Abstract Title

Impact of exogenous hyaluronan on dendritic cell activation and trafficking from skin

Presenter Name

Jessica Hersh

RAD Assignment Number

1201

Abstract

Introduction: Hyaluronan (HA) is a polysaccharide used as dermal filler in cosmetic dermatology. A minority of patients (

Purpose: This study is to determine whether HA at different molecular weights (M.W.) can be degraded into biologically active fragments inducing migration of APCs to draining lymph nodes causing contact hypersensitivity in mice.

Materials and Methods: Mice, divided into 3 groups of 4, were subcutaneously injected in their ears with Lifecore Biomedical HA of graded Molecular Weights (M.W.) (Group 1: 851 kDa-1.19 MDa, Group 2: 66kDa-90 kDa, Group 3:kDa). The positive control of HA tetrasaccharide (Group 4) and negative control of PBS (Group 5) were from Sigma-Aldrich. All antibodies (Ab) were from BD Pharmingin. Female BALB/c mice were injected with 200 µg HA/ear or the contralateral ear was injected with PBS (40 µL/ear). Mice were sacrificed 24 hours post-injection, auricular lymph nodes were collected, and HA and PBS draining lymph nodes were pooled separately. Ears were harvested after lymph node collection, and stored in PBS wetted gauze at -20ºC. Pooled lymph nodes were homogenized; cells were counted using a hemocytometer. Cells were incubated for 30 minutes on ice with Fc blocking Ab, stained with a phycoerythrin labeled MHC II specific Ab, or phycoerythrin labeled isotype control IgG Ab for 30 minutes on ice. DCs in the auricular lymph nodes were assessed using flow cytometry. Epidermal sheets were prepared and stained with anti-MHC II specific Ab. The number and morphology of Langerhans cells were evaluated microscopically.

Results: The control Ab showed insignificant binding while anti-MHC II Ab showed significant binding to a subpopulation of APCs stained for MHC II. HA with M.W. > 5000 Da did not cause MHC II positive cells to migrate out of the ears. None of the HA injections resulted in significant morphological changes or differences in Langerhans cell densities

Conclusion: HA >5,000 Da did not cause migration of APCs to draining lymph nodes. None of the injected HA preparations cause morphological changes or emigration of Langerhans cells.

Presentation Type

Poster

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Impact of exogenous hyaluronan on dendritic cell activation and trafficking from skin

Introduction: Hyaluronan (HA) is a polysaccharide used as dermal filler in cosmetic dermatology. A minority of patients (

Purpose: This study is to determine whether HA at different molecular weights (M.W.) can be degraded into biologically active fragments inducing migration of APCs to draining lymph nodes causing contact hypersensitivity in mice.

Materials and Methods: Mice, divided into 3 groups of 4, were subcutaneously injected in their ears with Lifecore Biomedical HA of graded Molecular Weights (M.W.) (Group 1: 851 kDa-1.19 MDa, Group 2: 66kDa-90 kDa, Group 3:kDa). The positive control of HA tetrasaccharide (Group 4) and negative control of PBS (Group 5) were from Sigma-Aldrich. All antibodies (Ab) were from BD Pharmingin. Female BALB/c mice were injected with 200 µg HA/ear or the contralateral ear was injected with PBS (40 µL/ear). Mice were sacrificed 24 hours post-injection, auricular lymph nodes were collected, and HA and PBS draining lymph nodes were pooled separately. Ears were harvested after lymph node collection, and stored in PBS wetted gauze at -20ºC. Pooled lymph nodes were homogenized; cells were counted using a hemocytometer. Cells were incubated for 30 minutes on ice with Fc blocking Ab, stained with a phycoerythrin labeled MHC II specific Ab, or phycoerythrin labeled isotype control IgG Ab for 30 minutes on ice. DCs in the auricular lymph nodes were assessed using flow cytometry. Epidermal sheets were prepared and stained with anti-MHC II specific Ab. The number and morphology of Langerhans cells were evaluated microscopically.

Results: The control Ab showed insignificant binding while anti-MHC II Ab showed significant binding to a subpopulation of APCs stained for MHC II. HA with M.W. > 5000 Da did not cause MHC II positive cells to migrate out of the ears. None of the HA injections resulted in significant morphological changes or differences in Langerhans cell densities

Conclusion: HA >5,000 Da did not cause migration of APCs to draining lymph nodes. None of the injected HA preparations cause morphological changes or emigration of Langerhans cells.