MAdCAM-1 costimulates T cell proliferation exclusively through integrin α4β7, whereas VCAM-1 and CS-1 peptide use α4β1: evidence for “remote” costimulation and induction of hyperresponsiveness to B7 molecules

We have analyzed the effects of the α⁴ integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the α⁴ β₁ and α⁴ β₇ integrins, medicate T cell costimulation exclusively through integrin α⁴ β₁, but not through α⁴ β₇. The inability of VCAM-1-Fc to costimulate via α⁴ β₇ suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via α⁴ β₇, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody (“remote” costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc > ICAM-1-Fc > MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.     

Distinct binding specificities of integrins {alpha}4{beta}7 (LPAM-1), {alpha}4{beta}1 (VLA-4), and {alpha}IEL{beta}7

Integrin receptors are Important for regulating lymphocyte reclrculatlonand recruitment to sites of inflammation. Transfoctants of theB cell lymphoma 38C13 were generated that differ exclusivelyin the expression of integrin ß1 or ß7 subunltsallowing for a functional comparison of lymphocyte Peyer's patchHEV adhesion molecule 1 (LPAM-1) (4ß7) and very lateantigen 4 (VLA-4) (4ß1) in an Identical cellular environment.Whereas 38-ß7 transfectants bound to purified andcellular mucosal addressin cell adhesion molecule (MAdCAM-1),unstlmulated 38-ß1 cells failed to bind MAdCAM-1.Treatment of 38-ß1 cells with Mn²⁺ but not with PMAinduced low level binding to MAdCAM-1. MAdCAM-1 adhesion of38-ß7 cells was constitutive and not enhanced by Mn²⁺treatment. Similarly, MAdCAM-1-dependent adhesion to mucosalhigh endothellal venules was shown for 38-ß7 but notfor 38-ß1 cells. The results therefore establish theLPAM-1 - MAdCAM-1 Interaction as the functionally dominant adhesionpathway for regulating lymphocyte homing to mucosal sites. Nonetheless,the activated VLA-4 on some lymphocytes may be involved in MAdCAM-1recognition or promote binding to MAdCAM-1 In other tissues.By contrast, 38-ß7 and 38-ß1 transfectantsdid not differ in their binding capacity for vascular cell adhesionmolecule 1 (VCAM-1) or fibronectin and LPAM-1 did not displayany preference for interacting with either MAdCAM-1 or VCAM-1.LPAM-1 may therefore contribute significantly to cellular functionspreviously attributed to VLA-4. Interestingly, functional analysisof the intraepithellal lymphocyte integrin IELß7 whichIs structurally related to LPAM-1 did not reveal detectablebinding activity for MAdCAM-1, VCAM-1, or fibronectin.     

The low molecular weight Dextran 40 inhibits the adhesion of T lymphocytes to endothelial cells

Dextrans are complex colloidal macromolecules widely used as haemorrheologic substances and anti-thrombotic agents. Here we describe a novel function of Dextran 40 by demonstrating an inhibition of T lymphocyte adhesion to endothelial cells (EC). We applied an established microassay in which constitutive and tumour necrosis factor-alpha (TNF-α)-induced binding of mouse T lymphoma cells (TK-1) to mouse endothelioma (eEND.2) cells is mediated by the interaction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on EC with their counter-receptors the LFA-1 heterodimer (CD11a/CD18) and VLA-4 on T cells. Dextran 40 in therapeutically achievable levels (2–32 mg/ml) reduced both constitutive and TNF-α-stimulated TK-1 adhesion to eEND.2. Selective preincubation of eEND.2 or TK-1 revealed that Dextran 40 acted exclusively on the T cells. To explore further the mechanisms by which Dextran 40 interfered with TK-1 adhesion, their LFA-1 and VLA-4 expression was analysed by FACS. The surface expression levels of neither receptor were affected by Dextran 40. However, confocal microscopy revealed that Dextran 40 interfered with the activation-dependent capping and clustering of LFA-1 and VLA-4 on the surface of TK-1. We conclude that Dextran 40 inhibits the capacity of TK-1 T cells to adhere to eEND.2 endothelial cells and thus may be useful for therapeutic intervention in diseases associated with enhanced T lymphocyte binding to microvascular endothelium.     

