Catherine Massacrier

Up-Regulation of Macrophage Inflammatory Protein-3α/CCL20 and CC Chemokine Receptor 6 in Psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3α, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-α/IL-1β, CD40 ligand, IFN-γ, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.

Dendritic cell biology and regulation of dendritic cell trafficking by chemokines

DC (dendritic cells) represent an heterogeneous family of cells which function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. Then, following inflammatory stimuli, they leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. The key role of DC migration in their sentinel function led to the investigation of the chemokine responsiveness of DC populations during their development and maturation. These studies have shown that immature DC respond to many CC and CXC chemokines (MIP-lα, MIP-iβ, MIP-3α, MIP-5, MCP-3, MCP-4, RANTES, TECK and SDF-1) which are inducible upon inflammatory stimuli. Importantly, each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells migrate selectively to MIP-3α (via CCR6), blood CDllc+ DC to MCP chemokines (via CCR2), monocytes derived-DC respond to MIP-lα/β (via CCR1 and CCR5), while blood CDllc- DC precursors do not respond to any of these chemokines. All these chemokines are inducible upon inflammatory stimuli, in particular MIP-3α, which is only detected within inflamed epithelium, a site of antigen entry known to be infiltrated by immature DC. In contrast to immature DC, mature DC lose their responsiveness to most of these inflammatory chemokines through receptor down-regulation or desensitization, but acquire responsiveness to ELC/MIP-3β and SLCASCkine as a consequence of CCR7 up-regulation. ELC/MIP-3(3 and SLC/6Ckine are specifically expressed in the T-cell-rich areas where mature DC home to become interdigitating DC. Altogether, these observations suggest that the inflammatory chemokines secreted at the site of pathogen invasion will determine the DC subset recruited and will influence the class of the immune response initiated. In contrast, MIP-3β/6Ckine have a determinant role in the accumulation of antigen-loaded mature DC in T cell-rich areas of the draining lymph node, as illustrated by recent observations in mice deficient for CCR7 or SLC/6Ckine. A better understanding of the regulation of DC trafficking might offer new opportunities of therapeutic interventions to suppress, stimulate or deviate the immune response.

The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface

We generated monoclonal antibody (mAb) DCGM4 by immunization with human dendritic cells (DC) from CD34+ progenitors cultured with granulocyte‐macrophage colony‐stimulating factor and TNF‐α. mAb DCGM4 was selected for its reactivity with a cell surface epitope present only on a subset of DC. Reactivity was strongly enhanced by the Langerhans cell (LC) differentiation factor TGF‐β and down‐regulated by CD40 ligation. mAb DCGM4 selectively stained LC, hence we propose that the antigen be termed Langerin. mAb DCGM4 also stained intracytoplasmically, but neither colocalized with MHC class II nor with lysosomal LAMP‐1 markers. Notably, mAb DCGM4 was rapidly internalized at 37 °C, but did not gain access to MHC class II compartments. Finally, Langerin was immunoprecipitated as a 40‐kDa protein with a pI of 5.2 – 5.5. mAb DCGM4 will be useful to further characterize Langerin, an LC‐restricted molecule involved in routing of cell surface material in immature DC.

The Inducible CXCR3 Ligands Control Plasmacytoid Dendritic Cell Responsiveness to the Constitutive Chemokine Stromal Cell–derived Factor 1 (SDF-1)/CXCL12

The recruitment of selected dendritic cell (DC) subtypes conditions the class of the immune response. Here we show that the migration of human plasmacytoid DCs (pDCs), the blood natural interferon α–producing cells, is induced upon the collective action of inducible and constitutive chemokines. Despite expression of very high levels of CXCR3, pDCs do not respond efficiently to CXCR3 ligands. However, they migrate in response to the constitutive chemokine stromal cell–derived factor 1 (SDF-1)/CXCL12 and CXCR3 ligands synergize with SDF-1/CXCL12 to induce pDC migration. This synergy reflects a sensitizing effect of CXCR3 ligands, which, independently of a gradient and chemoattraction, decrease by 20–50-fold the threshold of sensitivity to SDF-1/CXCL12. Thus, the ability of the constitutive chemokine SDF-1/CXCL12 to induce pDC recruitment might be controlled by CXCR3 ligands released during inflammation such as in virus infection. SDF-1/CXCL12 and the CXCR3 ligands Mig/CXCL9 and ITAC/CXCL1 display adjacent expression both in secondary lymphoid organs and in inflamed epithelium from virus-induced pathologic lesions. Because pDCs express both the lymph node homing molecule l-selectin and the cutaneous homing molecule cutaneous lymphocyte antigen, the cooperation between inducible CXCR3 ligands and constitutive SDF-1/CXCL12 may regulate recruitment of pDCs either in lymph nodes or at peripheral sites of inflammation.

