CXCR 3 activation promotes lymphocyte transendothelial migration across human hepatic endothelium under fluid flow

T cells infiltrating the inflamed liver express high levels of CXCR 3 and show enhanced migration to CXCR 3 ligands in chemotactic assays. Moreover, CXCR 3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR 3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR 3(high) lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR 3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR 3 ligands secreted by the endothelium or by exogenous CXCR 3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR 3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver.     

MEMD, a new cell adhesion molecule in metastasizing human melanoma cell lines, is identical to ALCAM (activated leukocyte cell adhesion molecule).

From a differential mRNA display comparing a non- and a highly metastasizing human melanoma cell line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma cell lines of a larger panel. The encoded protein, MEMD, is identical to activated leukocyte cell adhesion molecule (ALCAM), a recently identified ligand of CD6. ALCAM is involved in homophylic (ALCAM-ALCAM) and heterophylic (ALCAM-CD6) cell adhesion interactions. We have studied MEMD/ALCAM cell-cell interactions between human melanoma cells. The expression of this cell adhesion molecule not only correlates with enhanced metastatic properties and with aggregational behavior of human melanoma cells as tested by FACS analysis, but transfection experiments also make clear that MEMD/ALCAM expression is essential for cell-cell interaction of the investigated human melanoma cells. As the melanoma cell lines analyzed are all CD6 negative, these results strongly suggest that MEMD/ALCAM is an adhesion molecule mediating homophylic clustering of melanoma cells. MEMD/ALCAM expression is not restricted to subsets of leukocytes and melanoma cells, it can also be found in healthy organs and in several other malignant tumor cell lines. Besides, MEMD/ALCAM is also expressed in cultured endothelial cells, pericytes and melanocytes, in xenografts derived from the radial and vertical growth phase and in 4 of 13 melanoma metastasis lesions. The potential role is discussed of MEMD/ALCAM mediated cell-cell interactions in migration of mobile cells (ie, activated leukocytes, metastasizing tumor cells) through tissues.     

Anti-adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: Correlation with B cell differentiation stage

We show that major histocompatibility complex (MHC) class II molecules on B cells transmit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-γ-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphopro-liferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.     

The role of sialylated Lewis antigens on hematogenous metastases of human pancreas carcinoma cell lines in vivo

Previous studies have shown that sialyl Lewis a (SLea) and sialyl Lewis x (SLex) correlated to hematogenous metastasis of human cancers. Although SLea/SLex and E-selectin act as a set of adhesion molecules in vitro, it is not clear whether the in vivo correlation is exclusively mediated by the adhesion function. To address this issue, we investigated whether or not the role of SLea/SLex antigens on hematogenous metastasis to the liver in SCID mice was exclusively mediated by adhesion by using antibodies for these antigens and SLea/SLEx-negative, human pancreas adenocarcinoma cell line PCI-6. The absence of SLea/SLex expression was supported by the absent flow cytometric detection of the antigens as well as by the absent attachment augmentation to activated endothelial cells. PCI-6 cells are xenotransplantable to nude and SCID mice and produce vascular endothelial cell growth factor (VEGF) in a significant amount. PCI-6 cells, 1 x 10(6), were injected into the spleens of SCID mice, and resultant liver metastases were evaluated six weeks later. We observed an inhibitory effect on the establishment and growth of metastatic colonies when anti-SLea or anti-SLex antibody was administered. This indicates that SLea/x antigens have an important in vivo role, even in the metastasis of SLea/SLex-negative tumor cells. This implies that there may be an in vivo function of SLea/x antigens other than that of the attachment between tumor and endothelial cells.     

The Rap GTPases mediate CXCL13- and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes

The localization of B cells to lymphoid organs where they can become activated and differentiate into antibody-secreting plasma cells is controlled by multiple chemoattractants that promote cell migration and integrin-mediated adhesion. CXCL13 and sphingosine 1-phosphate (S1P) are two important chemoattractants that control the trafficking of B cells. CXCL13 directs B lymphocytes to lymphoid follicles where they receive survival signals and, if activated, undergo a germinal center response. In contrast, S1P allows B cells and plasma cells to exit lymphoid organs and re-enter the circulation. The Rap1 GTPase is a key regulator of cell adhesion and cell migration in a number of systems. We now show that Rap activation is required for CXCL13 and S1P to induce B cell migration as well as adhesion to ICAM-1 and VCAM-1. We also show that Pyk2, a tyrosine kinase involved in cytoskeleton rearrangements and B cell migration, is a downstream target of both CXCL13 and S1P signaling and that Rap activation is important for CXCL13 and S1P to stimulate tyrosine phosphorylation of Pyk2, a modification that increases Pyk2 kinase activity. This suggests that the ability of CXCL13 and S1P to direct the trafficking and localization of B cells in vivo may be dependent on Rap activation.     