Construction and Adhesive Properties of a Soluble MAdCAM-1-Fc Chimera Expressed in a Baculovirus System: Phylogenetic Conservation of Receptor-Ligand Interaction

MAdCAM-1 is a high endothelial venule adhesion molecule composed of immunoglobulin and mucin-like domains which binds the leucocyte integrin LPAM-1 (α4β7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mouse MAdCAM-1 which is normally membrane bound has been produced by joining the extracellular region of the receptor to the Fc domain of human IgGl. The MAdCAM-1-Fc cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda insect cells infected with the recombinant virus produced MAdCAM-1-Fc as a disulfide-linked homodimer of 82kDa polypeptides, which was secreted into the culture medium at > 1 μg/ml. The product purified by Protein G-Sepharose was identified as authentic MAdCAM-1-Fc by the anti-MAdCAM-l monoclonal antibody (MoAb) MECA-367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in supporting the binding of mouse α4β1⁺α4β7⁺ mesenteric lymph node lymphocytes, and the α4β1^?αβ7⁺ TK1 T cell lymphoma. Binding was enhanced by Mn⁺⁺ -induced integrin activation, and specifically blocked by anti-integrin α4 subunit and anti-MAdCAM-1 MoAbs. Binding was blocked by pretreatment of cells with sodium azide, and EDTA, indicating that binding is an energy-dependent process which requires divalent cations. Thus the mouse MAdCAM-1-Fc chimera produced in insect cells retains certain functional properties that typify the native receptor, and should be valuable in analysing the role of MAdCAM-1 in lymphocyte recirculation and emigration. However it was not sialylated despite being post-translational modified with N- and O-linked carbohydrate moieties, suggesting that the ability of MAdCAM-1 to support cell adhesion under static conditions is sialylation-independent. A rabbit polyclonal antibody raised against the entire cytopiasmic domain of the human integrin β7 subunit recognized LPAM-1-like molecules in human, rat, and mouse cells, suggesting a high degree of conservation of the MAdCAM-1 receptor across species. In agreement with this notion MAdCAM-1-Fc immobilized on glass was fully functional in supporting the cation-dependent binding of peripheral blood or spleen cells from a range of other species including human, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by o4 integrins.     

Cross talk between αvβ3 and α4β1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand α4β1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between α4β1 and VCAM-1 in vivo. Here we show that the lymphocyte αβ3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte αvβ3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of α4β3 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This αvβ3-mediated enhanced migration of lymphocytes via α4β1 is likely to depend on the interaction of αvβ3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between αvβ3 integrin and α4β1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.     

The small GTP-binding proteins Rho and Rac induce T cell adhesion to the mucosal addressin MAdCAM-1 in a hierarchical fashion.

Here we report that an activator (AIF4-) of heterotrimeric GTP-binding proteins (G-proteins) and inhibitors (lovastatin and C3 exoenzyme) of small GTP-binding proteins regulate the induction of alpha4beta7-mediated adhesion of TK-1 T lymphoma cells (alpha4+beta7+beta1-) to the mucosal addressin cell adhesion molecule MAdCAM-1. Activation of cell adhesion by AIF4- was abrogated by lovastatin, thereby establishing a link between heterotrimeric G-proteins and small GTP-binding proteins in the regulation of alpha4beta7-mediated cell adhesion. Increased numbers of cells bound MAdCAM-1-coated microspheres following activation with AIF4-, discounting an obligatory role for cell spreading in alpha4beta7-mediated cell adhesion. MAdCAM-1-Fc dimers triggered ligand-induced clustering of alpha4beta7 in response to AIF4- and Mn2+-induced activation of integrins. Hence alpha4beta7 cluster formation may be responsible, at least in part, for inducing cell adhesion in response to both extracellular and intracellular signals that impact on integrin function. Electroporation of constitutively active V14RhoA and V12Rac1 recombinant proteins into TK-1 cells revealed that both RhoA and Rac1 induce alpha4beta7 adhesion to MAdCAM-1. Activation is hierarchical since Rac1 is unable to directly activate alpha4beta7, but induces cell adhesion via RhoA, whereas the transient induction of cell adhesion mediated by RhoA is dependent on the activities of protein tyrosine kinases and protein kinase(s) C.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄ epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Integrin α chain cytoplasmic tails regulate “antibody-redirected” cell adhesion,independently of ligand binding

