Carlos Cabañas

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

Chemokine stromal cell-derived factor-1α modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.

CXCR7 impact on CXCL12 biology and disease

It is known that the chemokine receptor CXCR7 (RDC1) can be engaged by both chemokines CXCL12 (SDF-1) and CXCL11 (I-TAC), but the exact expression pattern and function of CXCR7 is controversial. CXCR7 expression seems to be enhanced during pathological inflammation and tumor development, and emerging data suggest this receptor is an attractive therapeutic target for autoimmune diseases and cancer. CXCR7/CXCR4 heterodimerization, β-arrestin-mediated signaling, and modulation of CXCL12 responsiveness by CXCR7 suggest that the monogamous CXCR4/CXCL12 signaling axis is an oversimplified model that needs to be revisited. Consequently, research into CXCR7 biology is of great interest and further studies are warranted. This review summarizes recent findings about the CXCR7 receptor and analyses its impact on understanding the roles of CXCL12 biology in health and disease.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well‐established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin‐dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti‐CD9‐specific antibodies resulted in (i) increased β1 integrin‐mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF‐α, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF‐α, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration

Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5β1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5β1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

Immunological synapse formation inhibits, via NF-κB and FOXO1, the apoptosis of dendritic cells

The immunological synapse (IS) is a cell–cell junction formed between CD4+ T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-κB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

α4β1-Integrin Activation Is Necessary for High-Efficiency T-Cell Subset Interactions with VCAM-1 under Flow

Objective: The purpose of this study was to examine the relationship between α4β1‐integrin state of activation on CD4+ T‐cell subsets and their adhesive interaction to VCAM‐1 under flow.

Activation‐dependent changes in soluble fibronectin binding and expression of β1 integrin activation epitopes in T cells: relationship to T cell adhesion and migration

The relationship between activation‐dependent changes in β1 integrin conformation, T cell adhesion to immobilized fibronectin, and T cell migration in vitro was analyzed in this study. Stimulation of Jurkat T cells and peripheral T cells with Mn2+, the activating β1 integrin‐specific monoclonal antibody (mAb) TS2 / 16, CD2, or CD28 stimulation led to increased adhesion, soluble fibronectin (FN) binding and expression of the activation epitope defined by the β1 integrin mAb HUTS‐21. Phorbol 12‐myristate 13‐acetate treatment increased adhesion, but not soluble FN binding or HUTS‐21 epitope expression. In peripheral T cells, CD3 or CD7 stimulation also led to increased adhesion, soluble FN binding and HUTS‐21 epitope expression. Soluble FN blocked peripheral T cell adhesion induced by Mn2+ or TS2 / 16, but had no effect on adhesion induced by the other integrin‐activating signals. In contrast, migration induced by TS2 / 16, CD2, CD3, CD7 or CD28 stimulation was blocked by excess soluble FN. Phosphoinositide 3‐OH kinase (PI 3‐K) inhibitors blocked receptor‐mediated increases in cell adhesion, but not soluble FN binding or HUTS‐21 expression. Migration was similarly unaffected by PI 3‐K inhibitors, with the exception of CD7‐ and CD28‐induced migration, which was specifically blocked by LY294,002. These results suggest that activation‐dependent changes in β1 integrin conformation are PI 3‐K‐independent and are involved in T cell migration but not adhesion.