Human Langerhans Cells Express a Specific TLR Profile and Differentially Respond to Viruses and Gram-Positive Bacteria

Dendritic cells (DC) are APCs essential for the development of primary immune responses. In pluristratified epithelia, Langerhans cells (LC) are a critical subset of DC which take up Ags and migrate toward lymph nodes upon inflammatory stimuli. TLR allow detection of pathogen-associated molecular patterns (PAMP) by different DC subsets. The repertoire of TLR expressed by human LC is uncharacterized and their ability to directly respond to PAMP has not been systematically investigated. In this study, we show for the first time that freshly purified LC from human skin express mRNA encoding TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10. In addition, keratinocytes ex vivo display TLR1–5, TLR7, and TLR10. Accordingly, highly enriched immature LC efficiently respond to TLR2 agonists peptidoglycan and lipoteichoic acid from Gram-positive bacteria, and to dsRNA which engages TLR3. In contrast, LC do not directly sense TLR7/8 ligands and LPS from Gram-negative bacteria, which signals through TLR4. TLR engagement also results in cytokine production, with marked differences depending on the PAMP detected. TLR2 and TLR3 ligands increase IL-6 and IL-8 production, while dsRNA alone stimulates TNF-α release. Strikingly, only peptidoglycan triggers IL-10 secretion, thereby suggesting a specific function in tolerance to commensal Gram-positive bacteria. However, LC do not produce IL-12p70 or type I IFNs. In conclusion, human LC are equipped with TLR that enable direct detection of PAMP from viruses and Gram-positive bacteria, subsequent phenotypic maturation, and differential cytokine production. This implies a significant role for LC in the control of skin immune responses.

Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides

Toll‐like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single‐stranded RNA (ssRNA) act as TLR7 agonists in a sequence‐independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.

Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment: possible role at inflamed epithelial surfaces

To reach the site of antigen deposition at epithelial surfaces, dendritic cells (DC) have to traverse the endothelial barrier, progress through the tissue (i.e. dermis) and cross the dermo‐epithelial junction (basal membrane). In the present study, we demonstrate that (1) circulating blood DC and monocytes express high levels of CCR2 and primarily respond to monocyte chemotacticprotein (MCP) and not to macrophage inflammatory protein (MIP)‐3α/CCL20; (2) while the CD34+ hematopoietic progenitor cells (HPC)‐derived CD1a+ precursors committed to Langerhans cell differentiation primarily respond to MIP‐3α/CCL20, the HPC‐derived CD14+ precursors respond to both MCP and MIP‐3α/CCL20; (3) in concordance with the sequential expression of CCR2 and CCR6, the HPC‐derived CD14+ precursors initially acquire the ability to migrate in response to MCP‐4/CCL13 and subsequently in response to MIP‐3α/CCL20; and (4) in vivo, in inflamed epithelium, MCP‐4/CCL13 and MIP‐3α/CCL20 form complementary gradients, with MCP‐4/CCL13 expressed in basal epithelial cells at the contact of blood vessels, while MIP‐3α/CCL20 expression is restricted to epithelial cells bordering the external milieu. These observations suggest that the recruitment of DC to the site of infection is controlled by the sequential action of different chemokines: (i) CCR2+ circulating DC or DC precursors are mobilized into the tissue via the expression of MCP by cells lining blood vessels, and (ii) these cells traffic from the tissueto the site of pathogen invasion via the production of MIP‐3α/CL20 by epithelial cells and the up‐regulation of CCR6 in response to the tissue environment.

Regulation of dendritic cell recruitment by chemokines

Dendritic cells (DC) are a heterogeneous family of cells that function as sentinels of the immune system. This article summarizes observations suggesting that inflammatory chemokines secreted at the site of pathogen invasion determine the DC subset recruited and influence the class of the immune response initiated. Langerhans cells are selectively recruited by MIP-3&agr;/CCL20. In contrast, CCR7 ligands have a key role in the accumulation of antigen-loaded mature DC in T cell-rich areas of the draining lymph node. Improved understanding of the regulation of DC trafficking might offer new opportunities for therapeutic interventions to control immune responses.

RESPECTIVE INVOLVEMENT OF TGF-BETA AND IL-4 IN THE DEVELOPMENT OF LANGERHANS CELLS AND NON-LANGERHANS DENDRITIC CELLS FROM CD34+ PROGENITORS

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor β (TGF‐β) for its development. In this study, we show that endogenous TGF‐β is required for the development of both LC and non‐LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF‐β polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E‐cadherin+, Lag+CD68−, and Factor XIIIa−LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF‐β displayed overlapping functions with CD1a+ precursor‐derived DC. In particular, unlike CD14+‐derived DC obtained in the absence of TGF‐β, they neither secreted interleukin‐10 (IL‐10) on CD40 triggering nor stimulated the differentiation of CD40‐activated naive B cells. Finally, IL‐4, when combined with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), induced TGF‐β‐independent development of non‐LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM‐CSF and IL‐4 was TGF‐β independent. Collectively these results show that TGF‐β polarized CD34+ HPC differentiation toward LC, whereas IL‐4 induced non‐LC DC development independently of TGF‐β. J. Leukoc. Biol. 66: 781–791; 1999.

TLR3 as a Biomarker for the Therapeutic Efficacy of Double-stranded RNA in Breast Cancer

The discovery of a targeted therapeutic compound along with its companion predictive biomarker is a major goal of clinical development for a personalized anticancer therapy to date. Here we present evidence of the predictive value of TLR3 expression by tumor cells for the efficacy of Poly (A:U) dsRNA in 194 breast cancer patients enrolled in a randomized clinical trial. Adjuvant treatment with double-stranded RNA (dsRNA) was associated with a significant decrease in the risk of metastatic relapse in TLR3 positive but not in TLR3-negative breast cancers. Moreover, we show the functional relevance of TLR3 expression by human tumor cells for the antitumor effects mediated by dsRNA in several preclinical mouse models carried out in immunocompromised animals. These 2 independent lines of evidence relied upon the generation of a novel tool, an anti-TLR3 antibody (40F9.6) validated for routine detection of TLR3 expression on paraffin-embedded tissues. Altogether, these data suggest that dsRNA mediates its therapeutic effect through TLR3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with TLR3-positive cancers.