Here we describe a novel “antibody-redirected cell adhesion” (ARCA) assay. This assay measures heterotypic cell-cell adhesion, resulting from antibody bridging between Fcγ receptors type II (CD32) on leukocytes, and clustered intergrins on adherent cell monolayers. This ARCA activity, facilitated by integrins αβ₁ or α⁴β₁, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the α⁴ cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although α⁴ tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM-1 to intact α⁴-transfected K562 cells. In conclusion, the integrin α chain tail can positively regulate integrin-dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand-binding considerations.     

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization

Ligation of very late antigen (VLA)-4 (α₄β₁ integrin) with a cross-linked anti-α₄ subunit monoclonal antibody (mAb) triggered a biphasic Ca²⁺ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca²⁺ response. Tumor necrosis factor-α-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca²⁺ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca²⁺ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca²⁺ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (～ 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca²⁺ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca²⁺ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca²⁺.     

Streptavidin blocks immune reactions mediated by fibronectin-VLA-5 recognition through an Arg-Gly-Asp mimicking site

Streptavidin is a biotin-binding analogue of egg-white avidin which is secreted by the bacterium Streptomyces avidinii. We have recently reported that streptavidin contains an Arg-Tyr-Asp-Ser (RYDS) sequence which exhibits structural homology to the Arg-Gly-Asp-Ser (RGDS) cell adhesion domain of fibronectin and other matrix-associated glycoproteins. Competition studies with RGD peptides indicated that streptavidin binds to cells via this site and that the binding is independent of biotin recognition. Since the RGD-containing peptide has been shown to play a key role in integrin-mediated cell adhesion, we assumed that streptavidin may utilize the RYDS site to bind to immune cells and thereby abrogate their adhesion-dependent functions. We now report that streptavidin modulates several atrix-dependent interactions of immune cells. In this context, immobilized streptavidin was found to support activated human CD4⁺ T cell adhesion in an RGD-specific, α₅β₁ dependent manner. In addition, soluble streptavidin (the commercially available or biotin-blocked forms) inhibited T cell adhesion to fibronectin and interfered with its co-stimulatory effect on tumor necrosis factor-a secretion by co-cultures of CD4+ T cells and macrophages. These results suggest that streptavidin is a novel example of a bacterial protein which utilizes RGD mimicry to interfere with integrin-mediated immune responses.     

L1 adhesion molecule on human lymphocytes and monocytes: expression and involvement in binding to αvβ3 integrin

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig-like domains. Besides the known L1-L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)-5 in the mouse and αvβ3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4⁺ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8⁺ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for αvβ3 on T lymphocytes, only a small proportion of cells were CD31⁺L1⁺ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of ∼220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD-containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1-Fc), we demonstrated the binding of human MED-B1 (αvβ3^hi, α5β1^lo) tumor cells and this binding was blocked by αv-specific mAb. In contrast, human Nalm-6 cells (αvβ3^lo, α5β1^hi) did not bind to the 6.L1-Fc fusion protein. MED-B1 cells could also be stained with the 6.L1-Fc fusion protein. Our results suggest that human L1 binds predominantly to αvβ3 and that its presence on leukocytes could be important for adhesion and migration.     

