Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo.

The adhesion receptors, LFA-1 and VLA-4, on lymphocytes mediate lymphocyte adherence to cytokine-activated endothelial cells (EC) in vitro. Based on our previous data, which suggested that the mAb TA-2 reacted with rat VLA-4, the effect of TA-2 on lymphocyte migration out of the blood was examined. Small peritoneal exudate lymphocytes (sPEL) preferentially migrate to cutaneous inflammatory reactions, whereas lymphocytes from peripheral lymph nodes (PLN) migrate poorly to inflammatory sites but home avidly to PLN. Treatment of sPEL with TA-2 inhibited sPEL migration to DTH, LPS, poly I:C, IFN-gamma, IFN-alpha/beta, and TNF-alpha by 35 to 65% and their accumulation in PLN by 50%. The homing of PLN lymphocytes to PLN was not inhibited by TA-2. Spleen T cell migration to cutaneous inflammatory sites was inhibited but homing to PLN was not affected. Systemic treatment with TA-2 inhibited sPEL migration to inflamed or cytokine-injected skin by up to 70%. Similarly, TA-2 strongly inhibited the migration of Ag-stimulated PLN lymphoblasts to skin and to PLN. The migration of lymphocytes from all sources, including the peritoneum, spleen, PLN, mesenteric nodes, and Peyer's patches, to mesenteric lymph nodes and Peyer's patches was inhibited by 80% and 95%, respectively. In conclusion, our results suggest that VLA-4 and possibly other alpha 4 integrins mediate the migration of the inflammation-seeking sPEL and Ag-activated lymphoblasts to cutaneous inflammatory sites and lymph nodes but do not affect the homing of PLN lymphocytes to PLN. These integrins also appear to be necessary for the migration of all types of lymphocytes to Peyer's patches and mesenteric lymph nodes.     

Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine stimulated endothelium in the rat.

An important event in the migration of lymphocytes out of the blood is their adherence to endothelial cells (EC). In inflammatory sites cytokines activate EC and promote lymphocyte EC adherence and migration. Small peritoneal exudate lymphocytes (sPEL) preferentially migrate from the blood to cutaneous delayed-type hypersensitivity reactions and to sites injected with IFN-gamma, IFN-alpha/beta, and TNF-alpha, rather than to peripheral lymph nodes. The basis of this migration is sPEL adherence to cytokine-activated EC. To study this adhesion mAb to rat sPEL were screened for inhibition of sPEL adherence to IFN-gamma-stimulated EC. One mAb, TA-2, inhibited IFN-gamma-stimulated adherence to EC by 60%. This antibody had no effect on the baseline adherence of sPEL to unstimulated EC. Treatment of sPEL, but not EC, with TA-2-inhibited adhesion. TA-2 also inhibited adhesion to EC activated with mIL-1 alpha, TNF-alpha, and LPS, and the adhesion of spleen T cells to activated EC. The TA-2 Ag was expressed on virtually all lymph node, spleen, and sPEL lymphocytes but sPEL expressed two to three times higher levels than lymph node lymphocytes, and the highest levels were found on CD4+ and CD45R- memory T cells. TA-2 immunoprecipitated a group of four polypeptides with molecular mass of 150, 130, 83, and 66 kDa. Finally, TA-2 inhibited sPEL adhesion to TNF-alpha and IL-1 stimulated human umbilical vein EC to the same extent as an anti-human VCAM-1 mAb, and combinations of TA-2 and anti-VCAM-1 were not different from treatment with either antibody alone. Thus, TA-2 appears to recognize rat VLA-4 based on immunoprecipitation, immunofluorescence, and lymphocyte EC studies. VLA-4 mediates the adhesion of rat lymphocytes to rat microvascular EC stimulated with IFN-gamma, mIL-1 alpha, TNF-alpha, and LPS. VLA-4 is important in the increased adhesion of sPEL to EC and the enhanced sPEL migration to inflammation may in part be explained by increased expression of VLA-4 on these cells.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.     

Effect of T cell activation on lymphocyte-endothelial cell adherence and the role of VLA-4 in the rat.

