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.     

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.     

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.     

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.     

Identification of subsets of human T cells capable of enhanced transendothelial migration.

A critical step in immunologically mediated inflammation is the migration of T cells between endothelial cells of postcapillary venules and into the tissues. To determine whether specific cells are capable of transendothelial migration, T cells that had migrated through endothelial monolayers were retrieved and analyzed. To accomplish this, human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and incubated with human T cells. T cells that were nonadherent to the EC, those that bound to the endothelium, and cells that had migrated through the endothelial monolayer and into the collagen were individually harvested and characterized. After a 4-h incubation with EC, T cells distributed themselves such that 77 +/- 2% were nonadherent, 13 +/- 2% were bound to EC, and 10 +/- 1% had migrated into the collagen. The CD4+ T cells that had migrated into the collagen were predominantly CD29bright/CD45RObright and CD45RA-. CD8+ T cells demonstrated a greater transendothelial migratory capacity than the CD4+ T cells. The migrated CD8+ T cells were mainly CD29bright but CD45RA+. Additional phenotypic analysis of the migrating cells indicated that they contained fewer cells that expressed L-selectin. Moreover the surface expression of CD7 was less dense in the T cells that had migrated than in the nonadherent T cells. Finally the T cells that migrated were not enriched for CD45RBdim T cells. Prolonging the incubation with EC to 36 h increased the number of T cells that migrated but did not alter the predominance of CD29bright T cells in the migrated population. Stimulation of EC with IL-1 or IFN-gamma also increased the number of adherent and migrating T cells, respectively, but did not alter the phenotype of the migrating cells. These results indicate that the capacity for transendothelial migration is an intrinsic ability of certain subpopulations of T cells and is related to their stage of differentiation as identified by their surface phenotype.     

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.     

Lovastatin and phospholipase Cgamma regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen

We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.     

The nonintegrin laminin binding protein (p67 LBP) is expressed on a subset of activated human T lymphocytes and, together with the integrin very late activation antigen-6, mediates avid cellular adherence to laminin.

A search for genes expressed in activated T cells revealed that the nonintegrin, 67-kDa laminin binding protein (p67 LBP) is expressed on the surface of a subset (10-15%) of activated peripheral blood T cells. Surface p67 LBP expression is detectable by FACS using the anti-p67 LBP mAb, MLuC5, within 6 h of T cell activation with phorbol dibutyrate and ionomycin, peaks 18-36 h postactivation, and persists for 7-10 days. The subset of T cells expressing p67 LBP is composed of mature, single-positive cells (85% CD4+8-, 15% CD4-8+) of memory cell phenotype (100% CD45 RO+/CD45 RA-). The p67 LBP+ T cells also express the integrin alpha6 chain (CD49f), which is known to associate with p67 LBP on tumor cells. In addition, the p67 LBP+ T cells express the integrin beta1, which associates with alpha6 in the laminin-specific integrin receptor very late activation Ag (VLA)-6 (alpha6beta1). Expression of an exogenous cDNA encoding the 37-kDa LBP precursor (p37 LBPP) confers p67 LBP surface expression on a p67 LBP-negative Jurkat T cell line (B2.7). Expression of p67 LBP induces B2.7 transfectants to adhere to laminin, but avid laminin binding depends on coexpression of VLA-6. Taken together, these data indicate that p67 LBP is an activation-induced surface structure on memory T cells that, together with VLA-6, mediates cellular adherence to laminin.     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

Stat6 signaling suppresses VLA-4 expression by CD8+ T cells and limits their ability to infiltrate tumor lesions in vivo

VLA-4 plays a critical role in T cell trafficking into inflammatory sites. Our recent studies have suggested that VLA-4 expression on CD8+ T cells is negatively controlled by IL-4 and serves as a functionally distinguishing variable for why Type-1, but not Type-2, CD8+ T cells are able to traffic into tumors. In this study, using in vitro culture of murine CD8+ T cells under Type-1 and Type-2 cytokine conditions, we show that IL-4-mediated down-regulation of VLA-4 expression is completely abrogated in Stat6-deficient CD8+ T cells. Conversely, CD8+ T cells expressing a constitutively active mutant form Stat6 (Stat6VT) failed to express VLA-4 even in the absence of IL-4-stimulation. Notably, Type-2 CD8+ T cells developed from Stat6-/- but not wild-type mice were competent to migrate into tumor lesions in vivo. These results suggest that Stat6-signaling is necessary and sufficient to restrict CD8+ T cell expression of VLA-4 (by IL-4), thereby serving as a regulator for CD8+ T cell infiltration into tumors.     