Adhesion molecules from the LFA-1/ICAM-1, 3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves' and Hashimoto's thyroid glands

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations. In this study, we examined by flow cytometry and immunohistochemical techniques, the expression of integrin receptors and their counter-receptors by infiltrating lymphocytes and vascular endothelium in thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). A high proportion of GD intrathyroidal T lymphocytes expressed the CD69 and gp95/85 (Ea2) activation antigens as well as an increased number of LFA-α_L, VLA-α1, -α4, -α5, and -β1 integrin receptors, as compared with peripheral blood T lymphocytes from the same patients. The expression of intercellular adhesion molecule (ICAM)-1 was increased in EC from GD and HT thyroids. In addition, an up-regulated de novo expression of vascular cell adhesion molecule (VCAM)-1 was found in EC in GD and HT thyroids, with no reactivity in control thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM-1 and VCAM-1. In addition, most of intrathyroidal mononuclear cells expressed the ICAM-3 adhesion molecule. This enhanced expression of ICAM-1 and VCAM-1 by thyroid EC in GD and HT may reflect their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. Our data suggest that both the LFA-1/ICAM-1, ICAM-3 and VLA-4/VCAM-1 pathways could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.     

Increased Expression of Intercellular Adhesion Molecule-1 and Mucosal Adhesion Molecule α4β7 Integrin in Small Intestinal Mucosa of Adult Patients with Food Allergy

The mechanisms of adverse reactions to foods in the gastrointestinal tract are poorly understood. Previous studies of other atopic diseases and animal models suggest that adhesion molecules and mucosal lymphocytes may be implicated in the pathogenesis of food allergy (FA). The aim of our study was to investigate the expression of adhesion molecules and mucosal lymphocytes in duodena of patients with food allergies and of controls. Ten patients with FA to cereals (wheat, oats, and rye) or cow's milk and 9 control patients were included in the study. Quantitative analysis and immunohistochemical stainings for two pairs of adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), α₄β₇ integrin, and mucosal addressin cell adhesion molecule (MAdCAM-1) and lymphocyte markers on endoscopic duodenal biopsy specimens were performed. The villous structure and density of LFA-1-positive cells were normal in every biopsy specimen, but the patients had significantly more α₄β₇⁺ cells in the intraepithelial space (P = 0.01). The expression of ICAM-1 in the lamina propria of patients with FA was also substantially increased (P = 0.003); however, staining with MAdCAM showed no intergroup difference. Moreover, we found significantly increased CD4⁺ and HLA-DR⁺ cells in the lamina propria of patients, in comparison to the controls, P = 0.05 and P = 0.04, respectively. The densities of CD3, CD8, HLA-DP, T cell receptor αβ⁺ and γδ⁺ cells and IgA-, IgA₁-, and IgA₂-containing cells did not differ in the two groups studied. Our results suggest that the increased expression of ICAM-1 and α₄β₇ integrin may play an important role in the pathogenesis of food hypersensitivity and with the elevation of CD4- and HLA-DR-positive cells reflect a stage of inflammation in the structurally normal intestines.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Sphingosine‐1‐phosphate activates chemokine‐promoted myeloma cell adhesion and migration involving α4β1 integrin function

Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1‐dependent myeloma cell adhesion is up‐regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine‐1‐phosphate (S1P) regulates immune cell trafficking upon binding to G‐protein‐coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up‐regulated the α4β1‐mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high‐affinity α4β1 that efficiently bound the α4β1 ligand VCAM‐1, a finding that was associated with S1P‐triggered increase in talin‐β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1‐dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2‐Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1‐dependent adhesion and migration of myeloma cells by CXCL12‐S1P combined activities might have important consequences for myeloma disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.     

Integrin α1β1 (VLA-1) mediates adhesion of activated intraepithelial lymphocytes to collagen

