Natividad Longo

DC-SIGN (CD209) Expression Is IL-4 Dependent and Is Negatively Regulated by IFN, TGF-β, and Anti-Inflammatory Agents

Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a monocyte-derived dendritic cell (MDDC)-specific lectin which participates in dendritic cell (DC) migration and DC-T lymphocyte interactions at the initiation of immune responses and enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to generate a high level of DC-SIGN mRNA and cell surface expression on immature MDDC. IL-4 was capable of inducing DC-SIGN expression on monocytes without affecting the expression of other MDDC differentiation markers. By contrast, IFN-α, IFN-γ, and TGF-β were identified as negative regulators of DC-SIGN expression, as they prevented the IL-4-dependent induction of DC-SIGN mRNA on monocytes, and a similar inhibitory effect was exerted by dexamethasone, an inhibitor of the monocyte-MDDC differentiation pathway. The relevance of the inhibitory action of dexamethasone, IFN, and TGF-β on DC-SIGN expression was emphasized by their ability to inhibit the DC-SIGN-dependent HIV-1 binding to differentiating MDDC. These results demonstrate that DC-SIGN, considered as a MDDC differentiation marker, is a molecule specifically expressed on IL-4-treated monocytes, and whose expression is subjected to a tight regulation by numerous cytokines and growth factors. This feature might help in the development of strategies to modulate the DC-SIGN-dependent cell surface attachment of HIV for therapeutic purposes.

Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1α (SDF-1α). Using Matrigel as a model, we show that SDF-1α promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1α was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1α triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1α, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-β1, a Matrigel component, whereas anti-transforming growth factor-β antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1α. The identification of SDF-1α as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

Migration of human blood dendritic cells across endothelial cell monolayers: adhesion molecules and chemokines involved in subset-specific transmigration

Distinct subsets of dendritic cells (DCs) are present in blood, probably “en route” to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c+) and plasmacytoid (CD123+) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c+ subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123+ DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell‐derived factor‐1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein‐1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node‐derived chemokine CCL21 (secondary lymphoid‐tissue chemokine) in response to microbial stimuli [viral double‐stranded (ds)RNA or bacterial CpG‐DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c+ DCs to CCL21, whereas a longer stimulation with CpG‐DNA was needed to trigger CD123+ DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule‐1 to move across activated endothelium. CD123+ DCs required β2 and β1 integrins to transmigrate, whereas CD11c+ DCs may use integrin‐independent mechanisms to migrate across activated endothelium.

Maturation-Dependent Expression and Function of the CD49d Integrin on Monocyte-Derived Human Dendritic Cells

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/β7) was induced/up-regulated during TNF-α- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and β7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-α treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N-acetylcysteine, which inhibits NF-κB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.

Clinical significance of CXCR3 and CXCR4 expression in primary melanoma.

Tumor cell migration involved in metastases is a tightly regulated, nonrandom process. Chemokines have been identified as critical molecules guiding cell migration. We performed a prospective study to analyze a possible association between the expression of chemokine receptors CXCR3 and CXCR4 by primary melanoma and clinical outcome. Forty primary melanomas were available for analysis; 57% of the tumors expressed CXCR3 and 35% expressed CXCR4 by melanoma cells. At initial diagnosis, 5 patients had subclinical lymph node involvement and after a median follow‐up time of 32 months, 2 additional patients developed regional lymph node metastases and 5 patients developed distant metastases. The expression of CXCR4, but not CXCR3, by melanoma cells in primary lesions was significantly associated with the presence of ulceration, increased tumor thickness, a greater risk of developing regional and distant metastases and a higher mortality rate. Our study underscores the value of CXCR4 expression as a useful marker for predicting outcome in patients with localized melanoma. In addition, our findings support that, among chemokine receptors, CXCR4 might be an appropriate therapeutic target for adjuvant therapy in patients at risk for metastatic disease.

