Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells.

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.

Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line.

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) +/- interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF +/- IL-4 suppressed T cell responses. Treatment of FSDC with IFN-gamma reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4+ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.

Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin.

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR-mycMH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2d,b+, I-Ad,b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER-MP 12-, Sca-1+, Sca-2+, NLDC-145-, B7.2+, B7.1-, J11d-, B220-, Thy-1-, and CD3-. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-gamma (IFN-gamma); in contrast, stem cell factor, IL-1 alpha and tumor necrosis factor-alpha had no effect. Preculture with IFN-gamma was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.

Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity.

We have developed a method to generate immortalized phagocytic and dendritic cell clones from various mouse tissues such as spleen, thymus, brain and bone marrow. The clones were phenotypically characterized and shown to retain the ability to respond to immune or inflammatory signals, e.g., IFN-gamma. Functional cytokine activity and nitric oxide production were maintained in activated macrophages, microglial and dendritic cell clones. Immune functions, such as antigen presentation was exhibited by all clones whereas tissue-specific properties such as the ability to respond to corticotropin-releasing hormone and produce beta-endorphin was shown in microglial cell clones but not in macrophage cell clones, indicating that heterogeneity of cells of the mononuclear-phagocytic lineage can be maintained in vitro after the immortalization procedure. Moreover, the continuous proliferation of the clones could be inhibited by various stimuli and further differentiation of the cells could be achieved in vitro.