Cytomegalovirus-infected neuroblastoma cells exhibit augmented invasiveness mediated by beta1alpha5 integrin (VLA-5)

Previously, experimental in vivo results showed that the productively and persistently human cytomegalovirus (HCMV)-infected neuroblastoma cell line UKF-NB-4AD169 exhibits a more malignant phenotype than the non-infected variant UKF-NB-4. To prove the assumption that enhanced malignancy may be due to enhanced invasive potential of the infected cells we studied interactions of both lines with monolayers of cultured endothelial cells. UKF-NB-4AD169 cells adhered to and transmigrated through endothelial monolayer to a significantly higher extent compared with UKF-NB4. Furthermore, the adhesion of UKF-NB-4AD169 but not of UKF-NB4 resulted in focal disruption of the monolayer integrity which facilitates tumor cell transmigration. Blocking antibodies directed against the beta1 integrin chain as well as beta1alpha5 on the tumor cells specifically inhibited adhesion in a concentration-dependent manner. When UKF-NB-4 were pretreated with a beta1 integrin activating antibody, focal disruption of the endothelial integrity also occurred. These findings lead us to suggest that HCMV infection activates beta1alpha5 in the host neuroblastoma cell which in turn enables these cells to tightly adhere to endothelial cells. In the presence of the protease inhibitor phenantroline, beta1alpha5-mediated adhesion was not impaired whereas UKF-NB4AD169-mediated endothelial monolayer permeabilization was dose dependently inhibited. We conclude that human cytomegalovirus infection contributes to augmented neuroblastoma invasiveness via adhesion of activated beta1alpha5 and subsequent matrix digestion by proteases.     

Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair

Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac-derived cells as three-dimensional cardiospheres may recapitulate a stem cell niche-like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self-assembled into stem cell niche-like structures in vitro in suspension culture, while exhibiting greater proportions of c-kit(+) cells and upregulated expression of SOX2 and Nanog. Pathway-focused polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunostaining revealed enhanced expression of stem cell-relevant factors and adhesion/extracellular-matrix molecules (ECM) in cardiospheres including IGF-1, histone deacetylase 2 (HDAC2), Tert, integrin-α(2), laminin-β(1), and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer-cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac-derived cells as cardiospheres mimics stem cell niche properties with enhanced "stemness" and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart.     

Aquaporin-3 expression in human fetal airway epithelial progenitor cells

Airway epithelium stem cells have not yet been prospectively identified, but it is generally assumed that both secretory and basal cells have the capacity to divide and differentiate. Previously, we developed a test for progenitor cells of the human airway epithelium, relying on the transplantation of fetal respiratory tissues into immunodeficient mice. In this study, we hypothesized that airway-repopulating epithelial progenitors can be marked with surface antigens, and we screened an array of such candidate markers, including lectin ligands, the CD44 and CD166 adhesion molecules, and the aquaporin-3 (AQP3) water channel. We observed that AQP3 is selectively expressed on the surface of basal cells, allowing the separation by flow cytometry of AQP3+ basal cells and AQP3- ciliated and secretory cells. Functional evaluation of sorted cells in vivo showed that AQP3+ cells can restore a normal pseudostratified, mucociliary epithelium as well as submucosal glands. AQP3- cells are also endowed with a similar potential, although faster engraftment suggests their inclusion of more committed progenitors. These results show that stem cell candidates in the human tracheo-bronchial mucosa can be positively selected with a novel marker but also, for the first time, that epithelial progenitors exist among both basal and suprabasal cell subsets within the human airway.     

Human mast cells release Interleukin-8 and induce neutrophil chemotaxis on contact with activated T cells

BACKGROUND: Mast cells have recently been shown to control neutrophil recruitment during T-cell mediated cutaneous DTH reaction in vivo through TNF-alpha and MIP-2, the functional murine analogue of human IL-8. Although the nature of signals transmitted from T cells which activate mast cells has not yet been defined, we hypothesized that a direct cross-talk (i.e. heterotypic adhesion) between these two cell populations exists, as has previously been reported. AIMS: The present study was aimed at gaining insight into the functional role of mast cell-T cell contact in expression and release of IL-8, and its effect on neutrophil chemotaxis. METHODS: The IL-8 gene expression was identified by Affymetrix GeneChip arrays, validated by RT-PCR and the protein measured by ELISA. Chemotaxis was evaluated by using a modified Boyden chamber assay. RESULTS: Mast cells were found to express and release significantly higher concentrations of IL-8 on incubation with membranes obtained from activated, as compared to resting T cells. Supernatants obtained from these activated mast cells induced significant neutrophil chemotaxis that was inhibited by neutralizing mAb to IL-8. CONCLUSIONS: Thus, activated T cells, on heterotypic adhesion to mast cells, deliver the necessary signals for the latter to release cytokines and chemokines necessary for cell migration to sites of antigen challenge, thereby facilitating T-cell mediated inflammatory processes.     

