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.     

Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1.

Lymphocyte function-associated Ag-1 (LFA-1) or CD11a/CD18 mediates lymphocyte adhesion to cultured vascular endothelial cells (EC). Thus, LFA-1 likely plays a major role in lymphocyte migration out of the blood, but there is little information on this in vivo. Small peritoneal exudate lymphocytes (sPEL) and lymph node (LN) lymphoblasts adhere to cytokine-activated EC and preferentially migrate to cutaneous inflammatory sites. The role of LFA-1 in the adherence and in vivo migration of these T cells was determined. Because of a lack of anti-rat LFA-1, mAb were prepared to rat T cells. One mAb, TA-3, inhibited homotypic aggregation; T cell proliferation to Ag, alloantigens, and mitogens; stained all leukocytes; and immunoprecipitated 170- and 95-kDa polypeptides from lymphocytes and neutrophils. TA-3 binding to lymphocytes also required Ca2+, but not Mg2+. Thus, TA-3 appears to react with rat LFA-1. TA-3 inhibited spleen T cell adhesion to unstimulated EC by 30% and to IFN-gamma, TNF-alpha, IL-1 alpha, and LPS stimulated EC by 50 to 60% but inhibited sPEL EC adhesion by only 10%. TA-3 also strongly inhibited anti-CD3-stimulated LN T cell adherence. The migration of spleen T cells to delayed-type hypersensitivity and skin sites injected with LPS, poly I:C, IFN-gamma, IFN-alpha/beta, and TNF was inhibited by 72 to 88% by TA-3, and was decreased by 50% to peripheral LN. TA-3 caused less but still 50 to 60% inhibition of sPEL migration to inflamed skin. Lymphoblast migration to skin was inhibited 40 to 80% and to PLN by 30%. Migration of lymphocytes from all sources to mesenteric LN was inhibited by 32 to 60%. In conclusion, LFA-1 mediates much of the adherence of spleen T cells and lymphoblasts to EC in vitro, most of the migration of these cells to dermal inflammation and about 50% of the homing of LN and spleen T cells to peripheral and mesenteric LN. sPEL are less dependent on LFA-1 for adhesion to EC in vitro and for migration to inflamed skin and LN in vivo.     

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 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.     

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.     

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.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

IL-4 acts synergistically with IL-1 beta to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1.

Adhesion of lymphocytes to endothelial cells (EC) is the requisite first element in the multistep process of transmigration from blood across the postcapillary venules. Selective expression of cell adhesion molecules (CM) by microvascular EC in lymphoid organs (e.g., lymph nodes) and during tissue inflammation modulates this traffic in a site-directed manner. CAM synthesis by EC is regulated in turn by cytokines released in the local microenvironment. Studies done largely with human umbilical vein EC have implicated IL-1, IFN-gamma, and TNF-alpha as cytokines which promote leukocyte adhesion to EC. In the work reported here, the responses of cultured microvascular EC derived from macaque lymph nodes to IL-1beta, IL-2, IFN-gamma, and IL-4 were examined. Increases in lymphocyte adhesion after preculture of microvascular EC in IL-1beta or IFN-gamma were typically 2-to 4-fold above controls and comparable to those reported for human umbilical vein EC. IL-2 had no effect. In contrast, IL-4 markedly enhanced adhesion to microvascular EC. IL-4-induced adhesion was observed as early as 4 h after induction, plateaued by 24 h, was stable through 72 h of culture, but decayed to basal levels within 72 h after removal of IL-4 from the cultures. IL-1beta, but not IL-2 or IFN-gamma, synergistically enhanced the action of IL-4 on cultured microvascular EC to promote lymphocyte binding. Adhesion triggered in this manner required de novo protein synthesis. However, the avidity of IL-4-activated microvascular EC for lymphocytes, and analyses of kinetics, cation and temperature dependence, and/or lack of blockade with mAb to endothelial leukocyte adhesion molecule-1, intra-cellular adhesion molecule-1, and MECA-79 indicated that these CAM were not central to the phenomenon. To aid identification of the relevant CAM, mAb specific to IL-4-induced microvascular EC were produced. One of these, 6G10, blocked up to 90% of lymphocyte adhesion to IL-4-induced microvascular EC, immunoprecipitated an IL-4-induced cell-surface molecule of 110-kDa molecular mass, and reacted specifically with Chinese hamster ovary cells transfected with human vascular cell adhesion molecule-1. Our results suggest that IL-4 may have potent effects on lymphocyte recirculation in vivo.     

Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes.