Intraepithelial lymphocytes (IELs) from human intestinal epithelium are memory CD8+ T cells that bind to epithelial cells through human mycosal lymphocyte (HML)-1 and to mesenchymal cells through very late activation antigen-4 (VLA-4). Their binding of extracellular matrix proteins and the mechanism involved were tested. Activated 51Cr-labelled lymphocytes were incubated in protein-coated microwells with various additives. After washing, the adherent cells were detected by radioactivity. The percentages of activated IELs that bound to collagen types I and IV were 20 and 31%, respectively; fewer bound to fibronectin or laminin. Compared to interleukin-2-activated peripheral blood CD8+ T lymphocytes, more IELs bound collagen IV and fewer bound fibronectin. IEL adhesion to collagen (but not fibronectin or laminin) was up-regulated by antibody ligation of CD2 or by protein kinase C stimulation by phorbol ester; staurosporine reduced binding, while herbimycin, phytohaemagglutinin and CD3 ligation had no effect. Antibody-blocking of integrin VLA-1 subunits alpha1 (CD49a) and beta1 (CD18) inhibited adhesion to collagen type I by 82+/-6% and to type IV by 94+/-1% (P<0.001), implicating VLA-1 as the main collagen receptor for IELs. Cell adhesion was dependent on extracellular divalent cations, a characteristic event of VLA-1 never before shown for IELs: manganese and magnesium ions supported binding in a dose-dependent manner; calcium ions inhibited their effectiveness. Therefore, IELs bind collagen through integrin alpha1beta1 after protein kinase C activation. Adhesion is modulated by divalent cations.     

A surrogate method for assessment of β2-integrin-dependent adhesion of human eosinophils to ICAM-1

We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by β₂-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene. The number of adherent eosinophils on BSA or ICAM-1 coated microplates was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA or ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for β₂-integrin; neither α-CD49d mAb directed against the α₄-chain or α-CD29 directed against the common β₁-chain of VLA-4 blocked adhesion to BSA or ICAM-1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to β₂-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.     

Loss of expression of alpha4beta7 integrin and L-selectin is associated with high-grade progression of low-grade MALT lymphoma.

Expression of adhesion molecule in low-grade B-cell mucosa-associated lymphoid tissue (MALT) lymphoma of the gastrointestinal tract has been reported in recent years, but these reports have primarily focused on low-grade gastrointestinal MALT lymphoma. In this study, we examined the lymphocytic homing receptor alpha4beta7 integrin, L-selectin, and VLA-4 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in low-grade lymphoma of the gastrointestinal tract and other organs such as the ocular adnexa and thyroid. We also observed changes in the expression pattern associated with high-grade transformation. Neoplastic cells in the gastrointestinal low-grade lymphoma and the low-grade component of high-grade MALT lymphoma were found to be alpha4beta7 integrin(+), L-selectin(+), whereas the gastrointestinal high-grade component and diffuse large B-cell lymphoma were found to be alpha4beta7 integrin(-), L-selectin(-). High endothelial venules in the gastric MALT lymphomas expressed MAdCAM-1. In the ocular adnexa low-grade MALT lymphoma, most cases were alpha4beta7 integrin(-), L-selectin(+); and in the thyroid, most cases of both low- and high-grade MALT lymphoma were alpha4beta7 integrin(-), L-selectin(-). These findings show that alpha4beta7 integrin and L-selectin may play an important role in the lymphocyte homing of gastrointestinal low-grade MALT lymphoma and in the loss of alpha4beta7 integrin expression throughout the course of high-grade progression.     

Human T lymphocytes bind to germinal centers of human tonsils via integrin α4/VCAM-1 and LFA-1/ICAM−1 and −2

Binding of T lymphocytes within the different compartments of the secondary lymphoid organs is crucial for the function of the cellular and the humoral immune response. It is still not known which adhesion molecules guide T cells to the distinct areas of the lymphoid microenvironment. In the current study an in situ adhesion assay was used to define the receptors for binding of T cells to human tonsils. The T cell lines Jurkat and MOLT-4 and normal, activated T cells were found to bind exclusively to germinal centers. Jurkat cells used the receptor pair integrin-α4 (VLA-4α)/VCAM-1, whereas activated MOLT-4 cells and normal T cells bound via both adhesion pathways, namely via integrin-α4/VCAM-1 and LFA-1/ICAM-1 and -2. It is suggested that these adhesion mechanisms are involved in the migration of T cells into the germinal centers of secondary lymphoid organs and that they influence the selection of B cells by apoptosis.