of VLA-4 for encephalitogenicity is not unique to cloned T cell lines, as similar results were obtained using myelin basic protein-primed lymph node T cells. alpha 4 integrin levels did not affect antigen responsiveness or production of the Th1 cytokines interleukin 2, interferon gamma, and lymphotoxin/tumor necrosis factor beta; and antibodies against alpha 4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of alpha 4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that alpha 4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.     

Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd,alpha4beta1 integrin/VCAM-1, and LFA-1 adhesion pathways

Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte-endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti-L-selectin, anti-PNAd, and anti-LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti-alpha4 integrin and anti-VCAM-1 mAbs inhibit homing by nearly 40%. alpha4beta7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against alpha4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of alpha4beta1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.     

Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.     

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.     

The alpha4beta1 and alpha5beta1 integrins mediate engraftment of granulocyte-colony-stimulating factor-mobilized human hematopoietic progenitor cells

BACKGROUND: The alpha4beta1 and alpha5beta1 integrins are major adhesion molecules of murine and human hematopoietic progenitor cells. Granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) are the most important source for clinical hematopoietic cell transplantation today. The contribution of alpha4beta1 and alpha5beta1 integrins to homing of PBPCs has not been studied yet. STUDY DESIGN AND METHODS: The expression of alpha4beta1 and alpha5beta1 integrins on purified human PBPCs was analyzed. Integrin function in adhesion to recombinant fibronectin and migration on fibronectin-coated transwells was assessed with fragments combining different adhesion domains and function-blocking antibodies. Finally, the function of those integrins in a transplantation model was investigated with repopulating cells of nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: More than 90 percent of all purified peripheral blood CD34+ cells express alpha4beta1 integrins, whereas only 10 to 15 percent express alpha5beta1. The alpha4beta1 integrin alone influences adhesion whereas alpha4beta1 and alpha5beta1 both mediate chemotaxis of clonogenic CD34+ progenitor cells on recombinant fibronectin. Importantly, antibodies against the integrins alpha4beta1 and alpha5beta1 independently reduce the repopulation of NOD/SCID mouse marrow after transplantation of human peripheral blood CD34+ cells. CONCLUSIONS: Alpha4beta1 and alpha5beta1 integrins are functional and critical adhesion receptors expressed on G-CSF-mobilized CD34+ hematopoietic blood progenitor cells with repopulating capacity mediating engraftment after transplantation.     

Aberrant activation of integrin alpha4beta7 suppresses lymphocyte migration to the gut

Integrin adhesion molecules mediate lymphocyte migration and homing to normal and inflamed tissues. While the ligand-binding activity of integrins is known to be modulated by conformational changes, little is known about how the appropriate balance of integrin adhesiveness is maintained in order to optimize the migratory capacity of lymphocytes in vivo. In this study we examined the regulation of the gut homing receptor alpha4beta7 integrin by manipulating at the germline level an integrin regulatory domain known as adjacent to metal ion-dependent adhesion site (ADMIDAS). ADMIDAS normally serves to raise the activation threshold of alpha4beta7, thereby stabilizing it in the default nonadhesive state. Lymphocytes from knockin beta7 (D146A) mice, which harbor a disrupted ADMIDAS, not only expressed an alpha4beta7 integrin that persistently adhered to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), but also exhibited perturbed cell migration along MAdCAM-1 substrates resulting from improper de-adhesion of the lymphocyte trailing edge. In vivo, aberrantly activated alpha4beta7 enhanced adhesion to Peyer's patch venules, but suppressed lymphocyte homing to the gut, diminishing the capacity of T cells to induce colitis. Our results underscore the importance of a proper balance in the adhesion and de-adhesion of the alpha4beta7 integrin, both for lymphocyte trafficking to the gut and for colitis progression.     

Leukocyte function-associated antigen 1 is an activation molecule for human T cells

The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.     

LFA-1和VLA-4参与人高增殖潜能内皮祖细胞与血管内皮的黏附和跨内皮迁移

为了了解淋巴细胞功能相关抗原1（lymphocyte function—associated antigen1，LFA-1）和极迟反应抗原4（very lateantigen 4，VLA-4）在高增殖潜能内皮祖细胞（high proliferative potential endothelial progenitor cells，HPP—EPCs）归巢过程中与血管内皮的黏附和跨内皮迁移中的作用，利用流式细胞术检测HPP—EPC中整合蛋白B1和B2的表达以及小鼠骨髓内皮细胞相应的受体的表达。利用体外黏附和迁移实验研究经过功能级别的中和抗体阻断VLA-4和LFA-1后HPP—EPC黏附和迁移细胞数目的变化。结果表明，HPP—EPC表达整合蛋白B1和B2，活化后小鼠骨髓内皮细胞表达细胞间黏附分子1（intercellular adhesion molecule1，ICAM-1）和血管细胞黏附分子1（vascular cell adhesion molecule1，VCAM-1）；加CDlla抗体组黏附细胞或CD49d抗体组黏附和迁移细胞均较同型对照抗体组少，而且加CDlla和CD49d两种抗体联用组黏附和迁移细胞明显减少，其细胞数较任何单一抗体组少。结论：LFA-1和VLA-4在HPP—EPC与血管内皮的黏附和跨内皮迁移中发挥了重要的作用。     

Role of integrin cross-regulation in parvovirus B19 targeting

Most viral vectors used for gene therapy lack the ability to target a defined cell population. Parvovirus B19 has a restricted tropism for human erythroid progenitor cells and uses activated alpha5beta1 integrins as coreceptors for entry [Weigel-Kelley, K.A., Yoder, M.C., and Srivastava, A. (2003). Blood 102, 3927-3933]. In this study we examined the role of coexpressed integrins in alpha5beta1 integrin coreceptor function. Antibody-mediated cross-linking of beta1, beta2, and beta3 integrins and the integrin-associated protein (IAP) increased parvovirus B19 entry into nontarget K562 cells. Functional silencing of one integrin group, however, reduced the virus uptake- promoting function of a subsequently activated integrin group, indicating that the three integrins did not operate in isolation but through shared signaling pathways. This was further corroborated by direct competition between simultaneously clustered beta2 and beta1 integrins that could be overcome by stabilizing clustered beta1 integrins in a high-affinity conformation. In contrast, parvovirus B19 entry into primary erythroid progenitor cells was characterized by strong clustering-induced beta1 integrin coreceptor activity that was not abolished by subsequent beta2 and beta3 integrin activation and was, in fact, substantially increased in the presence of preclustered beta2 and beta3 integrins. Thus, integrin function is regulated in a cell type-specific manner through coexpressed integrins and preferential parvovirus B19 entry into erythroid progenitor cells is promoted by a robust beta1 integrin response that is enhanced through stable preclustering of coexpressed integrins. These results have implications for other viral vectors that use integrins as receptors/coreceptors and for gene therapy of hematopoietic progenitor cells using parvovirus B19 vectors.     

Protein tyrosine kinase p56-Lck regulates lymphocyte function- associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell [published erratum appears in J Exp Med 1995 Jan 1;181(1):442]

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL- 2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function- associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA- ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2- Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin- mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1- mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR- CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin- mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1- dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.