José Luis Rodríguez-Fernández

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.

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.

BOMBESIN, VASOPRESSIN, LYSOPHOSPHATIDIC ACID, AND SPHINGOSYLPHOSPHORYLCHOLINE INDUCE FOCAL ADHESION KINASE ACTIVATION IN INTACT SWISS 3T3 CELLS

Treatment of quiescent Swiss 3T3 cells with bombesin rapidly increased focal adhesion kinase (FAK)-associated tyrosine kinase activity in immune complexes. The effect was rapid (maximum at 2.5 min) and dose dependent (half-maximum response at 0.05 nm). Addition of vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine also elicited a rapid increase in FAK-associated tyrosine kinase activity. Addition of the selective Src inhibitor pyrazolopyrimidine directly to the in vitrokinase assay potently inhibited Src kinase activity induced by bombesin but did not affect the kinase activity of FAK measured by autophosphorylation or by synthetic substrate phosphorylation in paralell assays. In addition, Src activity was not detected in FAK immunoprecipitates using an optimal Src peptide substrate. Thus, agonist-induced tyrosine kinase activity measured in FAK immunoprecipitates is mediated by FAK activation rather than by co-immunoprecipitating Src. Bombesin-induced FAK activation is not dependent either on protein kinase C or Ca2+ mobilization but was completely blocked by treatment with cytochalasin D or by placing the cells in suspension. These findings indicate that FAK activation requires an intact actin cytoskeleton. Our results demonstrate that agonists that act via 7-transmembrane domain receptors stimulate FAK kinase activation.

Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)1/2, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells.

Background: The mechanisms used by chemokine CXCL12 to regulate the functions of mature dendritic cells are unknown. Results: CXCL12 controls chemotaxis and survival in these cells using CXCR4 and bifunctional signaling molecules. Conclusion: CXCR4 uses a redundant signaling pathway to control chemotaxis and survival in mature dendritic cells. Significance: CXCR4 may use a specific signaling signature to regulate the functions of dendritic cells. Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes Gi/Gβγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.

CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3β

Chemokine receptor CCR7 regulates chemotaxis and survival in mature dendritic cells (DCs). We studied the role of glycogen synthase kinase-3β (GSK3β) in the regulation of CCR7-dependent survival. We show that GSK3β behaves as a proapoptotic regulator in cultured monocyte-derived human DCs and murine splenic DCs in vitro, and in lymph node DCs in vivo. In keeping with its prosurvival role, stimulation of CCR7 induced phosphorylation/inhibition of GSK3β, which was mediated by the prosurvival regulator Akt1, but it was independent of ERK1/2, a key regulator of chemotaxis. Stimulation of CCR7 also induced translocation of two transcription-factor targets of Akt, prosurvival NF-κB and proapoptotic FOXO1, to the nucleus and cytosol, respectively, resulting in DCs with a phenotype more resistant to apoptotic stimuli. We analyzed if GSK3β was able to modulate the mobilizations of these transcription factors. Using pharmacological inhibitors, small interfering RNA, and a construct encoding constitutively active GSK3β, we show that active GSK3β fosters and hampers the translocations to the nucleus of FOXO and NF-κB, respectively. Inhibition of GSK3β resulted in the degradation of the NF-κB inhibitor IκB, indicating a mechanism whereby GSK3 can control the translocation of NF-κB to the nucleus. GSK3β and FOXO interacted in vivo, suggesting that this transcription factor could be a substrate of GSK3. The results provide a novel mechanism whereby active GSK3β contributes to regulate apoptosis in DCs. They also suggest that upon stimulation of CCR7, Akt-mediated phosphorylation/inhibition of GSK3β may be required to allow complete translocations of FOXO and NF-κB that confer DCs an extended survival.

Detecting apoptosis of leukocytes in mouse lymph nodes

Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6–6.5 h for preparation and analysis plus an additional 34–40 h to allow apoptosis of the injected DCs in the PLN.