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.     

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.     

Phosphatidylinositol mannoside from Mycobacterium tuberculosis binds alpha5beta1 integrin (VLA-5) on CD4+ T cells and induces adhesion to fibronectin

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against alpha5 integrin chain and with RGD-containing peptides. Alpha5beta1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promoting adhesion to the ECM glycoprotein, FN. This is the first report of direct binding of a M. tuberculosis molecule to a receptor on human T cells resulting in a change in CD4+ T cell function.     

Phorbol ester-induced promyelocytic leukemia cell adhesion to marrow stromal cells involves fibronectin specific alpha 5 beta 1 integrin receptors.

The human promyelocytic cell line NB4 exhibited a weak adhesion capacity for bone marrow-derived stromal cells and their extracellular matrices (5-15% of adherent cells). Adhesion was enhanced by pulse-treatment of cells with phorbolester (PMA 10(-7) M). Adhesion was induced within minutes, was fibronectin-specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA-4 and VLA-5) at the membrane of untreated or PMA-treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti-VLA monoclonal antibodies demonstrated that the FN-specific adhesion triggered by PKC involved the alpha 5 beta 1 FN-specific receptors (VLA-5). We showed that the binding of NB4 cells to fibronectin was RGD-dependent. PMA-induced adhesion was not correlated to phosphorylation of the VLA-5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stromal cells may arrest differentiation and explain the large number of leukemic cells in the circulation.     

Murine T-cell hybridomas that produce lymphokine with macrophage-activating factor activity as a constitutive product

Responder spleen cells primed to alloantigens in vivo could generate high degree of cytotoxicity against low- or nonimmunogenic stimulators such as thymocytes or uv light-treated spleen cells in vitro. However, a removal of adherent cells from primed responder cells remarkably reduced the cytotoxicity after stimulation with such low-immunogenic stimulators. Adding a small number of peritoneal adherent cells (PACs) also suppressed the cytotoxic activity of unseparated responders against low-immunogenic stimulators. These suppressive effects by PACs were blocked by indomethacin. By adding prostaglandin E₂, cytotoxic T lymphocyte (CTL) generation of primed unseparated responders against low-immunogenic stimulators was suppressed; however, cytotoxic activity against mitomycin C-treated stimulators was not suppressed. These results suggested that prostaglandins released from PACs selectively inhibited the function of splenic adherent cells that were required for CTL generation of primed responder spleen cells against low-immunogenic stimulators in vitro.     

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.     

Calpain 2 controls turnover of LFA-1 adhesions on migrating T lymphocytes

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

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adherence of Peripheral Blood Leukocytes to Cytomegalovirus-Infected Fibroblasts

Peripheral blood monocytes and B cells adhered to cytomegalovirus (CMV)-infected fibroblasts, whereas T cells and polymorphonuclear leukocytes did not adhere to either CMV-infected or uninfected fibroblasts. When T cells were activated with anti-CD3 antibody, activated T cells demonstrated adherence and cytotoxicity to both CMV-infected and uninfected fibroblasts. Adherence of peripheral blood mononuclear cells (PBMC) and cytotoxicity mediated by adherent activated T cells were blocked by treatment of CMV-infected fibroblasts with anti-ICAM-1 antibody and by treatment of leukocytes with anti-LFA-1 antibody. These data suggest that an interaction of ICAM-1 and LFA-1 is responsible for the adherence of leukocytes and for adherent activated T cell-mediated cytotoxicity against CMV-infected fibroblasts.     

Regulation of experimental allergic encephalomyelitis. II. Appearance of suppressor cells during the remission phase of the disease

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE). Most rats recover from paralysis and are subsequently resistant to the disease. In an adoptive transfer system, we found that lymph node cells (LNC) from rats that had recovered from EAE protect syngeneic recipients from the disease when the latter are challenged with encephalitogenic myelin basic protein and adjuvant after receiving donor cells. Suppression is antigen-specific and requires viable LNC. In contrast to the suppressor cells we previously studied in tolerized rats, which were nonadherent T lymphocytes, the suppressor cells found in rats that have recovered from EAE adhere to glass wool. However, they are not retained on Sephadex G-10 columns to which macrophages adhere. Suppressor activity is enriched in the nylon wool-adherent LNC population (which consists of approximately 80% Ig+ cells). Our findings suggest that activation of adherent suppressor cells may be implicated in recovery from EAE. These may be adherent T cells, or B cells that produce anti-BP blocking antibodies.