No abstract

SUMMARYAlthough it is known that dendritic cells (DC) migrate in response to inflammatory stimuli, there is little information about the expression of receptors for chemotactic factors on DC. The present study has demonstrated by double immunostaining and flow cytometry of Langerhan's cell (LC)-enriched epidermal cell suspensions that a small subpopulation (5-6%) of epidermal resident LC (rLC) expresses receptors for C5a (C5aR). Epidermal rLC positive for C5aR show a round-shape morphology, were located next to the basement membrane, and express HLA-DR molecules higher than C5aR negative rLC. These observations suggest that rLC would express C5aR as part of their process of maturation during tissue trafficking. To investigate whether epidermal LC up-regulate C5aR along their differentiation pathway, LC were differentiated in vitro after culture in epidermal cell suspensions supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, in vitro differentiated LC increased the expression of C5aR up to 69% of the DC population. In accordance with this observation, interdigitating DC of secondary lymphoid organs (lymph node and tonsil) also expressed C5aR. Migratory CD1a positive DC that spontaneously migrated out of dermal or split-skin organ explants were also positive for C5aR and were used for chemotaxis and chemokinesis assays in response to human recombinant C5a (rC5a). Optimum migration to rC5a was observed at 10 ÿ 8 M with a sigmoidal dose-response curve. Checkboard analysis demonstrated that locomotion in response to rC5a was chemotaxis and not chemokinesis.

SUMMARYIt is well established that granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and tumour necrosis factor-a (TNF-a) are involved in Langerhans' cell (LC) development and dendritic cell traffic. However, little is known about the pattern of cytokine receptors on human LC and their modulation during different stages of maturation. The expression of cytokine receptors was studied by flow cytometry on both freshly isolated LC (fLC) and 72-hr cultured LC (cLC). Epidermal cell suspensions enriched in LC were obtained after skin trypsinization and Ficoll-Hypaque gradient. LC were identified by their CD1a positivity. Although the majority of fLC were positive for the a chain of GM-CSF receptor (GM-CSFR), the b chain of GM-CSFR was detected only on 15% of CD1a cells. fLC were also positive for IL-1 receptor (IL-1R) type 1, IL-1R type 2, 75 000 molecular weight TNF receptor (TNFR) and interferon-g receptor (IFN-gR). IL-6R and its transducing signal gp130 were present in a subset of fLC. Granulocyte colony-stimulating factor receptor (G-CSFR), macrophage colony-stimulating factor receptor (M-CSFR), the a and b chain of IL-2R, IL-4R, IL-7R, IL-8R and 55 000 molecular weight TNFR were not detected on fLC. After culture, LC up-regulated the expression of both the a and b chains of GM-CSFR, IL-1R type 2, a and b chains of IL-2R, IL-6R and gp130. In contrast, IL-1R type 1 and 75 000 molecular weight TNFR were down-modulated and the expression of IFN-gR was not affected by culture. These results suggest that LC undergo changes in the cytokine receptor repertory during in vitro maturation.

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte-derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte-derived dendritic cells were obtained after culturing monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) (800 U/ml) and interleukin-4 (IL-4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen-DR (HLA-DR) and CD86. On day 5 of incubation, 20-67% of moDC cultured in the presence of HS (HS-moDC) expressed CD1a, b and c versus 94-97% when cultured in the presence of FBS (FBS-moDC). DC showed a differential gradient of adhesion to FN: FBS-moDC>HS-moDC>sDC approximately monocytes. Both FBS-moDC and HS-moDC were strongly positive for CD49e (alpha5-integrin) and CD29 (beta1-integrin) but negative for CD49d (alpha4-integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS-moDC and HS-moDC attached to endothelium (a 76% and 63% increase, respectively), only HS-moDC were able to migrate through non-activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS-moDC are less adhesive to FN and endothelial cells but more motile than FBS-moDC, and that alpha5beta1-integrin is the molecule involved in moDC adhesion to FN.