Regulated recruitment of DC-SIGN to cell- cell contact regions during zymosan-induced human dendritic cell aggregation

Zymosan is a β‐glucan, mannan‐rich yeast particle widely used to activate the inflammatory response of immune cells. We studied the zymosan‐binding potential of human dendritic cells (hDCs) by using specific carbohydrate inhibitors and blocking monoclonal antibodies. We show that DC‐specific intercellular adhesion molecule‐grabbing nonintegrin (DC‐SIGN) is a major nonopsonic recognition receptor for zymosan on hDCs. Indeed, blocking of DC‐SIGN inhibited the inflammatory response of DCs to zymosan. We compared the zymosan‐binding capacity of hDC‐SIGN to that of Dectin‐1 and complement receptor 3 (CR3), which are receptors involved in the nonopsonic recognition of these yeast‐derived particles. Dectin‐1‐ and DC‐SIGN‐K562 cells bound to zymosan particles, whereas CR3‐K562 cells did not. DC‐SIGN and Dectin‐1 were also expressed in COS cells to compare their ability to trigger particle internalization in a nonphagocytic cell line. DC‐SIGN transfectants were unable to internalize bound particles, indicating that DC‐SIGN is primarily involved in recognition but not in particle internalization. Zymosan induced a rapid DC aggregation that was accompanied by a dramatic change of DC‐SIGN distribution in the plasma membrane. Under resting conditions, DC‐SIGN was diffusely distributed through the cell surface, displaying clusters at the free leading edge. Upon zymosan treatment, DC‐SIGN was markedly redistributed to cell–cell contacts, supporting an adhesion role in DC–DC interactions. The mechanism(s) supporting DC‐SIGN‐mediated intercellular adhesion were further investigated by using DC‐SIGN‐K562 aggregation. DC‐SIGN was highly concentrated at points of cell–cell contact, suggesting a role for enhanced avidity during DC‐SIGN‐mediated intercellular adhesion.

The neuronal protein Kidins220 localizes in a raft compartment at the leading edge of motile immature dendritic cells

Kidins220, a protein predominantly expressed in neural tissues, is the first physiological substrate for protein kinase D (PKD). We show that Kidins220 is expressed in monocyte‐derived and in peripheral blood immature dendritic cells (im DC). Immature DC (im DC) migrate onto extracellular matrices changing cyclically from a highly polarized morphology (monopolar (MP) stage) to a morphologically symmetrical shape (bipolar (BP) stage). Kidins220 was localized on membrane protrusions at the leading edge or on both poles in MP and BP cells, respectively. CD43, CD44, ICAM‐3 and DC‐SIGN,and signaling molecules PKD, Arp2/3 were found at the leading edge in MP or on both edges in BP cells, showing an intriguing parallelism between morphology and localization of molecular components on the poles of the motile DC. F‐actin co‐localized and it was necessary for Kidins220 localization on the membrane in MP and BP cells. Kidins220 was also found in a raft compartment. Disruption of rafts with methyl‐β‐cyclodextrin induced rounding of the cells, inhibition of motility and lost of Kidins220 polarization. Our results describe for the first time the molecular components of the polesof motile im DC and indicate that a novel neuronal protein may be an important component among these molecules.

Mesenchymal Contribution to Recruitment, Infiltration, and Positioning of Leukocytes in Human Melanoma Tissues

To understand factors that regulate leukocyte entry and positioning within human melanoma tissues, we performed a multiparametric quantitative analysis of two separated regions: the intratumoral area and the peritumoral stroma. Using two mesenchymal markers, fibroblast activation protein (FAP) and CD90, we identified three subsets of mesenchymal cells (MCs): (i) intratumoral FAP(+)CD90(low/-) MC, (ii) peritumoral FAP(+)CD90(+) MC, and (iii) FAP(-)CD90(+) perivascular MC. We characterized CD90(+) MCs, which showed a stable CCL2-secretory phenotype when long-term expanded ex vivo, and heavily surrounded peritumoral Duffy antigen receptor for chemokine(+) (DARC) postcapillary venules, supporting a role for these vessels in peritumoral inflammatory leukocyte recruitment. Conversely, the intratumoral area was variably invaded by FAP(+)CD90(low/-) MCs that colocalized with a distinct extracellular matrix (ECM) network. A positive correlation was observed between intratumoral stromal cell/ECM networks and leukocyte infiltration among tumor cells (TCs), as well as in a stroma-dependent xenograft tumor model. Adoptively transferred T lymphocytes preferentially infiltrated tumors composed of TC+MC, compared with TCs only. Altogether, our results suggest that a variety of MCs contribute to regulate different steps of leukocyte tumor infiltration, that is, CD90(+) cells surrounding peritumoral vessels secrete CCL2 to recruit CCR2(+) leukocytes at the tumor periphery, whereas intratumoral FAP(+) cells organize a stromal scaffold that contact guide further invasion among densely packed tumor cells.