Analysis of antigen handling by epidermal Langerhans' cells during the early neonatal period

Antigen applied to the epidermis of 4-day-old mice results in antigen specific immune suppression and is linked to a decrease in the amount of antigen transported to the draining lymph node by epidermal Langerhans cells (LC). It was therefore proposed that a decrease in receptor-mediated endocytosis was responsible for the reduced antigen transport by the LC. Consequently the aims of this study were to characterise the expression of antigen uptake receptors during development of neonatal mice, evaluate their capacity to internalise antigens via receptor-mediated and fluid phase uptake and to assess antigen proteolysis and MHC-II complexing following exposure to maturation stimuli. Langerhans cells isolated from 4-day-old epidermis express reduced levels of the Ctype lectin receptors DEC-205 and Langerin in comparison to LC isolated from 6- week-old epidermis. Despite this reduction neonatal LC were efficient in the uptake of the mannosylated antigen FITC-dextran. When incubated in the presence of the macropinocytosis inhibitor wortmannin, antigen uptake was reduced to a greater extent in neonatal LC than adult counterparts. As such, neonatal LC preferentially utilise a wortmannin-sensitive fluid phase pathway, such as macropinocytosis, to internalise antigens such as FITC-dextran. Despite the presence of LAMP-1 +MHC-II+ vesicles within LC from neonatal epidermis, proteolysed DQ-OVA was localised to a MHC-II" compartment. This suggests that antigen was internalised via macropinocytosis and retained in a LAMPmacropinosome. As no differences were observed in DQ-OVA+ vesicle localisation, it was evident that neonatal and adult LC were associated with a comparable proteolytic activity. 24 hours following exposure to a maturationinducing dose of LPS, membrane MHC-II:peptide complexes were evident on adult LC, but not on neonatal LC. Although neonatal LC demonstrated both CD86 upregulation and MHC-II redistribution following LPS exposure, the transport of MHC-II:peptide complexes to the membrane was diminished. This reduced transport was not simply due to a defect in MHC-II recycling as LPS stimulation of LC caused a redistribution of MHC-II from an intracellular endosome to the cell surface. Therefore the reduced transport of MHC-II:peptide to the cell surface was most likely a consequence of a failure to associate with MHC-II in the cytoplasm. The capacity of neonatal LC to sample their external environment without inducing immunity has important biological consequences for the protection against inappropriate responses during the developmental period