Expression of leukocyte adhesion molecules by endothelial cells seeded on various polymer surfaces

Although endothelial cell seeding in small-diameter vascular prostheses significantly improves graft survival, the detachment of adherent endothelial cells after the restoration of circulation remains one of the major obstacles. Because in vivo experiments indicate that leukocyte infiltration is involved in endothelial cell loss, we hypothesize that seeded endothelial cells become activated and express leukocyte adhesion molecules and cytokines because of an interaction with the underlying polymer surface. The aim of this study was to investigate the expression of the leukocyte adhesion molecules ICAM-1, VCAM-1, PECAM-1, and E-selectin by cultured human umbilical vein endothelial cells (HUVECs) and human adipose microvascular endothelial cells (HAMVECs). The cells were seeded on tissue culture poly(styrene) and the vascular graft materials Dacron and Teflon. The results of this study indicate that the expression of leukocyte adhesion molecules by cultured endothelial cells is mainly affected by the endothelial cell origin, that is, umbilical vein or adipose tissue. Expressions of both ICAM-1 and E-selectin by HUVECs and HAMVECs are characterized by the presence of two cell populations with distinct levels of expression. With respect to endothelial cell seeding in vascular prostheses, the increased expression of E-selectin by microvascular endothelial cells deserves further attention.     

The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig

Integrin-mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10-kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL-2-stimulated T lymphocytes (IL-2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5 – 10 nM, resulting in three- to sixfold increase in adhesion of IL-2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN-γ and TNF-α. These chemokines induce IL-2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL-2 T cells roll and rapidly adhere to IP10/Mig-expressing HUVEC, and anti-CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine-induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN-γ as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN-γ-dependent pathologies.     

Activation induces rapid and profound alterations in the trafficking of T cells

Activation and differentiation of lymphocytes have profound effects on their trafficking. Whereas naive T cells recirculate through lymphoid organs, activated cells localize predominantly in other compartments. Here, we report that changes in migratory properties of T cells occur immediately upon activation via the TCR. One hour stimulation is enough to target T cells into lung and liver following i.v. injection. The high localization within lung and liver and the lack of recirculation through lymphoid tissues are key features of activated lymphocytes. the source, in vitro as well as in vivo activated lymphocytes show this behavior, which is not caused by increased cell size. Accumulation in the lung requires protein synthesis and is partly mediated by LFA-1, in contrast to the acquisition of liver "homing" properties. Intravital microscopy reveals firm adhesion of activated cells within periportal sinusoids of the liver. Selective homing to other organs, such as skin or mucosa, was not observed, regardless of the cell's origin. These data indicate that activation quickly switches the trafficking program of lymphocytes from recirculation to sequestration; it is tempting to speculate that especially the induced trapping in the liver has a distinct role in limiting systemic T cell responses.     

Low activated leukocyte cell adhesion molecule expression is associated with advanced tumor stage and early prostate-specific antigen relapse in prostate cancer

Activated leukocyte cell adhesion molecule (CD166) is a member of the immunoglobulin superfamily and is aberrantly expressed in different tumors, including prostate cancer. To learn more on the prevalence and clinical significance of activated leukocyte cell adhesion molecule expression in prostate cancer, a tissue microarray containing 3261 primary prostate cancers treated by radical prostatectomy was used. A total of 2390 different prostate cancers were analyzed by immunohistochemistry in a tissue microarray format. Activated leukocyte cell adhesion molecule immunostaining in cancers was compared with clinical follow-up, which was available for 1746 patients. Membranous activated leukocyte cell adhesion molecule immunostaining was recorded in 1663 (69.6%) of cases. High activated leukocyte cell adhesion molecule expression levels were significantly associated with favorable tumor features (pT: P = .0015; pN: P = .0008; preoperative prostate-specific antigen: P = .0057) and a lower risk of a biochemical recurrence (P = .0067). Cytoplasmatic activated leukocyte cell adhesion molecule staining was usually associated with membranous staining. The small number of cancers with pure cytoplasmatic staining did not reveal any particularities with respect to clinical outcome or tumor phenotype. It is concluded that activated leukocyte cell adhesion molecule protein is almost always expressed in prostate cancer and that decreased levels of activated leukocyte cell adhesion molecule expression may lead to an aggressive behavior of tumor cells. The abundant presence of activated leukocyte cell adhesion molecule and its membranous localization in prostate cancer epithelium make activated leukocyte cell adhesion molecule a potentially attractive structure for targeted therapy.     

Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.

