Use of Intravital Multi-Photon Microscopy to Study In Vivo Migratory Kinetics of Corneal

Bone Marrow-Derived Cells

Pedram Hamrah, M.D.Pedram Hamrah, M.D., Dimosthenis Mantopoulos, MD; Lixin Zheng, MD; Ulrich von Andrian, MD, PhD

Massachusetts Eye & Ear Infirmary, Department of Ophthalmology & Immune Massachusetts Eye & Ear Infirmary, Department of Ophthalmology & Immune Disease Institute, Harvard Medical SchoolDisease Institute, Harvard Medical School

Financial Disclosure: The authors have no financial disclosure related to this projectFinancial Disclosure: The authors have no financial disclosure related to this project

Support: Support: NEI K12-EY016335, New England Corneal Transplant Research Fund, Falk Medical Research Trust

Antigen-presenting Cells

Sentinels of the immune system

Dendritic cells, macrophages and B cells

Dendritic Cells and macrophages are the

professional antigen-presenting cells

(APC) of the cornea

Implicated in corneal transplantation and

allergic immunity, microbial keratitis, and

dry eye disease

Corneal, unlike limbal, APC are universally MHC class II-negative and immature in the epithelium and stroma

Limbus

Green = Class II (Maturation marker)

Limbus

Red = CD45 (Bone marrow marker)

Green = Class II

Periphery

Red = CD11c(DC marker)

Green = CD80 (B7 costimulatory marker)

Center

Red = CD11c

Green = CD80

Hamrah et al., Novel Characterization of MHC class II-negative Population of Resident Corneal Langerhans cell-type Dendritic Cells. Invest Ophthalmol Vis Sci, 2002; 43:639-646

Hamrah et al., The Corneal Stroma is Endowed with Significant Numbers of Resident Dendritic Cells. Invest Ophthalmol Vis Sci, 2003; 44:581-589

Hamrah and Dana, Immune homeostasis of the eye: Antigen Presenting Cells in the Eye and Ocular Surface. Encyclopedia of the Eye. Elsevier. In press

Advantages of Multi-Photon Microscopy

Deeper imaging into scattering specimens

Reduced out of plane photobleaching and

photodamage in optically thick specimens

Access to nonlinear signals other than

fluorescence such as second harmonic

scattering

Purpose and Methods

The purpose of this study was to dissect migratory properties of

antigen presenting cells by novel multi-photon intravital microscopy.

Multi-photon intravital microscopy (MPM) of the cornea was applied

to investigate localization and trafficking properties of corneal APCs

in transgenic mice in steady state and inflammation in vivo.

CD11c-eYFP

MHC class II-eGFPDay 5 Inflammation

MHC class II-eGFPDay 5 inflammation

Normal Inflamed

Results Intravital MPM studies of the normal cornea demonstrated that

APCs were sparsely distributed centrally and more dense in the

periphery

Epithelial and stromal APCs were distinguished by second

harmonic generation that visualizes stromal collagen

While APCs demonstrated continuous sampling motions in steady

state, cells generally did not migrate laterally

During inflammation, increased numbers of APCs were

demonstrated, exhibiting extreme morphological changes

An increase in lateral and vertical migration was shown particularly

in stromal subpopulations

Conclusions Our studies are the first to demonstrate long-term migratory kinetics of corneal

APCs in steady state and inflammation through high-resolution intravital multi-

photon microscopy.

Collectively, these models allow for dissecting molecular regulation of APC

recruitment to, and migration in the cornea.