Dendritic Cell Tracking from the Eye and Skin, and the Functional Effects Thereof, Are Strictly Regional

Abstract

Abstract: : Purpose:To determine the migratory route of murine bone-marrow-derived dendritic cell (BMDC) following subcutaneous (s.c) and subconjunctival (s.conj) injection in vivo Methods:DC were enriched from BM cultures and the phenotype determined by flow cytometry (FACS). After labeling with PKH2-GL green fluorescent dye in vitro, PBS or 10₆ DC were injected into the right side of the nape of neck (s.c.), or subconjunctivally (s.conj) in the right eye of syngeneic mice. Cervical lymph nodes were then separately dissected as submandibular (SM) and superficial cervical (SC) lymph node (LN). Labeled DC were detected by confocal microscopy on LN sections or FACS analysis of LN cells at 24h and 48h after injection. In addition, LN cytokine production was tested by ELISA in supernatants from SC or SM LN cells in MLR culture. Results:BMDC were CD11c_high, CD86_low, MHC ClassII_low, CD80_neg, CD40_neg, CD8a_neg myeloid cells. After s.c. injection, labeled DC were found bilaterally in SC LN at 48h, but not in SM LN. In contrast, after s.conj injection, DC accumulated unilaterally only on the injected side in SM LN at 24h and 48h. Approximately 1×10₃ DC per draining LN were found. Low levels of IFN-gama and high levels of IL-10 were present in MLR supernatant only from cultured SC LN after s.c. injection and only from cultured SM LN after s.conj. injection respectively. Conclusion:This study has shown DC tracking from the eye and skin is strictly regional. DC from the eye only go to the SM LN where they induce an immune response, despite the close co-localization of the SM and SC LN. This suggest that there is no lymphatic communication between lymph nodes based on their anatomical proximity, and this has implication for DC trafficking from the tissues and lymphocyte recirculation generally.

Keywords: 436 injection • 365 conjunctiva • 471 microscopy: confocal/tunneling