Lymphocyte migration from the blood is in part controlled by lymphocyte surface adhesion molecules, such as VLA-4 and LFA-1. Small lymph node lymphocytes from rats adhere poorly to rat microvascular endothelial cells (EC), while lymphoblasts from antigen-challenged lymph nodes have an enhanced adherence to EC and preferentially migrate to inflamed tissues. This lymphoblast adherence is partially inhibited by anti-VLA-4. The effects of in vitro activation of lymph node lymphocytes on lymphocyte-EC adhesion were examined. In vitro stimulation of T cells with concanavalin A or calcium ionophore and phorbol myristate acetate (PMA) for 1-4 hr caused a marked (7- to 12-fold) increase in lymphocyte adhesion to unstimulated and IFN-gamma- or LPS-treated EC. This adhesion was partially inhibited by anti-VLA-4, was not associated with increased VLA-4 expression, was partially inhibited at 4 degrees C, and was virtually eliminated at 4 degrees C with anti-VLA-4. Anti-CD3 or IL-2 stimulation of T cells also enhanced lymphocyte adhesion but required 2-3 days of culture. This adhesion was not inhibited by anti-VLA-4 and was almost totally inhibited at 4 degrees C, suggesting a primarily LFA-1-mediated adhesion. In conclusion, stimulation of T cells with Con A or calcium ionophore plus PMA caused a rapid enhancement of lymphocyte-EC adhesion mediated in part through VLA-4, while stimulation of T cells with anti-CD3 or IL-2 enhanced lymphocyte adhesion apparently independent of VLA-4.     

Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat.

Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen-stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF-alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS-treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4.     

T lymphocyte adhesion to endothelial cells: mechanisms demonstrated by anti-LFA-1 monoclonal antibodies

Adhesion of lymphocytes to vascular endothelium is the first event in the passage of lymphocytes into a chronic inflammatory reaction. To investigate molecular mechanisms of T-EC adhesion, monoclonal antibodies (Mab) against T cell surface antigens have been tested for inhibition of binding. Baseline and phorbol ester-stimulated adhesion were strongly inhibited by either Mab 60.3 (reactive with the beta-chain of the LFA-1, OKM1, and p150,95 molecules) or by Mab TS 1/22 (specific for the alpha-chain of LFA-1). Although the increased binding of phorbol ester-stimulated lymphocytes was inhibited by anti-LFA-1 antibody, there was no increased expression of LFA-1 on phorbol ester-stimulated T cells, as determined by FACS analysis. Maximal inhibition of unstimulated and phorbol ester-stimulated T-EC adhesion was seen at Mab concentrations of 1 microgram/ml. In contrast, LPS- and IL 1-enhanced T-EC adhesion were only weakly inhibited by these antibodies. Mab 60.3 and TS 1/22 did not stain either unstimulated EC or LPS- or IL 1-stimulated EC, as measured by FACS analysis; moreover, preincubation of EC alone with these antibodies did not lead to inhibition of T-EC binding. Adhesion was not affected by Mab against the sheep erythrocyte receptor (LFA-2), a nonpolymorphic HLA class 1 framework antigen, or against LFA-3, the alpha-chain of OKM1, or the alpha-chain of p150,95. These results suggest that the mechanism of binding of lymphocytes to unstimulated endothelium differs from that to stimulated endothelium. LFA-1 appears to be an important adhesion-related molecule for binding to unstimulated endothelium. However, the increased lymphocyte adhesion to IL 1- or LPS-stimulated EC observed in these experiments appears to be relatively independent of LFA-1. The increased adhesion to stimulated EC could be due either to an increase in the avidity or the density of the EC receptor molecules ordinarily involved in unstimulated T-EC binding or to the formation of alternative receptors on the stimulated EC that are not present on unstimulated cells.     

Endothelial cell lymphocyte function-associated antigen-3 and an unidentified ligand act in concert to provide costimulation to human peripheral blood CD4+ T cells.

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.     

Stimulation of lymphocyte migration by endotoxin, tumor necrosis factor, and interferon

Since several studies have demonstrated that lipopolysaccharide (LPS), tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhanced lymphocyte binding to endothelial cells in vitro, we examined the effects of these agents on lymphocyte migration in vivo. Small peritoneal exudate lymphocytes (sPEL), which perferentially migrate into inflammatory sites, were radiolabeled with 111In and injected iv into rats. The id injection of LPS was a strong stimulus for the migration of these cells into the skin. TNF alpha was also a good stimulator of lymphocyte migration, while TNF beta and IL-1 alpha were weak or nearly inactive. Kinetic analysis demonstrated that migration to TNF was rapid, with a peak at 6 hr, followed by a steady decline, while migration to LPS was sustained for 24 hr. TNF alpha, TNF beta, and IL-1 alpha, when combined with interferon-gamma (IFN-gamma) or IFN-alpha/beta produced striking synergistic increases in lymphocyte migration. Combinations of the TNFs and IL-1 had less than additive effects, as did combinations of the IFNs. Qualitatively similar migration responses were found when spleen T cells instead of sPEL were studied.     

Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.     

The role of E-selectin, P-selectin, and very late activation antigen-4 in T lymphocyte migration to dermal inflammation

T lymphocyte infiltration into inflamed tissues is thought to involve lymphocyte rolling on vascular endothelial cells. Because both selectin and alpha(4) integrin adhesion molecules can mediate leukocyte rolling, the contribution of these receptors to lymphocyte migration to inflammation was examined. The recruitment of (111)In-labeled spleen T cells to intradermal sites injected with IFN-gamma, TNF-alpha, LPS, poly inosine-cytosine, and Con A was measured in the rat, and the effect of blocking mAbs to E-selectin, P-selectin, very late activation Ag-4 (VLA-4), and LFA-1 was determined on this T cell migration in vivo. Anti-E-selectin and anti-P-selectin mAbs each inhibited 10-40 and 20-48%, respectively, of the T lymphocyte migration to the inflammatory sites, depending on the stimulus. Blocking VLA-4 inhibited 50% of the migration to all of the lesions except Con A. Treatment with both anti-VLA-4 and anti-E-selectin mAbs inhibited up to 85% of the lymphocyte accumulation, while P-selectin and VLA-4 blockade in combination was not more effective than VLA-4 blockade alone in TNF-alpha, IFN-gamma, LPS, and poly inosine-cytosine lesions. Inhibiting E-selectin, P-selectin, and VLA-4 together nearly abolished lymphocyte migration to all inflammatory sites. Anti-LFA-1 mAb strongly inhibited lymphocyte accumulation by itself, and this inhibition was not significantly further reduced by E- or P-selectin blockade. Thus, T cell migration to dermal inflammation is dependent on E-selectin, P-selectin, and VLA-4, likely because these three receptors are required for rolling of memory T lymphocytes, but VLA-4 and E-selectin are especially important for lymphocyte infiltration in these tissues.     

The VLA-4/VCAM-1 molecules participate in gamma delta cell interaction with endothelial cells.

The accumulation of T lymphocytes at the site of chronic inflammation depends on a number of factors including adherence of T cells to vascular endothelial cells (EC) and endothelial permeability. We examined the effects of human gamma delta + T lymphocytes on the permeability of EC to macromolecules and characterized the cell surface molecules that are involved in these interactions. In this model, the flux of [125I]albumin was measured across the EC monolayer after a short-term culture with cloned gamma delta cells. Our results show that coculture of activated, but not resting, gamma delta cells with EC enhances endothelial permeability by a cytolytic process. Pretreating gamma delta cells with monoclonal antibodies directed at either LFA-1 or VLA-4 molecules or pretreating EC with monoclonal antibodies directed against either ICAM-1 or VCAM-1 molecules significantly inhibited gamma delta cell-mediated enhancement in endothelial permeability. This indicated that VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways participate in gamma delta cell-EC interaction.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Two-way signalling through the LFA-1 lymphocyte adhesion receptor

T lymphocyte recognition of foreign antigens and migration throughout the body require the regulated adhesion of lymphocytes to diverse types of cells and to the extracellular matrix. The lymphocyte adhesion 'receptor' LFA-1, a member of the integrin family, interacts with ICAM-1 and other counter-receptors to mediate adhesion. The LFA-1/ICAM-1 interaction is regulated by signals transmitted from the cytoplasm to the extracellular space. Conversely, LFA-1 transmits signals from the extracellular space to the cytoplasm to regulate T lymphocyte activation. The observed properties of LFA-1 and related adhesion 'receptors' are incorporated into a general model for adhesion during immune surveillance and recognition of foreign antigens.     

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.     

IFN-gamma-inducible T cell alpha chemoattractant is a potent stimulator of normal human blood T lymphocyte transendothelial migration: differential regulation by IFN-gamma and TNF-alpha