The comparative roles of the endothelial cell (EC) adhesion receptors VCAM-1 and ICAM-1 during the adhesion and transendothelial migration of T cells were examined. The adhesion of T cells to IL-1-activated EC was markedly, but not completely, inhibited by mAb to VCAM-1 as well as to its counter-receptor, VLA-4, whereas, T cell binding to IL-1-activated EC was not blocked by mAb to ICAM-1 or to its counter-receptor, LFA-1. In contrast, LFA-1/ICAM-1, but not VLA-4/VCAM-1, mediated much, but not all, of the binding of T cells to unstimulated EC. Activation of T cells with phorbol dibutyrate and ionomycin alter the receptor-counter-receptor pairs used for binding to EC. Regardless of the activation status of the EC, the binding of activated T cells was not blocked by mAb to VLA-4 or VCAM-1. Moreover, the binding of activated T cells to EC was blocked to a lesser degree by mAb to LFA-1 than that of resting T cells, and mAb to ICAM-1 blocked binding only modestly. The role of VCAM-1 and ICAM-1 during the transendothelial migration of T cells was also examined. Regardless of the activation status of the T cells or the EC, VCAM-1 was never found to function during transendothelial migration, even when it mediated the binding of resting T cells to IL-1-activated EC. In contrast, ICAM-1 played an important role in transendothelial migration under all of the conditions examined, including situations when T cell-EC binding was not mediated by ICAM-1. Immunoelectron microscopic analysis of transendothelial migration supported the conclusion that ICAM-1 but not VCAM-1 played a central role in this process. Thus, ICAM-1 was prominently and uniformly expressed at all EC membrane sites that were in contact with bound and migrating T cells, whereas VCAM-1 was localized to the luminal surface of IL-1-activated EC, but was often absent from the surface of the EC in contact with T cells undergoing transendothelial migration. These studies confirm that ICAM-1 and VCAM-1 play reciprocal roles in the binding of resting T cells to resting and IL-1-activated EC, respectively, but a less prominent role in the binding of activated T cells. Moreover, ICAM-1 but not VCAM-1 plays a role in transendothelial migration, regardless of the receptor-counter-receptor pairs used for initial binding.     

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.     

IL-2-activated human killer lymphocytes but not their secreted products mediate increase in albumin flux across cultured endothelial monolayers. Implications for vascular leak syndrome

When cultured with IL-2, human lymphoid cells acquire the ability to lyse various NK-resistant tumor targets. Due to their anti-tumor cytolytic effect, clinical trials with IL-2 alone or IL-2 + IL-2-activated killer (IAK) lymphocytes have been undertaken. However, infusion of therapeutically effective doses of IL-2 is associated with the development of systemic toxicity characterized by exaggerated endothelial permeability, also known as vascular leak syndrome. The present study was designed to examine the effects of IAK cells and their secreted products on vascular endothelial permeability by using an in vitro endothelial permeability model in which the flux of FITC-albumin across endothelial cell (EC) monolayers was measured. When endothelial monolayers were exposed to IAK cells for 2 h, significant increases in the transendothelial permeability to albumin were observed. Exposure of EC to lymphocytes cultured in the absence of IL-2 did not induce significant alteration in the endothelial permeability. In addition, neither culture supernatants of IAK cells nor purified recombinant cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-6, TNF-alpha, GM-CSF, M-CSF, and IFN-gamma, had any effect on endothelial permeability in this model. Prior activation of EC with TNF-alpha did not alter the increased permeability induced by IAK cells or lack of it by nonactivated lymphocytes. Dexamethasone treatment of IAK cells abolished their anti-tumor cytolytic effect but only partially inhibited their ability to induce increased endothelial permeability. Pretreatment of IAK cells with mAb directed at the CD11a/CD18 (LFA-1) adhesion complex, and that of EC with mAb directed at the ICAM-1 molecule, inhibited the IAK cell-induced increase in endothelial permeability. These results demonstrate that direct cell-to-cell contact between IAK cells and EC is necessary and sufficient to cause increased endothelial permeability in this model system, and may therefore be an important factor contributing to the development of the vascular leak syndrome observed clinically.     

Role of CD11/CD18 in adhesion and transendothelial migration of T cells. Analysis utilizing CD18-deficient T cell clones.

The role of leukocyte function-associated Ag-1 (LFA-1) (CD11a/CD18) in T cell-endothelial cell (EC) interactions was assessed by utilizing CD11a/CD18-deficient T cell clones generated from a patient with leukocyte adhesion deficiency (LAD). The ability of these clones to bind to and migrate through monolayers of EC in vitro was compared with that of clones generated in a similar manner from normal controls. The LAD clones bound to EC to a similar extent as the controls. The contribution of other cell surface adhesion molecules was assessed with mAb blocking experiments. It was found that part of the EC binding by these CD11a/CD18-deficient clones was mediated by an interaction of very late Ag-4 (VLA-4) with vascular cell adhesion molecule-1 (VCAM-1) on the EC. In contrast to their normal ability to bind to EC, the capacity of the LAD clones to migrate through EC monolayers was significantly less than that of the control clones. This impairment in migration was not related to decreased intrinsic motility. Moreover, neither phorbol ester stimulation of the LAD clones nor IL-1 stimulation of the EC increased the capacity of the clones to migrate through EC monolayers, although binding to EC was augmented by both treatments. Only a minimal percentage of the migration of either control or LAD clones was inhibited by mAb to VLA-4 or VCAM-1. These data demonstrate that LFA-1 plays a central role in the transendothelial migration of T cells. In the absence of LFA-1, T cells retain the ability to bind to EC because of the activity of other receptor/ligand pairs, including VLA-4/VCAM-1. Finally, it is likely that, during both binding and transendothelial migration of T cells, additional cell surface molecules play a role.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

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.     