Vascular cell adhesion molecule-1 (VCAM-1), a mononuclear leukocyte adhesion molecule, is expressed in cultured vascular endothelial cells activated by cytokines and is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet. We now demonstrate that vascular smooth muscle cells can also express VCAM-1 in rabbit atherosclerotic lesions in vivo and in response to cytokines in vitro. Immunohistochemical staining of aortas from rabbits fed a 0.3% cholesterol-containing diet revealed that a portion of smooth muscle cells within intimal foam cell-rich lesions expressed VCAM-1. The intimal VCAM-1-expressing cells localized predominantly in regions above the internal elastic lamina. These VCAM-1-positive cells had the typical spindle shape of smooth muscle cells but had reduced alpha-actin expression in comparison to normal medial smooth muscle cells, and did not bear markers for endothelium, macrophages, and T cells. In culture, rabbit aortic smooth muscle cells expressed VCAM-1 mRNA and protein in a time- and concentration-dependent fashion when exposed to interferon-gamma or Gram-negative bacterial lipopolysaccharide. Cultured human vascular smooth muscle cells also expressed VCAM-1 mRNA and protein in response to lipopolysaccharide, interferon-gamma, and interleukin-4. The monokines interleukin-1 alpha and tumor necrosis factor-alpha did not induce VCAM-1 expression in either rabbit or human vascular smooth muscle cells. Inducible VCAM-1 expression by vascular smooth muscle cells in vivo during hypercholesterolemia and in vitro in response to certain cytokines suggests a broader range of VCAM-1 functions in vascular biology than heretofore appreciated.     

Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim

At the end of the T cell response, the majority of the activated T cells die. We activated Vbeta8(+) T cells with staphylococcal enterotoxin B (SEB) in vivo and monitored the expansion and deletion of Vbeta8(+) T cells. We found that, in response to SEB, activated T cells died in vivo in the absence of Fas or TNF-R signaling but not when they overexpressed human Bcl-2. We also found that Vbeta8(+) T cells from Bim-deficient mice are resistant to SEB-induced deletion. While Bim levels did not change, endogenous Bcl-2 levels within Vbeta8(+) T cells decrease following SEB injection. Thus, the death of superantigen-stimulated T cells in vivo is mediated by Bim and may be modulated by a decrease in Bcl-2.     

CD8(+)CD28(-) T lymphocytes from HIV-1-infected patients secrete factors that induce endothelial cell proliferation and acquisition of Kaposi's sarcoma cell features.

Kaposi's sarcoma (KS) develops more frequently in human immunodeficiency virus type 1 (HIV-1)-infected patients. In this study, we report that molecules released by CD8(+)CD28(-) T lymphocytes from HIV-1-infected patients promote endothelial-cell (EC) growth and induce ECs to acquire spindle cell morphology and upregulation of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular endothelial cell growth factor receptor-3 (VEGFR-3) (a typical feature of the KS cell phenotype). The effects observed on ECs cocultured with in vivo activated CD28(-) cells were partly reproduced when ECs were grown in medium containing interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). At concentrations similar to those found in the supernatant of in vivo activated CD28(-) cells, the two proinflammatory cytokines sustained EC growth and survival only when combined. We, therefore, conclude that CD28(-) T lymphocytes from HIV-1-infected patients exert their effect on ECs through a mechanism involving both IFN-gamma and TNF-alpha. This finding may have wide implications for our basic understanding of the immunopathology of KS.     

Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro

A prevalent belief in epidermal biology is that stem cells are highly clonogenic; that is, they have the ability to produce many large colonies in vitro. However, it has been well-established in hematology, and recently suggested in epithelial biology, that short-term in vitro clonogenic assays may not be reliable predictors of long-term in vivo repopulating ability. Numerous groups have shown that rapid adhesion to collagen selects for highly clonogenic keratinocytes, but it has not been demonstrated whether this subpopulation is enriched in stem cells as defined by long-term repopulating ability in vivo. We found that although rapid adhesion to collagen (within 5 minutes) selected for cells with increased short-term colony forming ability in vitro, these cells were not enriched in long-term proliferative ability in vitro or in repopulating ability in vivo after 9 weeks. Conversely, keratinocytes that did not adhere to collagen (after 20 minutes) were less clonogenic in short-term assays but possessed equivalent long-term proliferative ability in vitro and superior long-term repopulating ability in vivo. Both the rapidly adherent cell and not rapidly adherent cell populations contained small, noncomplex basaloid cells, expressed integrin alpha2 (a collagen IV receptor), and expressed the putative epidermal stem cell phenotype integrin alpha6(hi)CD71(lo). Our results indicate that the superior short-term colony forming ability of collagen-adherent murine keratinocytes does not correlate with long-term repopulating ability in vitro or in vivo and that proliferation in vitro is not a reliable surrogate for stem cell behavior in vivo.     

Activation of the small GTPase Rac2 via the B cell receptor regulates B cell adhesion and immunological-synapse formation

The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.