Previous studies have shown that the CXC chemokine, IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), was chemotactic for IL-2-activated human T lymphocytes, which express abundant CXCR3. However, because most memory T lymphocytes are also CXCR3(+), the ability of I-TAC to promote the migration of normal human blood T cells across HUVEC monolayers in Transwell chambers was examined. I-TAC induced a marked (4- to 6-fold) increase in transendothelial migration (TEM) of T cells across unstimulated HUVEC from 5.6 to 28% of input T cells and was substantially more active than IFN-gamma-inducible protein-10, another CXCR3 ligand. I-TAC significantly enhanced TEM of T cells across TNF-alpha, but not across IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC. IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC produced substantial amounts of I-TAC, in contrast to TNF-alpha-treated EC. Both CD4(+) and CD8(+) T cells migrated in response to I-TAC to a similar extent, while memory T cells migrated several fold better than naive T cells. Blockade of LFA-1 strongly inhibited I-TAC-induced T cell TEM across unstimulated HUVEC, and approximately 50-60% of the TEM across cytokine-activated HUVEC. However, blocking both LFA-1 and very late Ag-4 abolished I-TAC induced T cell TEM. In vivo significant levels of I-TAC were detected in arthritic synovial fluid. Thus, I-TAC is one of the most potent chemoattractants of normal human blood CD4 and CD8 T cell TEM and is likely a major mediator of blood memory T lymphocyte migration to inflammation.     

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.     

The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the beta2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the beta2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.     

Evidence for involvement of lymphocyte function-associated antigen 1 in T cell migration to epidermis.

Although it is well known that in various T cell-mediated skin diseases T cells migrate preferentially to epidermis, no direct evidence has been presented in which molecules on T cells are important in directing T cell traffic to epidermis. We have previously established CD4+ autoreactive cloned T cells with a special tropism for epidermis in vitro as well as in vivo. Antibody inhibition studies demonstrated that only anti-lymphocyte function associated Ag 1 (anti-LFA-1) mAb completely inhibited the in vitro migration of the T cells toward the epidermis, whereas mAb against other T cell surface molecules had little or no effect. Monovalent F(ab) fragment of the anti-LFA-1 mAb, although less efficient, also inhibited the T cell migration. The apparent dependency of the inhibition on the anti-alpha-chain mAb suggested a major role for the alpha-chain of LFA-1 in T cell migration to epidermis. The relevance of an LFA-1-dependent mechanism to the epidermotropic migration of T cells was further strengthened by the findings that the T cell migration to epidermis was inhibited by divalent cation depletion, cytochalasin B, and low temperature. These findings indicate that the LFA-1 molecule, which is thought to be primarily involved in cell-to-cell adhesions, also plays an important role in directing T cell migration to epidermis.     

Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing

In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.     

Human T lymphocyte adhesion to endothelial cells and transendothelial migration. Alteration of receptor use relates to the activation status of both the T cell and the endothelial cell

An in vitro model of T cell adhesion to human umbilical vein endothelial cells (HUVEC) and transendothelial migration was used to determine whether the activation state of the T cell or cytokine exposure of the HUVEC altered T cell-HUVEC interactions or receptor utilization. Stimulation of T cells with the activator of protein kinase C, phorbol dibutyrate (PDB) alone or in combination with the calcium ionophore, ionomycin increased their binding to HUVEC. Much of the binding of control and activated T cells to HUVEC was mediated by leukocyte function-associated Ag-1 (LFA-1) (CD11a/CD18), because mAb to either chain of this molecule inhibited binding substantially, but not completely. Activation of HUVEC with IL-1 also increased binding of T cells. Binding of control T cells to IL-1-stimulated HUVEC, however, was found to be LFA-1 independent, because mAb to CD11a/CD18 failed to block the interaction. In contrast, binding of activated T cells to IL-1-stimulated HUVEC was partially inhibited by mAb to LFA-1. Binding of activated T cells to IL-1-stimulated HUVEC also involved CD44 because this interaction was partially blocked by mAb to this determinant. When T cell migration was analyzed, it was found that the migration of PDB-activated T cells was three to four-fold more than that of control T cells. Migration through HUVEC and random migration were both enhanced by PDB stimulation. However, when the T cells were costimulated with PDB and ionomycin, migration was not increased above that of control T cells. PDB-activated T cells appeared to use LFA-1 for migration regardless of the activation status of the HUVEC, because mAb to CD11a/CD18 partially blocked their migration after binding to HUVEC. There was also a modest inhibition of PDB-activated T cell migration by mAb to CD44. In contrast, migration of control T cells involved neither LFA-1 nor CD44. Finally, binding of control T cells to high endothelial venules of peripheral lymphoid tissue was found to be CD11a/CD18 and CD44 independent, and completely inhibited by activation with either PDB or the combination of PDB and ionomycin. These results demonstrate that T cells use LFA-1 and CD44 as well as other as yet unidentified adhesion receptors for interactions with HUVEC, and that use of these adhesion receptors is mutable and related to the activation state of the T cell and cytokine stimulation of the HUVEC.