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.     

Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-gamma-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against beta(1) and beta(2) integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-gamma, TNF-alpha, and IFN-gamma plus TNF-alpha-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4(-), but not CD4(+), T cell TEM, suggesting a role for tTG in recruitment of CD8(+) T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8(+) T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

Divalent cation substitution reveals CD18- and very late antigen-dependent pathways that mediate human neutrophil adherence to fibronectin.

We investigated the mechanisms by which Mn2+ alters human neutrophil (PMN) adherence to various connective tissue proteins. Substitution of Mn2+ for Ca2+ and Mg2+ significantly increased adhesion of human PMN to plastic well coated with fibronectin, fibrinogen, and laminin but not gelatin. Anti-CD18 mAb almost completely blocked adherence to laminin, partly blocked adherence to fibrinogen, but did not inhibit adhesion to fibronectin at all. In contrast, anti-very late antigen (VLA)-5 mAb antibodies significantly reduced Mn(2+)-mediated PMN adherence to fibronectin, but not to laminin or fibrinogen, demonstrating that VLA-5-mediated PMN adherence to fibronectin, but not to fibrinogen or laminin. This was supported by experiments in which synthetic GRGDSP peptide significantly inhibited Mn(2+)-mediated adherence to fibronectin, but not to laminin or fibrinogen. Activation of PMN with phorbol ester or C5a stimulated VLA-5-mediated adhesion to fibronectin, but the contribution of VLA-5 to the forces mediating adherence could only be detected when CD18 function was either blocked with mAb, or when CD18 was congenitally absent. VLA-5 mediated adhesion was also more transient than CD18-dependent adhesion. These data further confirm the presence of PMN VLA integrins and demonstrate that PMN VLA-5 contributes to stimulated PMN adherence to fibronectin.     

Transforming growth factor-beta 1 and IL-4 regulate the adhesiveness of Peyer's patch high endothelial venule cells for lymphocytes.

The adhesion of lymphocytes to endothelial cells lining the postcapillary high endothelial venules (HEV) is the first step in their emigration from the bloodstream into lymph nodes and Peyer's patches (PP). We have recently shown that the adhesiveness of cultured rat lymph node and PP HEV cells for thoracic duct lymphocytes can be increased significantly by pretreatment with TNF-alpha, IFN-gamma, and IL-4. In the present study we investigated the role of transforming growth factor-beta 1 (TGF-beta) on the adhesiveness of nonstimulated and cytokine-stimulated PP HEV cells for rat lymphocytes. The results indicated that at picomolar concentrations, TGF-beta significantly (p less than 0.001) decreased the ability of PP HEV cells to adhere 51Cr-labeled rat lymphocytes. Maximal inhibition was observed with a TGF-beta dose of 0.5 ng/ml and an incubation time of 6 to 12 h. TGF-beta did not affect the morphology of HEV cells and had no adverse effect on their viability. Moreover, the decrease in HEV adhesiveness by TGF-beta was reversible, with lymphocyte binding returning to control level 24 h after removal of the cytokine. The specificity of TGF-beta was confirmed by the ability of neutralizing anti-TGF-beta 1 antibody, but not control serum, to abolish the inhibitory properties of the cytokine. In addition, TGF-beta completely abrogated the increased adhesiveness of PP HEV cells normally induced by TNF-alpha or IFN-gamma. In contrast, TGF-beta had no effect on the stimulating effects of IL-4. Moreover, preincubation of PP HEV cells with TGF-beta did not alter the ability of these cells to respond to IL-4. Importantly, the adhesion of rat lymphocytes to IL-4-stimulated PP HEV cells can be blocked by pretreatment of lymphocytes with the PP-homing receptor-specific 1B.2.6 antibody whereas pretreatment of human mononuclear cells with anti-very late activation antigen-4 alpha antibody inhibited only partially the binding of these cells to the IL-4-stimulated PP HEV monolayers. Taken together, these findings strongly suggest that TGF-beta and IL-4 play important regulatory roles in lymphocyte-HEV adhesion and that the stimulatory effect of IL-4 is mediated at least in part through the increased expression of organ-specific ligands on HEV cells.